Interaction of vasoactive intestinal peptide (VIP) with human peripheral blood lymphocytes: Specific binding and cyclic AMP production

Gen. Pharmac. Vol. 17, No. 2, pp. 185 189, 1986

0306-3623/86 $3.00 + 0.00 Copyright ¢(" 1986 Pergamon Press Ltd

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INTERACTION OF VASOACTIVE INTESTINAL PEPTIDE (VIP) WITH HUMAN PERIPHERAL BLOOD LYMPHOCYTES: SPECIFIC BINDING AND CYCLIC AMP PRODUCTION J. R. CALVO,* J. M. GUERRERO,P. MOLINERO,R. BLASCO and R. GOBERNA Department of Biochemistry, School of Medicine, Avda. Sfinchez Pizju/m 4, 41009 Sevilla, Spain (Received 14 May 1985)

Abstract--1. VIP binding sites and cyclic AMP production by the peptide have been studied in human blood mononuclear cells before and after selective depletion of or enrichment for T-lymphocytes, B-lymphocytes-K-NK cells and monocytes. 2. The specifically bound ~25I-labelled VIP correlated significantly with the presence of B-lymphocytes and/or cells of K-NK system. 3. The stoichiometric data were compatible with the existence of two classes of binding sites. 4. T-lymphocytes and monocytes did not show binding of the tracer. 5. The cyclic AMP production stimulated by VIP correlated significantly with the presence of B-lymphocytes and/or K-NK cells.

INTRODUCTION Vasoactive intestinal peptide (VIP) is an octacosapeptide isolated from the gut (Said and Mutt, 1970) that fails to behave as a typical hormone and now is considered as a locally acting substance or a neuromodulator released by VIP-containing nerve (Giachetti et aL, 1977). In this context, the physiological significance of VIP levels in blood remains unknown, and it has been suggested that they only reflect overflow of VIP released from nerve terminals (Laburthe et aL, 1979). We have shown (Guerrero et aL, 1981) the presence of binding sites with high affinity and specificity for VIP as well as an adenylate cyclase system highly sensitive to VIP in mononuclear cells isolated from human peripheral blood. Besides, VIP stimulates cyclic A M P - d e p e n d e n t protein kinase activity in these cells (Guerrero et al., 1984). These data suggest that VIP could be important in the regulation of mononuclear cell activity. Blood mononuclear cells, however, include different subpopulations such as T-lymphocytes, B-lymphocytes, monocytes and cells of uncertain origin ( K - N K cells). The cell type that specifically binds VIP in this mixture of lymphocytes and monocytes has not been clearly identified. In the present work, we address ourselves directly to this problem. By various depletion and enrichment experiments, we show that specific VIP binding and cyclic AMP-stimulating effect of VIP correlates with the presence of B-lymphocytes and/or cells of K - N K system and not with the presence of T-lymphocytes, monocytes or other cell types.

MATERIALS AND M E T H O D S

Cell preparations

Human peripheral blood was obtained from healthy adult volunteers and diluted 1:1 with 0.15 M NaCI. Mononuclear cells (MNC) were isolated by Ficoll-Hypaque density gradient (Boyum, 1968). The isolated ceils (about 65% T-lymphocytes, 20% B-lymphocytes, 10% monocytes and 5% K-NK cells) were resuspended in 0.15 M NaC1 and used for binding studies and for obtaining other mononuclear subpopulations. T-lymphocytes (TL) were isolated from MNC by sheep erythrocyte rosette formation (Weiner et al., 1973). After haemolysis of sheep erythrocytes, T-lymphocytes (99% TL) were resuspended in 0.15 M NaCI and used for experiments. The supernatant, consisting of B-lymphocytes (BL---60%), monocytes (M--30%) and K-NK cells (10%), was also used for experiments (BL-M-K-NK). Nonadherent lymphocytes (BL-TL-K-NK) and monocytes (M) were obtained from MNC by the technique of adherence as described by Arenson et al. (1980). Nonadherent cells (70% TL, 23% BL, 1% M and 6% K-NK cells) and monocytes (80% M) were resuspended in 0.15 M NaCI and used for experiments. Viability, as determined by trypan-blue exclusion, was always greater than 90%. TL were identified by sheep erythrocyte rosette formation (Weiner et al., 1973), BL by the use of fluorescein-conjugated rabbit antihuman immunoglobulin (Dickler and Kunkel, 1972) and monocytes by morphological criteria (Wintrobe, 1974) in cytocentrifuge smears stained with Giemsa's stain and nonspecific esterase staining (Yam et al., 1971). Cells of the K-NK system were identified by exclusion of B and T-lymphocytes. Erythrocytes were also isolated by Ficoll-Hypaque density gradient (Boyum, 1968) and platelets as described by Costa et al. (1977). Binding studies

