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Peripheral benzodiazepines enhance the respiratory burst of macrophage-like P388D1 cells stimulated by arachidonic acid
Int. J. lmmunopharmac., Vol. 9, No. 3, pp. 269-274, 1987. Printed in Great Britain.
0192-0561/87 $3.00+ .00 International Society for Immunopharmacology.
PERIPHERAL BENZODIAZEPINES ENHANCE THE RESPIRATORY BURST OF MACROPHAGE-LIKE P388D1 CELLS STIMULATED BY ARACHIDONIC ACID FLORA ZAVALA and MARYSE LENFANT Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France (Received 29 July 1986 and in final form 23 October 1986).
Abstract - - P388D~, a murine cell with macrophage properties, responds to exogenous arachidonic acid with
superoxide anion production. This oxidative burst is enhanced by peripheral and mixed type benzodiazepines and this stimulation is specifically reversed by the peripheral antagonist PK 11195. In contrast, PK 11195 is unable to antagonize a stimulation caused by a non-benzodiazepine ligand such as the chemotactic peptide fMet-Leu-Phe. The optimal concentrations were close to 10 nM and corresponded to the affinities of the compounds for the peripheral benzodiazepine receptor detected on these cells. Compared to other tissues where peripheral benzodiazepines acted only at micromolar concentrations, the macrophage with its functional receptor appears as a privileged site of action for these molecules.
Benzodiazepine molecules (BZD) have been divided into three main classes (central, peripheral and mixed types) according to their pharmacological properties as well as to the localization of their receptors. Central type molecules exhibit at low doses anticonvulsant, anxiolytic and sedative effects, which are well correlated with their binding to receptors in the central nervous system (CNS), mostly localized at synapses. Peripheral type molecules bind in the CNS to non-neuronal sites (Schoemaker, Bliss & Yamamura, 1981) and to several peripheral organs and cell types (Del Zompo, Bocchetta, Corsini, Tallman & Gessa, 1983; Martin, 1984; Haefely, Kyburz, Gerecke & Mohler, 1985). The pharmacological relevance of the peripheral binding sites is not clearly established since only large doses ( 5 - 2 0 mg/kg) of Ro 5-4864, a specific peripheral ligand, induce in vivo an anxiogenic and convulsant response (File, Green, Nutt & Vincent, 1984). Mixed type benzodiazepines share the properties of both central and peripheral type molecules, and bind to both types of receptors. In a previous study (Zavala, Haumont & Lenfant, 1984), we disclosed a hitherto unknown in vivo pharmacological activity which is restricted to peripheral and mixed type benzodiazepines, namely their ability to stimulate in mice the humoral antibody response to sheep red blood cells. This
immunomodulating activity was observed when the compounds were injected at doses of 1 mg/kg i.p. one day after immunization, i.e. around the time of presentation of the antigen by the macrophage to immunocompetent cells. Furthermore, the respective degrees of this in vivo activity of the tested compounds correlated well with their in vitro binding affinities for mouse thioglycollate-elicited peritoneal macrophages. It was, therefore, suggested that the immunomodulation observed in vivo could be exerted through the binding of these molecules to their receptors on macrophages, which might result in a modulation of physiologicaI functions of these important ceils in the immune system. In the present study, we used P388Dt a murine cell line which exhibits many macrophage properties (Koren, Handwerger & Wunderlich, 1975); it is both adherent and phagocytic, it possesses surface receptors for IgG and C3b, whereas it bears neither immunoglobulin nor Thy-I antigen; it functions as an effector cell in ADCC (antibody-dependent cellmediated cytotoxicity), possesses non-specific esterase (a cytochemical marker for monocytes) and secretes lysozyme. We report here that P388D~ cells are able to produce superoxide anion when triggered with arachidonic acid. The activities of peripheral, mixed and central type benzodiazepines on this cellular
269
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F. ZAVALA and M. LENFANT
response are studied and the implication in this process of the high affinity BZD binding site, which we found to be present on these cells, is evaluated. EXPERIMENTAL PROCEDURES
8
oo 150 x tq
