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Vasoactive intestinal peptide enhances phorbol myristate acetate-induced chemiluminescence in human lymphocytes
Life Sciences, Vol. Printed in the U S A
51, pp.
1803-1810
Pergamon
Press
VASOACTIVE INTESTINAL PEPTIDE ENHANCES PHORBOL MYRISTATE ACETATE-INDUCED CHEMILUMINESCENCE IN HUMAN LYMPHOCYTES Miguel A. Lopez-Gonzalez, Juan M. Guerrero and Miguel Lucas * Departamento de Bioquimica M6dica y Biologfa Molecular, Hospital Universitario Virgen Macarena, Facultad de Medicina, Avda Sdnchez Pizju~n 4, 41009-Sevilla, Spain (Received
in final
form September
28,
1992)
Summary Phorbol-myristate-acetate (PMA) induced in lymphocytes the production or reactive oxygen intermediates in a process which was stimulated by the presence of vasoactive intestinal peptide (VlP) in a dose-dependent response at VIP concentrations in the range 1011-10 .8 M. The dissociation constant for the high-affinity receptors of VlP agreed with the ID5o of the activation of adenylate cyclase, and the IDso for the stimulation by VlP of PMA-induced chemiluminescence, which were close to 0.2 nM VlP. Forskolin produced in lymphocytes an effect quite similar to VlP. A comparison of the response to VlP and forskolin of lymphocytes and monocytes showed that, in contrast to forskolin, VIP failed to induce the above described effect in monocytes. A possible mechanism involving protein kinase C, which is activated by PMA, and an intracellular signal linked to VlP receptors is pointed out. This study further supports a role for VlP as a mediator in the neuroimmune system.
Vasoactive intestinal peptide (VIP) is a 28 amino acid neuropeptide that was isolated for the first time from porcine duodenum (1). VlP is now regarded as a member of the secretin-glucagon family of polypeptides (secretin, glucagon, growth hormone releasing factor, helodermin, GIP, VIP and PHI/PHM-27)(2-4) and is considered as a local hormone or a neuromodulator released by riP-containing nerves (5). There is increasing evidence that this neuropeptide plays a role in communication between the nervous and immune system (6-8). In this context, specific receptors for VlP have been demonstrated in human peripheral blood mononuclear cells (9-11), human blood monocytes (12, 13), mouse lymphocytes (14), rat lymphoid cells (15), rat peritoneal macrophages (16), and rabbit spleen lymphocytes (17). VlP has also been shown to activate adenylate cyclase in human lymphocyte membranes (18), to stimulate cyclic AMP accumulation and to activate cyclic AMP-dependent protein kinase in human peripheral blood mononuclear cells (9,11,19), and rat peritoneal macrophages (20). *To whom correspondence should be addressed
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1804
Chemiluminescence
in L y m p h o c y t e s
by V I P
Vol.
51, No.
23, 1992
On the other hand, VlP has been reported to inhibit the production of reactive oxygen compounds (respiratory burst) in monocytes activated by serum opsonized zymosan (21). In this paper, we demonstrate for the first time the enhancement by r I P of a process, as detected by luminol-dependent chemiluminescence, which is induced by PMA in human lymphocytes and leads to the production of reactive oxygen metabolites. A possible mechanism of action involving protein-kinase C modulation is pointed out. These results further support a role of VlP in the neuroimmune axis.