*Correspondence should be addressed to J. R. Calvo, Department of Biochemistry, School of Medicine, Avda. S~inchez Pizju~n 4, Sevilla, Spain.

In a standard assay, cells (1.5 x 106 MNC, TL, BL-MK-NK, BL-TL-K-NK and M/ml; 5 x 106 erythrocytes/ml; 20 × 106 platelets/ml) were incubated at 15°C in 0.5ml of 185

186

J . R . CALVO et al.

3 5 m M Tris-HCl buffer (pH 7.5)/50mM N a C l / l . 4 % (w/v) bovine serum albumin/l mg/ml bacitracin/45 pM 125Ilabelled VIP in the absence or presence of increasing concentrations o f native peptide (0.1 100 nM). After 90 min incubation cell-bound peptide was separated by centrifugation (Freychet et al., 1971), then washed and radioactivity was determined. Data are reported as specific binding, i.e. total tracer bound minus the a m o u n t of tracer that was not displaced by 1 0 p M VIP (about 1% total radioactivity added).

Table 1. 12Sl-labelled VlP binding (%B) to human mononuclear cells (MNC), B-lymphocytes + monocytes + K-NK cells (BL-MK-NK), B-lymphocytes + T-lymphocytes + K-NK cells (BL-TLK-NK), T-lymphocytes (TL), monocytes (M), erythrocytes and platelets. Results of 12SI-VIP binding (%B) are expressed as the mean _+SEM of 5 or 3 separate experiments. Values reported were obtained by Scatchard analysis (Scatchard, 1949) of the data shown in Fig. 1 (Kd is the dissociation constant, nM. B.C. is the binding capacity, fmol/10 6 cells)

cA M P stimulation

MNC (n = 5)

Kd

In a standard assay, cells (0.5 × l06 M N C , TL, B L - M K - N K , B L - T L - K - N K and M/ml; 5 x l06 erythrocytes/ml) were incubated at 15°C in 0.5 ml o f 35 m M Tris-HC1, pH 7 . 5 / 5 0 m M N a C l / l . 4 % (w/v) bovine serum albumin/ l mg/ml bacitracin/0.2 m M 3-isobutyl- l-methyl-xanthine in the absence or presence of increasing concentrations of porcine VIP ( 0 . l - 1 0 0 n M ) . After 45 rain incubation the reaction was stopped by the addition of 2.5 ml methanol. The precipitate was removed by centrifugation, aliquots of the superuatant were evaporated and cyclic A M P was measured by a protein-binding assay (Gilman, 1970).

80 800

4.8 + 0.6

0.20 5.0

80 590

3.0 + 0.5

Kd

0.16

4.0

36 450

3.2 + 0.3

B.C.

Kd B.C. BL-TL-K-NK (n = 5)

TL (n = 5)

K~

B.C. M (n = 5)

K~

B.C.