I00 P388D~ cells were a gift from Dr. A. Adam (Orsay, France). They were maintained in ascitic form in DBA2/J female mice (Iffa Credo, France) by weekly i.p. transfer of 107 cells. 02 production was measured by the reduction of cytochrome c (Cyt c) (Cohen & Chovaniec, 1978): P388D~ cells were suspended at 10 6 cells/ml in Hanks-balanced salt solution (HBSS) supplemented with Hepes 20 mM and containing 80 /aM Cyt c. The assay was conducted at 37°C in plastic cuvettes in a total volume of 1 ml. Cells were preincubated 15 min with the test compounds before stimulation with arachidonate (AA). At given time intervals, the absorbance at 550 nm was measured; 20,000/M/cm was used as the extinction coefficient for reducedminus-oxidized Cyt c at 550 nm. The reference cuvette received concomitantly an equivalent volume of AA solvent (EtOH/H20, 60/40). Controls also included Cyt c and the test compounds in the absence of cells. The maximal concentration of EtOH used in the experiments (0.1°70) had no effect on the reduction of Cyt c and did not affect cell viability. The influence of superoxide dismutase (SOD) and catalase are presented under Results. The ICs0values for displacement of [3H] Ro 5-4864 specifically bound to P388D~ cells by Ro 5-4864, PK 11195, diazepam, and clonazepam, were assessed as follows: P388Dl cells (106 cells/ml) were incubated in HBSS with 2 nM [3H] Ro 5-4864 and various concentrations of unlabeled drugs. After a 30 min incubation at 0 ° - 4 ° C , the reactions were terminated by rapid vacuum filtration over Whatman GF/B glass fiber filters, previously moistened with 3 ml ice cold HBSS. The filters were further rinsed twice with the same amount of buffer and then counted for radioactivity in a/3-scintillation counter. Non-specific binding was defined as the binding observed in the presence of 10/aM unlabeled Ro 5-4864. Specific binding was defined as total binding minus non-specific binding and represented 84°70 of total binding. All experiments were performed three times in triplicate.
Materials Ro 5-4864, diazepam and clonazepam were obtained from the Research Division of HoffmannLa Roche (Basel, Switzerland). PK 11195 was from
| 50
o
o
2o
40
60
80
Incubation time (mln)
Fig. 1. Kinetic curves of the response of P388D, cells to various concentrations of arachidonic acid. To P388Dt cells (106/ml) suspended at 37°C in 80/aM Cyt c in Hanks were added at to 100 /aM (A), 150 /aM ((3) or 200 /aM (o) arachidonic acid. The reduction of Cyt c was measured by the increase of absorbance (AA) (1 munit = 0.001 AA) at 550 nm at different time intervals. Results are expressed as the mean of triplicate measurements in a representative experiment. Pharmuka, Rh6ne-Poulenc Sant6 (Gennevilliers, France). Fmet-Leu-Phe (FMLP) was purchased from Bachem AG (Switzerland), catalase from Boehringer Mannheim (France), arachidonate, cytochrome c, superoxide dismutase, indomethacin from Sigma, [3HI RO 5-4864 (76.6 Ci/ml) from Amersham, France. Nafazatrom and BW 755C were a gift from Dr. J. L. Robin, RhOne-Poulenc Sant6 (Vitry, France).
RESULTS
Oxidative burst o f P388DI cells P388D~ cells responded only poorly to phorbol myristate acetate (PMA) stimulation. In contrast, like human neutrophils (Curnutte, Badwey, Robinson, Karnovsky & Karnovsky, 1984) and guinea pig peritoneal macrophages (Pick & Bromberg, 1983), P388D~ cells (after 7 days in ascites in the mice) reproducibly responded in vitro to arachidonate stimulation with superoxide anion production as monitored by Cyt c reduction. A kinetic curve at three concentrations is given in Fig. 1, showing that, in those cells, the response was obtained only at an A A concentration about twice as high as that necessary for the former cells. At an intermediary AA concentration (150 /aM), 5 - 6 nmoles of Cyt c were reduced in the first 5 min. This reduction of Cyt c was inhibited by 8007o when superoxide dismutase (SOD) 30/ag/ml and catalase
Peripheral Benzodiazepines Enhance the Respiratory Burst I
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150
I00
S
E
[ 0
50
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0
0
0
I0 20
40
60
0
80
Fig. 2. Kinetics of O~ production by P388D, cells. Cells (106/ml) were preincubated for 15 min with control solvent (0), SOD 30 ug/ml (A), catalase 40/ag/ml (IS]), SOD 30tag/ ml + catalase 40/ag/ml (&), or Ro 5-4864 10 nM ( o ) . At to, 150/aM AA were added and the reduction of Cyt c was measured at 550 nm. Results are expressed as the mean of triplicate measurements in a representative experiment.