Material and Methods
Lymphocyte Isolation Solution was purchase from Lagitre SRL (Milano, Italy). Nycodenz Monocytes Solution was from Nycomed AS (Oslo). Synthetic rat r I P was from Peninsula Laboratories Europe (Merseyside, UK). Concanavalin A (con A), Forskolin, Luminol (5-amino-2,3-dihydro- 1,4-phthalazinedione),and PMA (4b-phorbol12b-myristate-13a-acetate) were from Sigma Chemical Co (St Louis, MO, USA). Cyclic AMP kit was from Amersham, U.K.. Others reagents were of analytical grade, and obtained from commercial sources. Human lymphocytes were prepared from freshly venesected blood of healthy donors according to (22). Briefly, blood was diluted 1/2 with 0.9% (w/v) NaCI and 6 ml aliquots were layered over 3 ml of lymphocytes isolation solution (HypaqueFicoll) and centrifuged (4°C, 20 rain, 600g) to obtain mononuclear leucocytes which were washed by low speed centrifugation (4°C, 5 min, 200g) in 0.9% NaCI. Thereafter, to eliminate monocytes of this preparation of mononuclear leucocytes, 5 ml of the suspension, 3xlOe/ml, were poured on a Petri disk and incubated at 37°C for 60 rain. Non-adhered cells were collected and washed by low speed centrifugation in 0.9% NaCI. Finally, cells were resuspended in PBS supplemented with 1.2 mM CaCI 2 and 1.2 mM MgCI 2. The cells were almost 100% lymphocytes, as they were identified by light microscopy after Giemsa staining. Human monocytes were prepared from leucocyte-rich plasma obtained by dextran sedimentation of freshly venesected blood according to (23). In brief, leucocyte-rich plasma, 6 ml, was layered over 3 ml Nycodenz monocytes solution, and centrifuged for 15 min at 600g. The band of monocytes at the interphase was collected and washed in 0.9% NaCI. Finally, monocytes were resuspended in PBS supplemented with 1.2 mM CaCI 2 and 1.2 mM MgCI 2. Monocytes were identified by light microscopy with Giemsa's stain. Oxygen radical production was analyzed by measuring luminol-dependent chemiluminescence according to (24) by means of a Berthold LB 9500 C luminometer, attached to a chart-recorder, modified to enable the injection of reagents via a microsyringe through a light-tight septum. All experiments were carried out at 37°C. Samples for chemiluminescence determination were prepared by adding aliquots of the human lymphocyte or monocyte suspensions to PBS buffer supplemented with 1.2 mM CaCI 2, 1.2 mM MgCI 2, 1 0 p M luminol, 2 Units/ml horseradish-peroxidase, and 100 pM sodium azide. Final volume was 1 ml and contained 2x105 lymphocytes or 4x104 monocytes. Further details are described in the legends to figures. The reduction of ferricytochrome c, 50 pM final concentration, was determined by the increase in the absorbance at 546 nm. The addition of 20 pg/ml superoxide dismutase to the reaction mixture inhibited by 100% the reduction of cytochrome induced by the effectors used in the present work. Appropriate control assays demonstrated no
Vol.
51, No. 23, 1992
Chemiluminescence
in Lymphocytes
by VIP
1805
spontaneous changes either in the chemiluminescence reaction or in the reduction of cytochrome c by any of the stimuli in the absence of cells. Besides, the solvents of the stimuli did not modify the activity of the cells in the above described tests.
Results In a set of experiments we studied the effect of PMA on lymphocytes and a dose-dependent induction of a chemiluminescence reaction was obtained. Maximal stimulation was reached at 50 nM PMA. VlP, by itself, failed to modify resting chemiluminescence of lymphocytes but a strong stimulation of PMA-induced chemiluminescence was produced when lymphocytes were preincubated with 10 .8 M VlP and thereafter activated with PMA, even at saturating PMA concentrations. The degree of stimulation by VlP approached 100% of the maximal response to PMA (see Fig. 1). The time lag required for VlP to produce the above described enhancement was close to 5 seconds and the best responses were obtained after 0.5-2 rain preincubation of lymphocytes in the presence of VlP. Longer preincubations failed to improve the response of lymphocytes; on the contrary a slightly decreased response was observed. We obtained a dose-dependent response of lymphocytes to VlP on the PMAinduced chemiluminescence which correlated rather well with the kinetic of cAMP production. In fact, maximal rate in both processes were obtained at VlP concentrations in the range 1-10 nM which induced cAMP production values close to 105 + 4 pmol /107 cells and a chemiluminescence stimulation of 8 0 - 1 0 0 % of the maximal response to PMA (see Fig. 2). IDso values were 0.2 nM and 0.18 nM VlP for the production of cAMP and chemiluminescence stimulation respectively.
Fig. 1 .4J ¢O E-
200
Dose-dependent effect of PMA on chemiluminescence reaction in lymphocytes. r 100 -r-I Lymphocytes (2x10 s) E were incubated for 2 rain at 37°C in the (1) J~ absence (-o-) and in the O I presence (-e-) of 10 nM O 50 100 VlP. PMA was added at PMA (nmol/1) the indicated final concentrations and the chemiluminescence rate was calculated from the actual traces of the chartrecords and the digital outputs of the luminometer. Values were normalized within each cell preparation as percent of the maximal rate, considering 100% the rate obtained in lymphocytes incubated in the absence of VlP and stimulated with 50 nM PMA. Results are the mean _+ SEM of experiments from at least three separate preparation of lymphocytes. C) r (t) (J U~
}
1806
Chemiluminescence
in
Lymphocytes
by
Vol.
VIP
51,
No.