Synthetic h u m a n VIP was purchased from Peninsula Laboratories (San Carlos, CA, U.S.A.); bacitracin, bovine serum albumin and Ficoll from Sigma (St Louis, MO, U.S.A.); 3-isobutyl- l -methyl-xanthine from Aldrich (Milwaukee, WI, U.S.A.); carrier-free Na )25I (IMS 30, 100 mCi/ml), 2,8[3H]adenosine 3',5'-cyclic phosphate ( T R K 498, 3 0 - 5 0 C i / m m o l ) from the Radiochemical Centre (Amersham, B U C K S , U.K.). H u m a n VIP was radioiodinated by using the modification of the chloramine T procedure described by Laburthe et al. (1977). The specific radioactivity of 125I-labelled VIP was about 250 Ci/g and had binding properties identical to those of native VIP (Prieto et al., 1979). All other chemicals were reagent grade.

Erythrocytes (n = 3)

K~

m

B,C.

m

Kd

B.C.

m

and the number of B-lymphocytes, T-lymphocytes or monocytes were determined. These include unseparated cell population (MNC) as well as populations impoverished of and enriched in T-lymphocytes and monocytes. Figure 1 shows the ):SI-labelled VIP binding to MNC (Fig. 1A), B L - T L - K - N K (Fig. 1B) and B L - M - K - N K (Fig. 1C) in absence and presence of increasing concentrations of native peptide. Maximal binding was similar in all subpopulations and it was

l:5l-labelled VIP binding to nonadherent peripheral blood lymphocytes and monocytes Figure 1 and Table 1 are a compilation of all the experiments in which both 125I-labelled VIP binding

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RESULTS

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Chemicals

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VIP binding sites High affinity Low affinity

Cell type

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Fig. 1. Competitive displacement of 125I-labelled VIP by unlabelled VIP from mononuclear cells (MNC) (A), B-lymphocytes + T-lymphocytes + K - N K cells ( B L - T L - K - N K ) (B) and B-lymphocytes + monocytes + K - N K cells ( B L - M - K - N K ) (C). Cells (1.5 x l06 cells/ml) were incubated with 45 pM VIP at 15°C for 90 rain in the absence or presence o f unlabelled VIP. Results are the m e a n +_ SEM of five separated experiments, determinations are made in triplicate. Scatchard analysis o f the data are shown in the inset.

Interaction of VIP with human blood lymphocytes "-'I0 o

• : - O. 3 3 7 6

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•

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: 0_

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4

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Monocyte

concentration

(% total

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Fig. 2. Correlation between the ~25I-labelledVIP binding to mononuclear cells (MNC) and the number of monocytes: monocytes were quantified by morphological criteria in cytocentrifuge smears stained with Giemsa's stain and with nonspecific esterase staining. Mononuclear cells (1.5 x 106 cells/ml) were incubated with 45 pM labelled VIP at 15°C for 90 min. Correlation coefficient (r) was calculated under the assumption that the paired data points were a random sample from a bivariate normal distribution. not related to the absence of monocytes (Fig. 1B) or T-lymphocytes (Fig. 1C). The presence of B-lymphocytes and cells of K - N K system was, however, constant. The Scatchard plots (1949) of the binding data were curvilinear with upward concavities (Fig. 1, insets). Since cooperative interactions between binding sites appeared to be absent (Guerrero et al., 1981), this result indicates the existence of a complex mixture of receptors. Two classes of VIP receptors could be defined with similar stoichiometric characteristics in MNC, B L - T L - K - N K , and B L - M - K - N K (Table 1): a high-affinity class (Kd = 0 . 1 - 0 . 2 n M ) with low

187

capacity (4-5 fmol/106 cells), and a low-affinity class ( K d = 3 6 - 8 0 n M ) with a high binding capacity (450-800 fmol/106 cells). Other experiments with Tlymphocytes isolated by sheep erythrocyte rosette formation and monocytes by technique of adherence did not show binding of the tracer (Table 1). Similar results were obtained with erythrocytes and platelets (Table 1). In the same way, Fig. 2 shows that the concentration of monocytes in MNC does not affect the ~:5I-labelled VIP binding. There was no correlation with the number of monocytes. Thus, of the two major cell types in blood mononuclear cells, nonadherent cells, and specially B-lymphocytes and cells of K - N K system, rather than monocytes account for the most of the VIP binding.

