Dual effect of vasoactive intestinal peptide on the mitogenic response of rabbit spleen lymphocytes

Regulatory Peptides, 27 (1990) 117-126

117

Elsevier REGPEP 00861

Dual effect of vasoactive intestinal peptide on the mitogenic response of rabbit spleen lymphocytes Sylviane Peuriere 1, Christiane Susini 2, J e a n - P i e r r e Esteve 2, Nicole Vaysse 2 and Louis E s c o u l a 1 IlNRA and 21NSERM, UI51, Toulouse (France)

(Received 19 January 1989;revised version received 27 July 1989; accepted 31 August 1989) Key words: Vasoactive intestinal peptide; Lymphocyte proliferation; Rabbit

Summary The effects of vasoactive intestinal peptide (VIP) have been investigated on the mitogenic response of rabbit spleen cells. Specific binding of 125I-VIP to these mononuclear cells is rapid and saturable. Analysis of binding reveals two classes of binding sites, a class of high-affinity binding sites with K D = 0.93 + 0.11 nM and maximal binding capacity of 2000 + 560 sites/cell, and a class of low-affinity binding sites with K D = 225 + 58 nM and maximal binding capacity of 280,000 + 60,000 sites/cell. The VIP regulatory effect on mitogen-stimulated rabbit spleen cell proliferation appears to be timedependent and bimodal. When VIP was added simultaneously with mitogens, it induced an inhibition of the proliferative response. With concanavalin A (Con A) or pokeweed mitogen (PWM), addition of 1 0 - S M VIP resulted in a maximal 30~o inhibition of [3H]thymidine incorporation after 96 h of culture. This inhibitory effect was significant at concentrations from 10- s - 1 0 - 6 M and half-maximal inhibition was obtained with 1.2.10 - 9 M VIP. By contrast, when rabbit spleen cells were preincubated for 18 h with VIP alone, the lymphocyte proliferative response to Con A was increased. However, this increase was mitogen-selective, since it was only observed when the T-cell mitogen Con A was used. The maximal response was obtained after 96 h of culture in the presence of Con A. The VIP stimulatory effect was dose-dependent with a maximal effect at 10- 7 M and a half-maximal effect at 1 . 7 . 1 0 - 9 M VIP. The effect of VIP was also time-dependent, since a 6 h preincubation was sufficient to induce a significant increase in the proliferative response which was maximal after an 18 h preincubation.

Correspondence: L. Escoula, INRA, BP3, 180 chemin de Tournefeuille,31931 Toulouse, Cedex, France.

0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)

118 Introduction

Recent information has suggested a relationship between neuroendocrinology and immunology [1-6] and particularly, numerous studies have implicated vasoactive intestinal peptide (VIP) as an immunoregulatory peptide in both mouse and man [7,8]. Specific receptors for VIP have been found on human peripheral blood mononuclear cells [9,10], on mouse [ 11 ] and on a human T-cell-derived culture line (Molt 4b) [ 12,13 ]. In vitro, incubation of mouse T-cells in the presence of VIP leads to dose-dependent alterations in the expression of VIP receptors and changes in the ability of the treated T-cells to localize in mesenteric lymph nodes and Peyer's patch lymphoid tissues [ 14]. An inhibitory effect of VIP on human natural killer cell function [15] and on the proliferative response of mouse lymphocytes to the T-cell mitogens concanavalin A (Con A) and phytohaemagglutinin (PHA) [11,16,17] has also been shown. The aim of the studies reported here was to demonstrate the presence of VIP receptors on rabbit spleen lymphocytes and to characterize the modulatory effects of VIP on lymphocyte mitogenic response.