100
-g u 0
50 E
n
° O II
'
'
'
-11 -10 -9 Log [VIP] (mol/l)
-8
'
'
I00 f-(D
0 C (D 0 (D ~, C -,--i E 221
50
I1)
o
0 li
i
-12
I
i
i
t
Fig. 2 Dose-dependent effect of VlP on PMA-induced chemiluminescence, and correlation with activation by VlP of cAMP production. In the upper figure, the activity of adenylatecyclase was determined in lymphocytes, 5x106/ml, incubated at 15°C for 60 rain in the presence of the indicated concentrations of rIP. Cyclic AMP was measured by radioimmunoassay. Results, given as pmol/107 cells, are the mean _+ SEM of three separate experiments. In the lower figure, the chemiluminescence reaction was assayed in either lymphocytes ( • ) or monocytes ( I3 ), which were incubated at 37°C in medium containing the indicated concentrations of VlP. After 2 rain incubation, 50 nM PMA was injected to trigger the chemiluminescence response. Values (mean + SEM of five separate experiments) are given as the increase in percent of the maximal rate obtained in the absence of VlP and stimulated with 50 nM PMA.
23,
1992
Vol.
51, NO. 23, 1992
Chemiluminescence
in Lymphocytes
by VlP
1807
In another set of experiments we studied the effect of VlP on the monocyte cell population prepared by density gradient centrifugation of mononuclear cells. VlP failed to produce in monocytes the above described effect on PMA-induced chemiluminescence in lymphocytes, in spite of the quite clear response induced by PMA alone. Only a minor effect, equivalent to a net increase of approximately 7% over the effect of PMA, was observed when monocytes were preincubated with 10 nM VlP (see Fig. 2) The effect of VlP on PMA-induced chemiluminescence in lymphocytes and monocytes was further studied in comparison to forskolin, a well-known stimulant of adenylate-cyclase. Fig 3 includes representative traces of the actual chemiluminescence records obtained in lymphocytes following preincubation with either VlP or forskolin and shows that forskolin also stimulated PMA-induced chemiluminescence and the degree of stimulation was almost identical to that produced by VlP. The same protocol was applied to monocytes where, in contrast to lymphocytes, we obtained a clear effect of forskolin whereas VlP failed to stimulate PMA-induced chemiluminescence (see Fig 3, lower panel). The results obtained in a number of experiments with the above mentioned agents and with concanavalin A are summarized in table I. Neither rIP, in a wide range of concentrations, forskolin, nor concanavalin A produced by themselves an increase of the resting chemiluminescence rate. Both forskolin and concanavalin A stimulated PMA-induced chemilunescence in monocytes. The most striking feature of these results was the stimulation by forskolin and concanavalin A of PMA-induced chemiluminescence in both cell types whereas the effect of VlP appears to be quite specific for lymphocytes.
Fig. 3
'min~_/b-~
d! \
oo i mlnQ
d-
Records of the chemiluminescence traces obtained from lymphocytes and monocytes stimulated with PMA, VIP, forskolin, and concanavalin A. 2x105 lymphocytes (upper panel), and 4x104 monocytes (lower panel) were incubated at 37°C in 1 ml PBS supplemented with 1.2 mM MgCI 2 and 1.2 mM CaCI 2 in the presence of the following agonists: none, a; l n M VIP, b; 100 nM forskolin, c and; 8 pg/ml concanavalin A, d. Incubation was in the thermostated chamber of the luminometer and after 2 rain PMA was added, see arrows, at a final concentration of 50 nM. Traces are hand-drawn copies of the actual records representative from at least three other cell batches.
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Table I
Stimulation of PMA-induced chemiluminescence by VIP, forskolin and concanavalin A
Chemiluminescence (% of resting rate)
Preincubation
Stimulus
LvmDhocvtes
None None None None r I P 1 nM Forskolin 100 nM Con A (8pg/ml)
PMA 50 nM r I P (0.001-10 nM) Forskolin 100 nM Con A (8 pg/ml) PMA 50 nM PMA 50 nM PMA 50 nM
100 0 0 2 _+ 1.5 188 ± 12 194 + 11 165 ± 33
Monocytes 100 0 0 0 100 + 10 133 ± 15 152 + 20
Lymphocytes (2 x 10 ~) and monocytes (4 x 103) were preincubated for 2 min at 37°C in 1 ml PBS supplemented with 1.2 mM MgCI 2 and 1.2 mM CaCI 2 in the presence of the indicated compounds. Stimuli were added and the chemiluminescence reaction continuously recorded (see Fig 3). Chemiluminescence values were normalized considering 100% the rate obtained following PMA stimulation of cells preincubated in the absence of any compound. Results are given as the mean ± SEM of five experiments.