Cyclic A M P production by VIP in nonadherent peripheral blood lymphocytes and monocytes Figure 3 shows cyclic AMP production by VIP in MNC, B L - T L - K - N K and B L - M - K - N K in the absence and presence of increasing concentrations of native peptide. Half-maximal stimulation (Table 2) was similar in all subpopulations and was elicited between 0.06 and 0.16 nM VIP and maximal stimulation at about 10 nM VIP. Greatest cyclic A M P production was reached in MNC. The value of the ratio between cyclic A M P production, however, in the presence or absence of VIP was similar (Table 2). The absence of T-iymphocytes ( B L - M - K - N K ) decreased the cyclic A M P production by VIP but a subpopulation with a high percentage of T-lymphocytes (TL) did not show any effect of the peptide. In the other cell type, the presence (M) or absence (BLT L - K - N K ) of monocytes was inversely related to the cyclic A M P production by VIP (Fig. 3; Table 2).

25 DISCUSSION

20

Specific binding sites for VIP, the stimulatory effect of this peptide on adenylate cyclase and specific activation of cyclic AMP-dependent protein kinase have been studied previously in human blood mononuclear cells (Guerrero et al., 1981, 1984). These cells, however, include different subpopulations such as T-lymphocytes, B-lymphocytes, monocytes and cells of uncertain origin, mainly null cells or K cells and natural killer or N K cells (Klain, 1983). The purpose of this study was to identify the VIP binding cell present in Ficoll-Hypaque preparations of mono-

T ~ ' ~ i

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Fig. 3. Effect of increasing concentrations of VIP on cyclic AMP levels in mononuclear cells (MNC, • • ) , Blymphocytes + T-lymphocytes + K-NK cells (BL-TL-K-NK, O ©), B-lymphocytes + monocytes + K-NK cells (BLM-K-NK, • l ) and T-lymphocytes (TL, [] •). Cells (0.5 x 106 cells/ml) were incubated at 15°C for 45 min in the absence or presence of VIP. Results are the mean + SEM of five separate experiments. Determinations were made in triplicate.

Table 2. Cyclic AMP production in mononuclear cells (MNC), B-lymphocytes + T-lymphocytes + K-NK cells (BI-TL-K-NK), Blymphocytes + monocytes + K-NK cells (BL-M-K-NK), T-lymphocytes (TL) and monocytes (M). Results are expressed as pmol of cAMP produced in 106 cells, as the ratio of cAMP production in the presence or absence of VIP ( + V I P / - V I P ) and the VIP concentration is capable of reaching the half-maximal stimulation (Kin). Each value of cAMP production is the mean _+ SEM of 5 separate experiments

MNC (n = 5) B L - T L - K - N K (n = 5) B L - M - K - N K (n = 5) TL (n = 5) M (n=5)