Materials and Methods

Animals Male New Zealand 3-month-old rabbits (SAGA-INRA, Toulouse) were used throughout these studies. Chemicals Synthetic VIP and PHI (peptide histidine-isoleucinamide) were a generous gift from Dr. P. Robberecht (Bruxelles, Belgium); Somatostatin 14 was a kind gift from Dr. L. Moroder (Mtlnchen, F.R.G.); Ultroser S F (serum substitute without steroids)was from I.B.F.; RPMI 1640-medium, glutamin, bicarbonate buffer, Con A and PWM from Eurobio (France), penicillin and streptomycin from bioMerieux (France), heparin from Roche (France), [ 3H]thymidine from CEA, S aclay (France), lipoluma from Kontron (France), albumin from Boehringer-Mannheim, (F.R.G.), bacitracin and soybean trypsin inhibitor from Sigma. Cell suspensions Single-cell suspensions from spleen were prepared at room temperature by gentle teasing of the tissues in RPMI-1640 medium supplemented with 1 ~ heparin (5000 IU), 1 ~o glutamin (200 mM), 2.8 ~ bicarbonate buffer, 0.5 ~o penicillin (50,000 U/ml), 0.5 ~o streptomycin (25 mg/ml) and 1~o ultroser SF. Cells were treated by 0.83 ~o ammonium chloride to lyse erythrocytes and washed three times with RPMI 1640. The isolated cells (about 44~o T-lymphocytes, 45~o of B-lymphocytes and 9~o macrophages [18,19]) were checked by trypan blue dye exclusion method and found to be greater than 96 ~o viable. Cells were adjusted to a concentration of 10 6 cells/ml and used either for the lymphocyte blastogenesis test or for binding studies.

119

Lymphocyte blastogenesis test Spleen cells (10 6 cells/ml) were suspended in RPMI-supplemented medium and cultured in microtiter plates for 48, 56, 72 and 96 h at 37 °C in a 5~o CO2 atmosphere. Cells were stimulated with concanavalin A (Con A, 10 #g/ml) or Pokeweed mitogen (PWM, 2 #g/ml) - control responses - and various concentrations of VIP. To estimate the amount of VIP degradation in the incubation medium during the 18 h VIP preincubation, 0.1 nM 125I-VIP was incubated at 37 °C in the presence of 0.1/~M VIP and 1 • 106 cells/ml. After 18 h of incubation, the degradation of ~25I-VIP was quantified by using trichloroacetic acid precipitation method and it was about 80~o. 18 h prior to harvesting, the cells were labeled by adding I/~Ci of methyl-[ 3H]thymidine to each well. Lymphocytes were harvested on glass fiber filter discs using a semi-automatic cell harvester (Skatron Lierbyen, Norway). Dried filter discs were transfered to scintillation vials containing 3 ml of Lipoluma. Samples were counted in automatic liquid scintillation counter. Cultures were performed in triplicate. Basal responses of lymphocytes in the absence of mitogens was substracted and data were expressed as ~ of control and calculated according to the formula: ~o of control =

cpm of lymphocytes with VIP cpm of lymphocytes without VIP

x 100

Statistical significance was evaluated by one-way analysis of variance.

Iodination of VIP VIP was radioiodinated by the chlorarnine-T method and purified by cellulose absorption and bovine serum albumin elution as previously described [20]. Its specific radioactivity was found to be about 840 Ci/mmol. Binding studies Spleen cells (2. 106cells/ml) were suspended in Krebs-Ringer-Hepes medium containing 103 mM NaC1, 4.8 mM KC1, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.5 mM CaC12, 5 mM glucose, 2 mM glutamin, 24.5 mM Hepes, 1~o albumin, 0.3 mg/ml soybean trypsin inhibitor and 0.5 mg/ml bacitracin. This binding medium was adjusted to pH7.4. 300/A of the cell suspension were incubated with 0.1nM ~25I-VIP (50,000 cpm/assay) at 25 °C for 60 min in the presence of various concentrations of unlabeled VIP. In separate experiments, radioactivity labeled VIP, unlabeled VIP, PHI and Somatostatin were added. At the end of the incubation period, cell suspension was added to a microcentrifuge tube containing 300 #1 cold binding medium and centrifuged with a Sigma (2 MK) microcentrifuge at 10,000 gfor 1 min. Cells were washed two times with binding medium at 4 °C to remove unbound label and radioactivity in the pellet was measured. Specific binding was defined as the excess of binding over that in blanks containing 1 #M VIP. Degradation of ~25I-VIP (assessed after precipitation with 10~ trichloroacetic acid) was less than 5 ~ at the end of the incubation time.