Discussion
As far as we know, this study provides evidence for the first time of a chemiluminescence response of human lymphocytes to PMA and the enhancement of this effect by rIP. NK cells are a subpopulation of lymphocytes which has been described to produce reactive oxygen metabolites in response to a number of stimuli except to PMA, as determined by luminol-enhanced chemiluminescence (see Ref. 25), indicating that this cell subpopulation can not be the target of the agonists described in the present work since PMA is required to trigger the chemiluminescence reaction. A putative target cell in our experiment could be contaminating monocytes, a well known phagocytic and reactive oxygen intermediate producing cell. In spite of we routinously checked the absence of monocytes we decided to analyze the behavior of the monocyte under the described experimental conditions. Indeed monocytes produced a chemiluminescence response to PMA, as should be expected from a phagocytic cell, which was enhanced by forskolin and by the lectin concanavalin A, but unmodified by VIP. This observation agree with previously described experiments demonstrating the lack of effect of VIP on PMA-induced respiratory burst in monocytes (21). Therefore, our results are consistent with a rather specific effect of r I P on the chemiluminescence induced by PMA in lymphocytes.
Vol.
51, No. 23, 1992
Chemiluminescence
in Lymphocytes
by VIP
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It is well known that receptors of VlP, present in human lymphocytes (9) and monocytes (12), are coupled to the adenylate cyclase in both cell types (9, 21 ). The dissociation constant for the high-affinity receptors of VlP in human lymphocytes is 0.24 nM (9) and agree quite well with ID~o values for cAMP production and PMAinduced chemiluminescence, 0.2 and 0.18 nM VlP respectively, described in the present work. Forskolin, which acts directly on the catalytic subunit of adenylate cyclase, also stimulated PMA-induced chemiluminescence response in lymphocytes. These considerations point to cAMP as the linking between receptor signaling by VlP and protein kinase C, the intracellular receptor for PMA. If this were the case, similar results should be expected in monocytes, but certainly VlP failed to modify the response of monocytes. The actual oxygen radical intermediate responsible for the chemiluminescence reaction in lymphocytes has not been elucidated in the present work. Nonetheless we approached this question by measuring superoxide anion production. We studied the reduction of cytochrome c in human lymphocytes by preincubation of 1 2 x l 06 cells/ml at 37°C for 5 min in the presence and in the absence of 100 nM VIP and stimulated with 50 nM PMA at 37°C for 30 min. Lymphocytes preincubated in the absence of r I P produced 2.6 pmoles/min/106 cells, whereas in the presence of VlP the rate increased up to 4.9 pmoles/min/106 cells. These results indicate that superoxide anion is an intermediate oxygen metabolite in the activation by PMA and VlP. In conclusion, we have found that human lymphocytes are activated by PMA producing reactive oxygen intermediates in a process which is stimulated by VlP. In spite of the cell type producing the chemiluminescence response is unclear, the activation of protein kinase C by PMA could explain the main mechanism, whereas the involvement of cAMP-dependent kinase or any other unknown signal linked to VlP receptors remains to be studied.
Acknowledgements Supported by grant n ° 92/399 from the Fondo de Investigaciones Sanitarias de la Seguridad Social, and n ° PB-0906 from the Comisi6n Interministerial de Ciencia y Tecnologfa.
References 1. 2. 3. 4.
5. 6.
S.I. SAID, and V. MUTT, Science, 1 6 9 1 2 1 7 - 1 2 1 8 (1970) K. TATEMOTO, and V. MUTT, Proc. Natl. Acad. Sci. USA 7_88 6603-6607 (1981) R. GUILLEMIN, P. BRAZEAU, P. BOHLEN, F. ESCH, N. LING, and W.B. WEBRENBERG, Science 2 1 8 5 8 5 - 5 8 7 (1982) A. VANDERMEERS, P. GOURLET, M-C. VANDERMEERS-PIRET, A. CAUVlN, P. DE NEEF, J. RATHI~, M. SVOBODA, P. ROBBERECHT, and J. CHRISTOPHE, Eur. J. Biochem. 1 6 4 3 2 1 - 3 2 7 (1987) A. GIACHETTI, S.I. SAID, R.C. REYNOLDS, and F.C. KONIGES, Proc. Natl. Acad. Sci. USA 7 4 3 4 2 4 - 3 4 2 8 (1977) M.S. O'DORISlO, C.L. WOOD, and T.M. O'DORISlO, J. Immunol, 135. 792-796 (1985)
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7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