-VIP

+VIP

5.6 _+ 2.2 3.3 ___0.8 2.1 + 0.6 1.3 _+0.2 1.0_+0.3

22.6 _ 3.5 15.7_+2.7 8.8 + 1.2 1.8 + 0.6 1.1_+0.7

+ VIP- V l P K,~ (nM) 4.0 4.7 4.7 1.3 1.1

0.09 0.06 0.02 ---

188

J. R. CALVOet al.

nuclear cells from human peripheral blood. Fractionation of this population by several techniques showed a highly significant positive correlation between VIP binding and the presence of B-lymphocytes and/or cells K-NK system, but no positive correlation with the presence of T-lymphocytes, monocytes, red blood cells or platelets. Depletion of T-lymphocytes (BLM - K - N K ) from the mixed population (MNC) by sheep erythrocyte rosette formation or depletion of monocytes ( B L - T L - K - N K ) by technique of adherence showed similar VIP binding. Other experiments with isolated T-lymphocytes, monocytes, red blood cells and platelets did not show binding of the tracer. Danek et al. (1983), however, show maximal VIP binding to nonadherent peripheral blood lymphocytes obtained by differential adherence to nylon wool columns. This cell preparation includes T-lymphocytes and at least 5% of K - N K cells ( O K T 3 - , O K T I I + , Leu7+). Our results clearly demonstrate no binding of VIP to an enriched population of T-lymphocytes (99%). With our technique (sheep erythrocyte rosette formation), K - N K cells are isolated together with B-lymphocytes. Because of that we cannot distinguish between VIP binding to B-lymphocytes or K - N K cells. But our results and the results of Danek et al. (1983), support the hypothesis that cells of the K - N K system may be the cell population that uniquely possesses VIP receptor. As suggested by Scatchard analysis two independent classes of VIP receptors can be defined with similar stoichiometric characteristics in unseparated (MNC) and separated subpopulations ( B L - T L K - N K and B L - M - K - N K ) : a class with high affinity (Kd=0.11q).20nM) and a class with low-affinity (Kd = 36-80 nM). This feature is common to all VIP receptors so far described (Christophe et al., 1976; Prieto et al., 1981, 1983) with the exception of rat brain membranes (Taylor and Pert, 1979) and enterocytes from guinea pig intestine (Binder et al., 1980), where a unique class of noninteracting receptor was found. The finite capacity of high-affinity binding sites (4-5 fmol/106 total cells) is much lower than that estimated in enterocytes (Prieto et al., 1979) or in the Molt 4b lymphoblastic cell line (Beed et al., 1983). A specific subpopulation bearing VIP receptors might explain the apparently lower number of binding sites in the total mononuclear cell population. The cyclic AMP production system present in mononuclear cells showed a high sensitivity to VIP. In unseparated (MNC) and separated subpopulations ( B L - T L - K - N K and B L - M - K - N K ) , this peptide stimulated cyclic A M P production at a concentration as low as 3 x 10 - H M, which is of the same order as that previously reported (Guerrero et al., 1981). Half-maximal stimulation of cyclic AMP production in the cell subpopulation studied (MNC, B L - T L K - N K and B L - M - K - N K ) was observed at 0.060.16 nM. This concentration of peptide is similar to the K~ (0.11-0.20 nM) of the high affinity binding sites. Furthermore, the concentration of peptide that determines the occupancy of all the high affinity binding sites and induces the maximal stimulation of cyclic A M P production are both 1 nM. These results suggest that only the coupling of VIP with the high affinity binding sites leads to stimulation of adenylate cyclase in mononuclear cells. Coupling of peptide