120

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Time (mln)

Fig. 1. Time-course of 125I-VIPbinding to spleen cells. Lymphocytes (2.106 cells/ml) were incubated with 0.1 nM~2SI-VIPat 25 °C in the presence or absence of 10 -6 M unlabeled VIP. At specified times, specific binding was determined. In each experiment, each value was determined in duplicate, and results given are means ( _+S.E.M.) of three separate experiments.

Results

Binding studies I n c u b a t i o n o f spleen cells with 0.1 n M ~25I-VIP resulted in a r a p i d increase in specific cell binding o f the labeled peptide (Fig. 1). A t 25 ° C, specific binding o f tzsI-VIP reached a plateau value after 50 min incubation a n d stable binding was o b s e r v e d between 50 a n d 120 min. A t equilibrium, specific binding represented about 2.3 + 0.2~o o f total radioactivity (mean + S.E.M., n = 3), c o r r e s p o n d i n g to 2.1 + 0.2 pg VIP. There was a p r o p o r t i o n a l relationship between the extent o f binding and the cell concentration in the incubation mixture up to 16.106 cells/ml ( d a t a n o t shown). In further binding experiments, 2 . 1 0 6 cells/ml were used. U n l a b e l e d V I P competitively inhibited specific binding o f 125I-VIP to spleen cells (Fig. 2). The concentration o f native V I P giving half-maximal inhibition o f binding

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Fig. 2. Competitive displacement of 12sI-VIP binding to spleen cells by native VIP. Cells (2.106 cells/ml) were incubated for 60 rain at 25 °C with 0.1 nM~25I-VIP and increasing concentrations of native VIP. Specific binding was calculated at each concentration of unlabeled peptide. Each value was determined in triplicate, and results shown are representative of two experiments (S.E.M. values are < 10%). Inset, corresponding Scatchard plot.

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tog [Pe~lael (M) Fig. 3. Specificity of '251-VIP binding to spleen cells. Cells (2. 106ceUs/ml) were incubated with 0.1 nMI2SI-VIP in the presence of increasing concentrations of VIP ( m ) , PHI ( x ) or Somatostatin ((3) for 60 min at 25 ° C. Results are expressed as the percentage of specifically bound radioactivity in the absence of peptide.

(IC5o) was 1.6 + 0.9 nM. Analysis of the data obtained from binding were performed using the curve-fitting Ligand program. Scatchard analysis was curvilinear (Fig. 2, inset), suggesting the presence of two classes of VIP binding sites on rabbit spleen cells: a class of high-affinity binding sites with K D of 0.93 + 0.11 nM and a Bmax of 1.64 + 0.4 fmol/assay, corresponding to 2000 + 560 sites/cell and a class of low-affinity binding sites with K D = 225 + 58 nM and a B m a x of 284 + 94 fmol/assay, corresponding to 280,000 + 60,000 sites/cell. The specificity of '25I-VIP binding to its receptor has been investigated by the use of peptides either structurally related or not to VIP (Fig. 3). PHI was approximately an order of magnitude less effective than VIP in inhibiting '25I-VIP binding (IC5o = 20 nM). Somatostatin was not able to inhibit '25I-VIP at all for concentrations up to 1/~M.

Time-course of the action of PIP on mitogen stimulation of spleen cells The control proliferative response of spleen lymphocytes stimulated for 48, 56, 72 and 96 h with the T-cell mitogen (Con A) or with the T-cell-dependent, T- and B-cell mitogen TABLE I In vitro kinetic mitogen control response of rabbit spleen cells Cells (1 • 106/ml) were cultured for different times in the presence of mitogens. Results are cpm of [3H]thymidine incorporated per culture expressed as the mean ( ± SEM) of six experiments. Culture time

Con A (10/ag/ml)

PWM (2 #g/ml)

48 h 56 h 72 h 96 h

38006 41104 75523 63741

14760 20301 27229 22404

± ± ± ±

5053 9955 7873 10113

± ± ± ±

3289 6760 7384 5557

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Time (hours)

Fig. 4. Effect of 10- 7 M VIP on the proliferative response of spleen cells. At the initation of the culture (t = 0), VIP was added simultaneously with the mitogen Con A (10/,g/ml, a) or PWM (2 #g/ml, b). The results are means ( + S.E.M.) of six experiments. Data are expressed as percentage of [3H]thymidine incorporation of control as described in Materials and Methods. (*P < 0.05).