Incubation
Indomethacin BW 755 C Nafazatrom
Concentration OaM)
070Modulation of control Cyt c reduction
0.25 25 1 10 1 10
+ 36% + 67 % + 54% + 141% - 42% +25%
The enzyme inhibitors were preincubated during 15 min at 37°C with P388D, cells (106/ml 80/aM Cyt c in Hanks) prior to stimulation by 150/aM arachidonate at to; the resultant Cyt c reductions were followed at different time intervals. Results are expressed as °70 of modulation of control response after 5 min of AA stimulation of P388D~ cells. 40 /~/gl were added. SOD a l o n e suppressed the r e d u c t i o n o f Cyt c by only 20°70 (Fig. 2). D u r i n g the c o n s u m p t i o n o f O 2 - , S O D produces H202 which in turn participates in the h y d r o p e r o x i d a t i o n o f the highly reactive A A leading to H P E T E metabolites. T h e latter A A derivatives are k n o w n to be p r o d u c e d in m a c r o p h a g e s (Scott, Znike, Hamill, K e m p e & C o h n , 1980) a n d to f u r t h e r stimulate 0 2 - p r o d u c t i o n in phagocytes (Smith & W e i d m a n n , 1980). Catalase a l o n e is inactive o n the reduction o f Cyt c by A A stimulated cells. However, w h e n a d d e d together with SOD, it c o n s u m e s H202
60
80
time (rain)
Fig. 3. Effect of Mn z÷ on the biphasic response of P388Dl cells to 150/aM arachidonate. Hanks buffer (©) or MnSO4 50/ag/ml in Hanks (A) were added to P388D~ cells prior to the addition of 150/aM AA. The reduction of Cyt c was measured at 550 nm at different time intervals. Results are expressed as the mean of triplicate measurements in a representative experiment.
Table 1. Modulation of the respiratory burst of AA stimulated P388D~ cells by cyclooxygenase and lipoxygenase inhibitors. Inhibitor
40
20
Incubation time (rain)
8O
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o
2
0
40
20
¢3 T
[0
9
8
7
6
e
- log r concentration (M]]
Fig. 4. Modulation of O2 production of P388D, cells by BDZ binding site ligands. P388D~ cells (l&/ml) were preincubated for 15 min with increasing concentrations of Ro 5-4864 ( e ) , PK 11195 (ll), diazepam (©), clonazepam (V) or solvent. At to, 150 /aM AA were added and the reduction of Cyt c at 550 nm was compared after 5 min of stimulation. Results are expressed as the mean _+ S.E. of triplicate measurements in a representative experiment. p r o d u c e d by SOD reaction a n d thus prevents the f o r m a t i o n o f H P E T E derivatives (otherwise f a v o r e d by the r a t h e r high A A c o n c e n t r a t i o n used) a n d the subsequent restimulation of cellular 02 p r o d u c t i o n . This explains why SOD a n d catalase together effectively inhibit the reduction of c y t o c h r o m e c which is t h e r e f o r e a t t r i b u t a b l e to O z initially p r o d u c e d by P 388D1 cells stimulated with AA.
272
F. ZAVALAand M. LENFANT
A
7a
B
+
-F
+
C
0
E
28
N RoS-4e64. PK II igO
RO5-4864 + PK 1119~
O~2epam PKIII96
Olazepam 4PK 11196
FMLP
PK I I I ~
F'MLP ÷ I=KIII95
Fig. 5. Specific antagonism by PK 11195 of the BDZs induced stimulation of AA triggered 02- production. P388Dj cells (10~/ml) were preincubated for 15 min with 10 nM concentrations of: (A) Ro 5-4864, PK 11195, Ro 5-4864 + PK 11195; (B) diazepam, PK 11195, diazepam + PK 11195; (C) FMLP, PK 11195, FMLP + PK 11195. At to, 150 gM AA were added and the 02 - productions were compared to control after 5 min of stimulation. Results are expressed as the mean _+S.E. of triplicate measurements in representative experiments.
i
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.
.
.
.
.
Nitta, 1982) resulted in the specific inhibition of the second phase of the response to AA (Fig. 3). The reoxidation of Cyt c occurring in the last minutes of the kinetics is apparently specific of P388D~ cells since it did not occur with thioglycollate-elicited mouse peritoneal macrophages nor BCG-elicited rabbit alveolar macrophages, stimulated with AA (data not shown). It possibly reflects the production of some oxidant factor by the stimulated tumoral cells.
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o
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,
,
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9 -
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8
,
7
log Econcentration
6
5
(M)-1
Fig. 6. Inhibition of [3H] Ro 5-4864 (2 nM) specific binding to P388D, ceils (106/ml) by increasing concentrations of PK 11195 (ll), Ro 5-4864 (e), diazepam (O) and clonazepam (V).