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in Lymphocytes
by VIP
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C.A. OTTAWAY, Ann. N.Y. Acad. Sci. 5 2 7 4 8 6 - 5 0 0 (1988) J. TSENG, and M.S. O'DORISIO, Neuroimmune networks: Physi01oav and Desease, E.F. Goetzl and N.H. Specter (eds), 105-111, Alan R. Liss, New York (1989) J.M. GUERRERO, J.C. PRIETO, F.L. ELORZA, R. RAMIREZ, and R. GOBERNA, Mol. Cell. Endocrinol. 2_! 151-160 (1981) A. DANEK, M.S. O'DORISIO, T.M. O'DORISIO, and J.M. GEORGE, J. Immunol. 1 3 1 1 1 7 3 - 1 1 7 7 (1983) J.R. CALVO, J.M. GUERRERO, P. MOLINERO, R. BLASCO, and R. GOBERNA, Gen. Pharmacol. 1 7 1 8 5 - 1 8 9 (1986) P. WlIK, P.K. OPSTAD, and A. BOYUM, Regul. Pept. 1__22145-153 (1985) P. WlIK, Regul. Pept. 3 5 1 9 - 3 0 (1991) C.A. OTTAWAY, and G.R. GRENBERG, J. Immunol. 1 3 2 4 1 7 - 4 2 3 (1984) J.R. CALVO, P. MOLINERO, J. JIMENEZ, R. GOBERNA, and J.M. GUERRERO, Peptides 7 1 7 7 - 1 8 1 (1986) J.J. SEGURA, J.M. GUERRERO, R. GOBERNA, and J.R. CALVO, Regul. Pept. 133-143 (1991) S. PEURIERE, CH. SUSINI, J-P. ESTEVE, N. VAYSSE, and L. ESCOULA, Regul. Pept. 2__77117-126 (1990) M.S. O'DORISIO, N.S. HERMINA, T.M. O'DORISIO, and P. BALCERZAK, J. Immunol. 1 2 7 2 5 5 1 - 2 5 5 6 (1981) J.M. GUERRERO, J.C. PRIETO, J.R. CALVO, and R. GOBERNA, Peptides 5 371-373 (1984) J.J. SEGURA, J.M. GUERRERO, R. GOBERNA, and J.R. CALVO, Regul. Pept. 3__7_7195-203 (1992) P. WlIK, Regul. Pept. 2.5187-197 (1989) A. BOYUM, Scand. J. Clin. Lab. Invest. 2__$1(Suppl. 97) 77-89 (1968) A. BOYUM, Scand. J. Immunol. 1__7_7429-436(1983) M. LUCAS AND F.SOLANO, Biochem. Pharmacol. 4 2 4 2 5 - 4 3 0 (1991) J.C. RODER, S.L. HELFAND, J. WERKMEISTER, R. McGARRY, T.J. BEAUMONT, and A. DUWE, Nature 2 9 8 5 6 9 - 5 7 2 (1982)
51, pp.
1803-1810
Pergamon
Press
VASOACTIVE INTESTINAL PEPTIDE ENHANCES PHORBOL MYRISTATE ACETATE-INDUCED CHEMILUMINESCENCE IN HUMAN LYMPHOCYTES Miguel A. Lopez-Gonzalez, Juan M. Guerrero and Miguel Lucas * Departamento de Bioquimica M6dica y Biologfa Molecular, Hospital Universitario Virgen Macarena, Facultad de Medicina, Avda Sdnchez Pizju~n 4, 41009-Sevilla, Spain (Received
in final
form September
28,
1992)
Summary Phorbol-myristate-acetate (PMA) induced in lymphocytes the production or reactive oxygen intermediates in a process which was stimulated by the presence of vasoactive intestinal peptide (VlP) in a dose-dependent response at VIP concentrations in the range 1011-10 .8 M. The dissociation constant for the high-affinity receptors of VlP agreed with the ID5o of the activation of adenylate cyclase, and the IDso for the stimulation by VlP of PMA-induced chemiluminescence, which were close to 0.2 nM VlP. Forskolin produced in lymphocytes an effect quite similar to VlP. A comparison of the response to VlP and forskolin of lymphocytes and monocytes showed that, in contrast to forskolin, VIP failed to induce the above described effect in monocytes. A possible mechanism involving protein kinase C, which is activated by PMA, and an intracellular signal linked to VlP receptors is pointed out. This study further supports a role for VlP as a mediator in the neuroimmune system.
Vasoactive intestinal peptide (VIP) is a 28 amino acid neuropeptide that was isolated for the first time from porcine duodenum (1). VlP is now regarded as a member of the secretin-glucagon family of polypeptides (secretin, glucagon, growth hormone releasing factor, helodermin, GIP, VIP and PHI/PHM-27)(2-4) and is considered as a local hormone or a neuromodulator released by riP-containing nerves (5). There is increasing evidence that this neuropeptide plays a role in communication between the nervous and immune system (6-8). In this context, specific receptors for VlP have been demonstrated in human peripheral blood mononuclear cells (9-11), human blood monocytes (12, 13), mouse lymphocytes (14), rat lymphoid cells (15), rat peritoneal macrophages (16), and rabbit spleen lymphocytes (17). VlP has also been shown to activate adenylate cyclase in human lymphocyte membranes (18), to stimulate cyclic AMP accumulation and to activate cyclic AMP-dependent protein kinase in human peripheral blood mononuclear cells (9,11,19), and rat peritoneal macrophages (20). *To whom correspondence should be addressed
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1804
Chemiluminescence
in L y m p h o c y t e s
by V I P
Vol.