with low-affinity binding sites appears to result in no stimulation of cyclic AMP production. Thus, the site with high affinity would be involved in peptide action and the other low-affinity would be a silent binding site, as described for other systems (Abramowitz et al., 1979). The presence or absence of T-lymphocytes and monocytes did not affect these results. The basal and the stimulated concentrations of cyclic AMP were, however, different in the subpopulations studied, although the average of stimulation was similar (4.0-4.7 above basal values). This decrease of cyclic AMP concentration in the separated subpopulation is probably due to the different technique of isolation used. Other experiments with isolated T-lymphocytes and monocytes did not show any increase in cyclic A M P production by VIP, with a basal value of cyclic A M P concentration in T-lymphocytes that was similar to that described by other authors (Atkinson et al., 1975; Niaudet et al., 1976). In conclusion, our results suggest that the VIP binding capacity and the cyclic A M P production stimulated by the peptide in mononuclear cells, is due to the presence of B-lymphocytes and/or cells of K - N K system. A definitive identification of the VIP binding cell requires further studies and should allow one better to define the role of VIP in the regulation of immunologic function. REFERENCES Abramowitz J., Iyengar R. and Birnbaumer L. (1979) Guanyl nucleotides regulation of hormonally-responsive adenyl cyclases. Mol. Cell. Endocrinol. 16, 129-146. Arenson E. B., Epstein M. B. and Seeger R. C. (1980) Volumetric and functional heterogeneity of human monocytes. J. clin. Invest. 65, 613-618. Atkinson J. P., Wedner H. J. and Parker C. W. (1975) Two novel stimuli of cyclic adenosine 3,5-monophosphate (cAMP) in human lymphocytes. J. Immunol. 115, 10231028. Beed E. A., O'Dorisio M. S., O'Dorisio T. M. and Gaginella T. S. (1983) Demonstration of a functional receptor for vasoactive intestinal polypeptide on Molt 4b T lymphoblasts. Regul. Pep. 6, 1-8. Binder H. J., Lemp G. F. and Gardner J. D. (1980) Receptors for vasoactive intestinal peptide and secretin on small intestinal epithelial cells. Am. J. Physiol. 238, GI90-GI96. Boyum A. (1968) Separation of leukocytes from blood and bone marrow. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. clin. Lab. Invest. 21, 51-76. Costa J. L., Murphy D. L. and Kafka M. S. (1977) Demonstration and evaluation of apparent cytoplasmic and vesicular serotonin compartments in human platelets. Biochem. Pharmac. 26, 517 522. Christophe J. P., Conlon T. P. and Gardner J. D. (1976) Interaction of porcine vasoactive intestinal peptide with dispersed pancreatic acinar cells from guinea pig. J. biol. Chem. 251, 4629~,634. Danek A., O'Dorisio M. S., O'Dorisio T. M. and George J. M. (1983) Specific binding sites for vasoactive intestinal polypeptide on nonadherent peripheral blood lymphocytes. J. lmmunol. 131, 1173-1177. Dickler H. B. and Kunkel H. G. (1972) Interaction of aggregated gammaglobulin with B lymphocytes. J. exp. Med. 136, 191-196. Freychet P., Roth J. and Neville D. M. Jr (1971) Insulin receptors in the liver: specific binding of ~25I-lnsulinto the

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AMP in human B and T lymphocytes. Eur. J. lmmunol. 6, 834-840. Prieto J. C., Laburthe M. and Rosselin G. 0979) Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cells from rat. I. Characterization quantative aspects and structural requirements of binding sites. Eur. J. Biochem. 96, 229-239. Prieto J. C., Guerrero J. M., De Miguel C. and Goberna R. (1981) Interaction of vasoactive intestinal peptide with a cell line (HeLa) derived from human carcinoma of the cervix: binding to specific sites and stimulation of adenylate cyclase. Mol. Cell. Biochem. 37, 167-176. Prieto J. C. and Carmena M. J. (1983) Receptors for vasoactive intestinal peptide on isolated epithelial cells of rat ventral prostate. Biochim. biophys. Acta. 763, 408-413. Said S. I. and Mutt V. (1970) Polypeptide with broad biological activity. Science 169, 1217-1218. Scatchard G. (1949) The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51, 66(~672. Taylor D. P. and Pert C. B. (1979) Vasoactive intestinal polypeptide specific binding to rat brain membranes. Proc. hath. Acad. Sci. U.S.A. 761, 660-664. Weiner M. S., Bianco C. and Nussenzweig V. (1973) Enhance binding of neuraminidase-treated sheep erythrocytes to human T lymphocytes. Blood 42, 939 946. Wintrobe M. M. (1974) Clinical Hematology, 7th edn, pp. 221 285. Lea and Febiger, Philadelphia. Yam L. T., Li C. Y. and Crosby W. H. (1971) Cytochemical identification of monocytes and granulocytes. Am. J. olin. path. 55, 283-290.