(PWM) is shown in Table I. Maximal response occurred at 72 h, whatever the mitogen and, as known for rabbit spleen lymphocytes, the response to Con A is of higher magnitude than the response to PWM [21]. As shown in Fig. 4, addition of 10 -7 M VIP to cell cultures induced an inhibition of the proliferative response to mitogens, whatever the time of culture, but the effect was only significant at 72 and 96 h. The inhibition reached 22 + 3.6~o at 72 h and 27 + 4 ~ at 96 h with Con A and 24 + 3~o and 27.5 + 2.5~o at 72 and 96 h, respectively, with PWM mitogen.

Effect of VIP concentrations on mitogen stimulation of spleen cell proliferation When cells were cultured for 96 h in the presence of Con A or PWM, the proliferative response of spleen cells was inhibited in a dose-dependent manner by addition of VIP at the start of the culture (Fig. 5). Whatever the mitogen, maximal inhibition was obtained from 10 - 8 M VIP and reached about 25 ~o. This effect remained constant for

higher concentrations of VIP, and half-maximal inhibition was obtained with 1.2. 10- 9 M VIP. a

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Fig. 5. Effect of VIP concentrations on the proliferative response of spleen cells stimulated with different mitogens. Cells were incubated for 96 h with or without Con A (10 #g/ml, a) or PWM (2 #g/ml, b) and different concentrations of VIP. The results are means ( + S.E.M.) of four experiments. Data are expressed as percentage of [3H]thymidine incorporation of control as described in Materials and Methods.

(*P < 0.05).

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Fig. 6. Effect of an 18 h preincubation with 10- 7 M VIP on the proliferation of spleen cells stimulated by Con A (10/~g/ml, a) or PWM (2 #g/ml, b). The results are means ( + S.E.M.) of six experiments. Data are expressed as percentage of [3H]thymidine incorporation of control as described in Materials and Methods. [3H]thymidine incorporation of control as described in Materials and Methods. (*P < 0.05).

Influence of VIP preincubation on the proliferative response W h e n spleen cells were exposed for 18 h to 10- 7 M VIP alone before addition of mitogens (Fig. 6), VIP enhanced the Con A stimulated proliferative response. This effect was culture-time-dependent since a significant increase (P < 0.05) was observed after 56 h incubation with Con A, and a maximal effect after 96 h incubation, this resulting in a 50~o increase in thymidine incorporation over control. This stimulatory effect of VIP was not due to an increase in the number o f viable ceils after VIPpreincubation as assessed by trypan blue dye exclusion method. At the end o f the 18 h preincubation, about 80yo o f VIP was degraded. On the other hand, V I P pretreatment o f the cells stimulated by P W M induced a slight increase of the cell proliferative response, but this effect was not significant (P > 0.05). VIP stimulatory effect on cell proliferation was dose-dependent. After 96 h o f culture with Con A, preincubation o f cells for 18 h with V I P induced a maximal and a halfmaximal increase in cell proliferation at 10 - 7 and 1.7.10 - 9 M VIP, respectively (Fig. 7). Furthermore, this effect was also preincubation-time-dependent. As shown in Fig. 8, a 6 h VIP pulsing period is sufficient to induce an increase of the Con A proliferative response, and a maximal effect was observed after a 18 h VIP preincubation. 180 0..J n,k-Z o o

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Fig. 7. Proliferative response of spleen cells stimulated with Con A (10 #g/ml) and preincubated for 18 h 11 _ 10-6 M concentrations of VIP.Then cells were incubated for 96 hwiththe mitogen.The results are means ( + S.E.M.) offour experiments. Data are expressed as percentage of [3H]thymidine incorporation of controls as described in Materials and Methods. (*P < 0.05).

with 10-

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Fig. 8. Cells were preincubated for 0, 1, 6, 18 and 24 h with 10 - 7 M VIP and then cells were cultured for 96 h with Con A (10/~g/ml). The results are m e a n s ( + S.E.M.) of four experiments. D a t a are expressed as percentage of [3H]thymidine incorporation of control as described in Materials and Methods.