51, No.
23, 1992
On the other hand, VlP has been reported to inhibit the production of reactive oxygen compounds (respiratory burst) in monocytes activated by serum opsonized zymosan (21). In this paper, we demonstrate for the first time the enhancement by r I P of a process, as detected by luminol-dependent chemiluminescence, which is induced by PMA in human lymphocytes and leads to the production of reactive oxygen metabolites. A possible mechanism of action involving protein-kinase C modulation is pointed out. These results further support a role of VlP in the neuroimmune axis.
Material and Methods
Lymphocyte Isolation Solution was purchase from Lagitre SRL (Milano, Italy). Nycodenz Monocytes Solution was from Nycomed AS (Oslo). Synthetic rat r I P was from Peninsula Laboratories Europe (Merseyside, UK). Concanavalin A (con A), Forskolin, Luminol (5-amino-2,3-dihydro- 1,4-phthalazinedione),and PMA (4b-phorbol12b-myristate-13a-acetate) were from Sigma Chemical Co (St Louis, MO, USA). Cyclic AMP kit was from Amersham, U.K.. Others reagents were of analytical grade, and obtained from commercial sources. Human lymphocytes were prepared from freshly venesected blood of healthy donors according to (22). Briefly, blood was diluted 1/2 with 0.9% (w/v) NaCI and 6 ml aliquots were layered over 3 ml of lymphocytes isolation solution (HypaqueFicoll) and centrifuged (4°C, 20 rain, 600g) to obtain mononuclear leucocytes which were washed by low speed centrifugation (4°C, 5 min, 200g) in 0.9% NaCI. Thereafter, to eliminate monocytes of this preparation of mononuclear leucocytes, 5 ml of the suspension, 3xlOe/ml, were poured on a Petri disk and incubated at 37°C for 60 rain. Non-adhered cells were collected and washed by low speed centrifugation in 0.9% NaCI. Finally, cells were resuspended in PBS supplemented with 1.2 mM CaCI 2 and 1.2 mM MgCI 2. The cells were almost 100% lymphocytes, as they were identified by light microscopy after Giemsa staining. Human monocytes were prepared from leucocyte-rich plasma obtained by dextran sedimentation of freshly venesected blood according to (23). In brief, leucocyte-rich plasma, 6 ml, was layered over 3 ml Nycodenz monocytes solution, and centrifuged for 15 min at 600g. The band of monocytes at the interphase was collected and washed in 0.9% NaCI. Finally, monocytes were resuspended in PBS supplemented with 1.2 mM CaCI 2 and 1.2 mM MgCI 2. Monocytes were identified by light microscopy with Giemsa's stain. Oxygen radical production was analyzed by measuring luminol-dependent chemiluminescence according to (24) by means of a Berthold LB 9500 C luminometer, attached to a chart-recorder, modified to enable the injection of reagents via a microsyringe through a light-tight septum. All experiments were carried out at 37°C. Samples for chemiluminescence determination were prepared by adding aliquots of the human lymphocyte or monocyte suspensions to PBS buffer supplemented with 1.2 mM CaCI 2, 1.2 mM MgCI 2, 1 0 p M luminol, 2 Units/ml horseradish-peroxidase, and 100 pM sodium azide. Final volume was 1 ml and contained 2x105 lymphocytes or 4x104 monocytes. Further details are described in the legends to figures. The reduction of ferricytochrome c, 50 pM final concentration, was determined by the increase in the absorbance at 546 nm. The addition of 20 pg/ml superoxide dismutase to the reaction mixture inhibited by 100% the reduction of cytochrome induced by the effectors used in the present work. Appropriate control assays demonstrated no
Vol.
51, No. 23, 1992
Chemiluminescence
in Lymphocytes
by VIP
1805
spontaneous changes either in the chemiluminescence reaction or in the reduction of cytochrome c by any of the stimuli in the absence of cells. Besides, the solvents of the stimuli did not modify the activity of the cells in the above described tests.