The biphasic kinetics of the curve suggested a sequential activation of enzymatic systems resulting in an activation NADPH-oxidase. Indeed, preincubation of P388D~ cells with cyclooxygenase and lipoxygenase inhibitors such as indomethacin, BW 755C and nafazatrom resulted, as previously observed on guinea pig peritoneal macrophages (Pick & Bromberg, 1983), in a modulation of the response to AA from the first minutes of the kinetics (Table 1). Preincubation with Mn 2÷ 50 /ag/ml, a concentration known to uncouple adenylate cyclase in P388D~ cells (Suzuki, Saito-Taki, Sadavisan and
I n f l u e n c e o f b e n z o d i a z e p i n e s on 0 2 P388D~ cells
p r o d u c t i o n by
A 15 min incubation of the cells with various concentrations of Ro 5-4864, PK 11195, diazepam and clonazepam did not induce production of superoxide. However, when this 15 min period was followed by addition of 150 /~M AA, the resulting 0 2 production was significantly enhanced in the presence of Ro 5-4864, a specific peripheral ligand of BZD sites (Fig. 2). This stimulating effect was apparent from the first minutes of 02 production and did not occur at optimal AA concentration (200 /aM). The addition of SOD and catalase to the reaction containing the BZD receptor ligands suppressed the Cyt c reduction to the same level as in the absence of ligands. Preincubation with a concentration range of Ro 5-4864 resulted in a bellshaped curve (Fig. 4) where maximal stimulation ( + 6 5 % ) was obtained at 10 nM. 1 nM and 100 nM concentrations produced intermediate stimulation whereas 0.1 nM as well as 1/aM did not significantly modulate the response to AA. A 53% stimulation
Peripheral Benzodiazepines Enhance the Respiratory Burst was obtained with 10 nM diazepam, a mixed type ligand, while the central type ligand clonazepam and the peripheral antagonist PK 11195, in a concentration range varying from 0.1 nM to 10/aM, showed no significant modulation of the production of 0 2 - following A A stimulation. Interestingly, the stimulatory effect of l0 nM Ro 5-4864 or diazepam was completely antagonized by addition of l0 nM PK 11195 during the 15 min preincubation with the cells (Fig. 5A, B); the resulting O2 production was not different from that of control cells triggered by A A alone. The specificity of this antagonism was established by the lack of activity of PK I 1195 on a non-BZD ligand induced stimulation: FMLP, a chemotactic peptide, did not trigger 0 2 production when incubated at 10 nM or 100 nM for 15 min with P388Dj cells, but at 10 nM, it was able to enhance 02' production ( + 61o70) triggered by A A in a way similar to that observed with the BZDs; however, equimolar or higher concentrations of PK 11195 were unable to antagonize the effect of FMLP (Fig. 5C). This observation clearly establishes the specificity of the antagonism by PK 11195, which is restricted to the actions mediated through the BZD receptor. Binding affinities o f benzodiazepines and P K 11195 on P388DI cells
The IC50values for the specific binding of [3HI Ro 5-4864 on P388Dj cells were evaluated for these compounds (Fig. 6). PK 11195 was the most effective ligand with an ICso value of 7.5 nM; Ro 5-4864 followed with an Ics0 of 45 nM; diazepam had an Ics0 of 200 nM; clonazepam did not reach 50% inhibition at concentrations up to 10 laM.