(*P < 0.05).

Discussion

The results presented here showed the presence of specific binding sites for VIP on rabbit spleen cells. The binding of VIP is time-dependent and saturable. As observed on several gastrointestinal and immune cells [22], rabbit spleen cells exhibit two classes of VIP binding sites with KD (0.93 nM and 225 nM, respectively) in the same range than those observed on human mononuclear cells [9,23]. The good agreement between the KD of high-affinity VIP receptors and the half-maximal values for VIP to modulate cell proliferation suggests that high-affinity VIP receptors could mediate the biological action of VIP on rabbit lymphocyte proliferation. As previously reported on mouse lymphocytes stimulated by Con A [16,17], when added at the initation of the mitogen response, VIP inhibited the proliferation of rabbit spleen cells stimulated by mitogens in a dose-dependent manner. This effect was seen with the T-cell mitogen Con A and with the T-cell-dependent T- and B-cell mitogen PWM. Previous work in mouse demonstrated that T-cells rather than B-cells principally account for specific VIP binding and that VIP did not affect the response oflymphocytes to the B-cell mitogen LPS [ 11 ], suggesting that a T-cell-mediated mechanism may be involved in the inhibitory effect of VIP on lymphocyte proliferation [7,11,24]. The molecular mechanisms by which VIP inhibits the mitogenic response of T-cells after binding to specific receptors are not fully understood, but the involvement of stimulation of adenylate-cyclase activity has been proposed [7]. VIP receptor-adenylate cyclase coupling has been demonstrated and accumulation of cAMP in lymphocyte membrane preparations can be elicited by VIP [12,25]. Interestingly, preincubation of rabbit spleen lymphocytes with VIP resulted in a significant increase of the Con A mitogenic response. This stimulation was dosedependent and reached 50~o with 1.9.10- 9 M VIP. Stimulation of proliferation by VIP was also function of time: a 6 h VIP preincubation was necessary and sufficient to obtain a significant stimulatory effect. Thus, our results demonstrate that VIP modulates rabbit lymphocyte proliferation in a bimodal manner: when it was added at the initiation of the mitogen-stimulated culture,

125 the peptide inhibited the cell proliferation, and when VIP was added before the mitogen initiation of the culture, it stimulated the proliferation o f rabbit spleen cells. This immunomodulation is not mediated by an alteration o f the basal response of lymphocytes in the absence of mitogens (data not shown). It is to be noted that this dual effect of VIP on lymphocyte proliferation is not found with mice spleen cells since VIP induced an inhibition of mice lymphocyte proliferation whatever the time of addition o f VIP (unpublished data). However, such a VIP bimodal effect was also observed on h u m a n spontaneous cytotoxicity ( N K C C ) . Exposure for 1 h before the assay o f h u m a n peripheral blood lymphocytes effector cells to VIP increased N K C C , but addition of VIP for the duration of the assay directly to admixtures of effectors and targets, induced an inhibition of the N K C C [15]. A same bimodal effects is obtained with the prostaglandins E 2 (PGE2), another adenylate cyclase stimulatory agent [26]. A dual effect of P G E 2 on IL 2 activity [27] has also been demonstrated. When added to the T-cell mitogen P H A , P G E 2 inhibits I L 2 activity of h u m a n tonsillar lymphocytes, whereas a preincubation for 16 h with P G E 2 before the addition o f P H A leads to a 2-fold increase in I L 2 activity. Although the role of c A M P formation induced by VIP in mediating modulatory effects of VIP on rabbit spleen lymphocytes could not be excluded, other cellular processes may be involved. It is likely that VIP acts differentially on the different spleen mononuclear cells. Indeed, specific VIP binding sites, not associated with adenylate cyclase activation, have been demonstrated on h u m a n monocytes [28]. Further studies will be necessary to elucidate VIP interaction with intraceUular signal systems involved in the VIP modulation of cell proliferation. In summary, our studies show that VIP has complex effects on the Con A-stimulated rabbit spleen cells and m a y act as an inhibitor or an activator according to culture conditions. The significance of these effects in in vivo situations remains to be investigated.