Results In a set of experiments we studied the effect of PMA on lymphocytes and a dose-dependent induction of a chemiluminescence reaction was obtained. Maximal stimulation was reached at 50 nM PMA. VlP, by itself, failed to modify resting chemiluminescence of lymphocytes but a strong stimulation of PMA-induced chemiluminescence was produced when lymphocytes were preincubated with 10 .8 M VlP and thereafter activated with PMA, even at saturating PMA concentrations. The degree of stimulation by VlP approached 100% of the maximal response to PMA (see Fig. 1). The time lag required for VlP to produce the above described enhancement was close to 5 seconds and the best responses were obtained after 0.5-2 rain preincubation of lymphocytes in the presence of VlP. Longer preincubations failed to improve the response of lymphocytes; on the contrary a slightly decreased response was observed. We obtained a dose-dependent response of lymphocytes to VlP on the PMAinduced chemiluminescence which correlated rather well with the kinetic of cAMP production. In fact, maximal rate in both processes were obtained at VlP concentrations in the range 1-10 nM which induced cAMP production values close to 105 + 4 pmol /107 cells and a chemiluminescence stimulation of 8 0 - 1 0 0 % of the maximal response to PMA (see Fig. 2). IDso values were 0.2 nM and 0.18 nM VlP for the production of cAMP and chemiluminescence stimulation respectively.
Fig. 1 .4J ¢O E-
200
Dose-dependent effect of PMA on chemiluminescence reaction in lymphocytes. r 100 -r-I Lymphocytes (2x10 s) E were incubated for 2 rain at 37°C in the (1) J~ absence (-o-) and in the O I presence (-e-) of 10 nM O 50 100 VlP. PMA was added at PMA (nmol/1) the indicated final concentrations and the chemiluminescence rate was calculated from the actual traces of the chartrecords and the digital outputs of the luminometer. Values were normalized within each cell preparation as percent of the maximal rate, considering 100% the rate obtained in lymphocytes incubated in the absence of VlP and stimulated with 50 nM PMA. Results are the mean _+ SEM of experiments from at least three separate preparation of lymphocytes. C) r (t) (J U~
}
1806
Chemiluminescence
in
Lymphocytes
by
Vol.
VIP
51,
No.
100
-g u 0
50 E
n
° O II
'
'
'
-11 -10 -9 Log [VIP] (mol/l)
-8
'
'
I00 f-(D
0 C (D 0 (D ~, C -,--i E 221
50
I1)
o
0 li
i
-12
I
i
i
t
Fig. 2 Dose-dependent effect of VlP on PMA-induced chemiluminescence, and correlation with activation by VlP of cAMP production. In the upper figure, the activity of adenylatecyclase was determined in lymphocytes, 5x106/ml, incubated at 15°C for 60 rain in the presence of the indicated concentrations of rIP. Cyclic AMP was measured by radioimmunoassay. Results, given as pmol/107 cells, are the mean _+ SEM of three separate experiments. In the lower figure, the chemiluminescence reaction was assayed in either lymphocytes ( • ) or monocytes ( I3 ), which were incubated at 37°C in medium containing the indicated concentrations of VlP. After 2 rain incubation, 50 nM PMA was injected to trigger the chemiluminescence response. Values (mean + SEM of five separate experiments) are given as the increase in percent of the maximal rate obtained in the absence of VlP and stimulated with 50 nM PMA.
23,
1992
Vol.
51, NO. 23, 1992
Chemiluminescence
in Lymphocytes
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In another set of experiments we studied the effect of VlP on the monocyte cell population prepared by density gradient centrifugation of mononuclear cells. VlP failed to produce in monocytes the above described effect on PMA-induced chemiluminescence in lymphocytes, in spite of the quite clear response induced by PMA alone. Only a minor effect, equivalent to a net increase of approximately 7% over the effect of PMA, was observed when monocytes were preincubated with 10 nM VlP (see Fig. 2) The effect of VlP on PMA-induced chemiluminescence in lymphocytes and monocytes was further studied in comparison to forskolin, a well-known stimulant of adenylate-cyclase. Fig 3 includes representative traces of the actual chemiluminescence records obtained in lymphocytes following preincubation with either VlP or forskolin and shows that forskolin also stimulated PMA-induced chemiluminescence and the degree of stimulation was almost identical to that produced by VlP. The same protocol was applied to monocytes where, in contrast to lymphocytes, we obtained a clear effect of forskolin whereas VlP failed to stimulate PMA-induced chemiluminescence (see Fig 3, lower panel). The results obtained in a number of experiments with the above mentioned agents and with concanavalin A are summarized in table I. Neither rIP, in a wide range of concentrations, forskolin, nor concanavalin A produced by themselves an increase of the resting chemiluminescence rate. Both forskolin and concanavalin A stimulated PMA-induced chemilunescence in monocytes. The most striking feature of these results was the stimulation by forskolin and concanavalin A of PMA-induced chemiluminescence in both cell types whereas the effect of VlP appears to be quite specific for lymphocytes.