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126 11 Ottaway, C.A. and Greenberg, G. R., Interaction of Vasoactive Intestinal Peptide with mouse lymphocytes: specific binding and the modulation of mitogen responses, J. Immunol., 132 (1984) 417-423. 12 Beed, E.A., O'Dorisio, M.S., O'Dorisio, T.M. and Gaginella, T.S., Demonstration of a functional receptor for Vasoactive Intestinal Polypeptide on Molt 4b T lymphoblasts, Regul. Pept., 6 (1983) 1-12. 13 Wood, C.L. and O'Dorisio, M.S., Covalent cross-linking of Vasoactive Intestinal Polypeptide to its receptors on intact human lymphoblasts, J. Biol. Chem., 260 (1985) 1243-1247. 14 Ottaway, C.A., In vitro alteration of receptors for Vasoactive Intestinal Peptide changes the in vivo localization of mouse T-cells, J. Exp. Med., 160 (1984) 1054-1069. 15 Rola-Pleszczynski, M., Bolduc, D. and St.-Pierre, S., The effects of Vasoactive Intestinal Peptide on human natural killer cell function, J. Immunol., 135 (1985) 2569-2573. 16 Stanisz, A.M., Befus, D. and 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 (1986) 152-156. 17 Krco, C.J., 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. 18 Mc Nicholas, J.M., Raffeld, M., Loken, M. R., Reiter, H. and Knight, K.L., Monoclonal antibodies to rabbit lymphoid cells: preparation and characterization of a T-cell specific antibody, Molec. Immunol., 18 (1981) 815-822. 19 Cavaillon, J.M., Udupa, T.N.S., Chou, C.T., Cinader, B. and Dubiski, S., Rabbit spleen B-lymphocytes as helper cells in lymphocyte activation by concanavalin A and phytohaemagglutinin, Scand. J. Immunol., 15 (1982) 49-54. 20 Robberecht, P., Coy, D.H., De Neef, P., Camus, J. C., Cauvin, A., Waelbroeck, M. and Christophe J., [D-Phe4] Peptide histidine-isoleucinamide ([D-Phe 4] PHI), a highly selective vasoactive intestinal peptide (VlP) agonist, discriminates VIP-preferring from secretin-preferring receptors in rat pancreatic membranes, Eur. J. Biochem., 165 (1987) 243-249. 21 Ozer, H. and Waksman, B.H., The response of rabbit lymphocytes to mitogens and alloantigens: evidence for T-cell heterogeneity, J. Immunol., 113 (1974) 1780-1792. 22 O'Dorisio, M.S., Biochemical characteristics of receptors for Vasoactive Intestinal Polypeptide in nervous, endocrine, and immune systems, Fed. Proc., 46 (1987) 192-195. 23 Wiik, P., Homologous regulation of adenylate cyclase-coupled receptors for Vasoactive Intestinal Peptide (VIP) on human mononuclear leucocytes, Regul. Pept., 20 (1988) 323-333. 24 Ottawa),, C.A., Selective effects of Vasoactive Intestinal Peptide on the mitogenic response of murine T-cells, Immunol., 62 (1987) 291-297. 25 O'Dorisio, M.S., Hermina, N.S., O'Dorisio, T.M. and Balcerzak, S.P., Vasoactive Intestinal Polypeptide modulation of lymphocyte adenylate cyclase, J. Immunol., 127 (1981) 2551-2554. 26 Kendall, R. A. and Targan, S., The dual effect ofprostaglandin (PGE2) and ethanol on the natural killer cytolytic process: effector activation and NK-cell-target cell conjugate lyric inhibition, J. Immunol., 125 (1980) 2770-2777. 27 Yamamoto, Y., Ohmura, T., Kawakami, K., Onoue, K. and Hidaka, H., Induction and regulation of human interleukin 2 gene expression: significance of protein kinase C activation, J. Biochem., 100 (1986) 333-340. 28 Wiik, P., Opstad, P.K. and Boyum, A., Binding of vasoactive intestinal polypeptide (VIP) by human blood monocytes: demonstration of specific binding sites, Regul. Pept., 12 (1985) 145-153.