Fig. 3
'min~_/b-~
d! \
oo i mlnQ
d-
Records of the chemiluminescence traces obtained from lymphocytes and monocytes stimulated with PMA, VIP, forskolin, and concanavalin A. 2x105 lymphocytes (upper panel), and 4x104 monocytes (lower panel) were incubated at 37°C in 1 ml PBS supplemented with 1.2 mM MgCI 2 and 1.2 mM CaCI 2 in the presence of the following agonists: none, a; l n M VIP, b; 100 nM forskolin, c and; 8 pg/ml concanavalin A, d. Incubation was in the thermostated chamber of the luminometer and after 2 rain PMA was added, see arrows, at a final concentration of 50 nM. Traces are hand-drawn copies of the actual records representative from at least three other cell batches.
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Table I
Stimulation of PMA-induced chemiluminescence by VIP, forskolin and concanavalin A
Chemiluminescence (% of resting rate)
Preincubation
Stimulus
LvmDhocvtes
None None None None r I P 1 nM Forskolin 100 nM Con A (8pg/ml)
PMA 50 nM r I P (0.001-10 nM) Forskolin 100 nM Con A (8 pg/ml) PMA 50 nM PMA 50 nM PMA 50 nM
100 0 0 2 _+ 1.5 188 ± 12 194 + 11 165 ± 33
Monocytes 100 0 0 0 100 + 10 133 ± 15 152 + 20
Lymphocytes (2 x 10 ~) and monocytes (4 x 103) were preincubated for 2 min at 37°C in 1 ml PBS supplemented with 1.2 mM MgCI 2 and 1.2 mM CaCI 2 in the presence of the indicated compounds. Stimuli were added and the chemiluminescence reaction continuously recorded (see Fig 3). Chemiluminescence values were normalized considering 100% the rate obtained following PMA stimulation of cells preincubated in the absence of any compound. Results are given as the mean ± SEM of five experiments.
Discussion
As far as we know, this study provides evidence for the first time of a chemiluminescence response of human lymphocytes to PMA and the enhancement of this effect by rIP. NK cells are a subpopulation of lymphocytes which has been described to produce reactive oxygen metabolites in response to a number of stimuli except to PMA, as determined by luminol-enhanced chemiluminescence (see Ref. 25), indicating that this cell subpopulation can not be the target of the agonists described in the present work since PMA is required to trigger the chemiluminescence reaction. A putative target cell in our experiment could be contaminating monocytes, a well known phagocytic and reactive oxygen intermediate producing cell. In spite of we routinously checked the absence of monocytes we decided to analyze the behavior of the monocyte under the described experimental conditions. Indeed monocytes produced a chemiluminescence response to PMA, as should be expected from a phagocytic cell, which was enhanced by forskolin and by the lectin concanavalin A, but unmodified by VIP. This observation agree with previously described experiments demonstrating the lack of effect of VIP on PMA-induced respiratory burst in monocytes (21). Therefore, our results are consistent with a rather specific effect of r I P on the chemiluminescence induced by PMA in lymphocytes.
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It is well known that receptors of VlP, present in human lymphocytes (9) and monocytes (12), are coupled to the adenylate cyclase in both cell types (9, 21 ). The dissociation constant for the high-affinity receptors of VlP in human lymphocytes is 0.24 nM (9) and agree quite well with ID~o values for cAMP production and PMAinduced chemiluminescence, 0.2 and 0.18 nM VlP respectively, described in the present work. Forskolin, which acts directly on the catalytic subunit of adenylate cyclase, also stimulated PMA-induced chemiluminescence response in lymphocytes. These considerations point to cAMP as the linking between receptor signaling by VlP and protein kinase C, the intracellular receptor for PMA. If this were the case, similar results should be expected in monocytes, but certainly VlP failed to modify the response of monocytes. The actual oxygen radical intermediate responsible for the chemiluminescence reaction in lymphocytes has not been elucidated in the present work. Nonetheless we approached this question by measuring superoxide anion production. We studied the reduction of cytochrome c in human lymphocytes by preincubation of 1 2 x l 06 cells/ml at 37°C for 5 min in the presence and in the absence of 100 nM VIP and stimulated with 50 nM PMA at 37°C for 30 min. Lymphocytes preincubated in the absence of r I P produced 2.6 pmoles/min/106 cells, whereas in the presence of VlP the rate increased up to 4.9 pmoles/min/106 cells. These results indicate that superoxide anion is an intermediate oxygen metabolite in the activation by PMA and VlP. In conclusion, we have found that human lymphocytes are activated by PMA producing reactive oxygen intermediates in a process which is stimulated by VlP. In spite of the cell type producing the chemiluminescence response is unclear, the activation of protein kinase C by PMA could explain the main mechanism, whereas the involvement of cAMP-dependent kinase or any other unknown signal linked to VlP receptors remains to be studied.
Acknowledgements Supported by grant n ° 92/399 from the Fondo de Investigaciones Sanitarias de la Seguridad Social, and n ° PB-0906 from the Comisi6n Interministerial de Ciencia y Tecnologfa.
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