DISCUSSION Our results show that P388D~ cells are able to respond by an oxidative burst to the stimulation by A A , thus sharing this property with human neutrophils (Curnutte et aL, 1984), and guinea pig peritoneal macrophages in which Pick & Bromberg (1983) have stressed the central role of A A in the regulation of the oxidative metabolism. As suggested by the latter authors, the resulting inhibition or stimulation of the cellular response to A A after preincubation with lipoxygenase and/or cyclooxygenase inhibitors possibly reflects the accumulation of either inhibiting PGEs or stimulating leukotrienes and thromboxanes. Further study is in progress to ascertain this point. We have
273
obtained similar results with BCG-elicited rabbit alveolar macrophages whose response to A A was also stimulated with peripheral BZD Ro 5-4864 (data not shown). The easy availability of large amounts of P388D~ cells and the reproducibility of their response, make these cells an interesting tool. Peripheral and mixed type BDZ did not stimulate the respiratory burst by themselves but specifically increased the response to AA. This stimulation was clearly shown to be mediated through the binding of these compounds to their specific high affinity receptor on P388D~ cells, since the Ic50 values of the BZD were close to the active concentrations of these compounds on the respiratory burst. The specific antagonism by PK 11195 further demonstrated the implication of this site in the observed activity. Interestingly, the affinity values measured on P388D~ cells were quite similar to those measured by Ruff et aL on human monocytes (Ruff, Pert, Weber, Wahl, Wahl & Paul, 1985) who reported that peripheral and mixed type molecules stimulated the chemotaxis of these cells. In the latter case, the chemotactic concentrations (1, 10 pM) were lower than I¢50 values. In the hitherto described in vitro activities of peripheral BZDs, such as inhibition of the in vitro growth of thymoma cells (Wang, Morgan & Spector, 1984), decrease of cardiac muscle contractility (Mestre, Carriot, Belin, Uzan, Renault, Dubroeucq, Gueremy & Le Fur, 1984), induction of melanogenesis in B 16/C3 melanoma cells (Matthews, Laskin, Zimmerman, Weinstein, Hsu & Englehardt, 1981), there was a discrepancy between the affinities of the molecules for their receptors on the corresponding cells, which were nanomolar, and their effective concentrations which were micromolar. In contrast, macrophages are affected by nearly nanomolar concentrations of peripheral BZDs, and appear thus as a privileged site of action for these molecules. Since O2'- production is an important biochemical correlate of the activity of macrophages in host's defense against infection and malignant cells (Cohn, 1978), the potential use of peripheral and mixed type BZDs and PK 11195 in the modulation of these functions as well as their pro- or anti-inflammatory properties, respectively, certainly deserve to be investigated. Acknowledgements -- We would like to thank Dr. G. H.
Werner, Dr. J. F. Petit and Dr. P. Potier for stimulating discussions and pertinent advice. We thank S. Guengard for typing the manuscript. This work was supported by Centre National de la Recherche Scientifique-Programme Interdisciplinairede Recherche sur les bases scientifiques du M6dicament (C.N.R.S.-P.I.R.M.E.D).
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F. ZAVALAand M. LENFANT
REFERENCES
COHEN, H. J. & CHOVANIEC, M. E. (1978). Superoxide generation by digitonin stimulated guinea-pig granulocytes. Basis for a continuous assay for monitoring superoxide production and for the study of the activation of the generation system. J. clin. Invest., 61, 1081- 1087. COHN, Z. A. (1978). The activation of mononuclear phagocytes: fact, fancy, and future. J. Immun., 121, 813- 816. CURNUTTE, J. Z., BADWAY, J. A., ROBINSON, J. M., KARNOVSKY,M. J. & KARNOVSKY, M. L. (1984). Studies on the mechanism of superoxide release from human neutrophils stimulated with arachidonate. J. biol. Chem., 259, 11851- 11857. DEE ZOMPO, M., BOCCHETTA, A., CORSINI, G. M., TALLMAN,J. F. t~ GESSA, G. L. (1983). Properties of benzodiazepine binding sites in peripheral tissue. In Benzodiazepine Recognition Site Ligands. Biochemistry and Pharmacology (eds Biggio, G. and Costa, E.) pp. 239-248. Raven Press, New York. FLEE, S. E., GREEN, A. R., NUTT, D. J. t~ VINCENT, N. O. (1984). On the convulsant action of Ro 5-4864 7-Chloro-5-4chlorophenyl-l,3-dihydro-l-methyl-2H-1,4benzodiazepine-2-one and the existence of a micromolar benzodiazepine binding site in rat brain. Psychopharmacology, 82, 199- 202. HAEFELY, W., KYBURZ, E., GERECKE, M. ,~ MOHLER, H. (1985). Recent advances in the molecular pharmacology of benzodiazepine receptors and in the structure- activity relationships of their agonists and antagonists. In: Advances in Drug Research (ed. Testa, B.) pp. 165- 322. Academic Press, London. KOREN, H. S., HANDWERGER, B. S. ~¢ WUNDERLICH, S. R. (1975). Identification of macrophage-like characteristics in cultured murine tumor line. J. Immun., 114, 894-897. MARTIN, I. L. (1984). The benzodiazepine receptor functional complexity. TIPS, 343 - 347. MATTHEWS, E., LASKIN, J. D., Z1MMERMANN, E. A., WEINSTEIN, J. B., Hsu, K. C. • ENGLEHARDT, O. L. (1981). Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells. Proc. natn Acad. Sci. U.S.A., 78, 3935-3939. MESTRE, M., CARRIOT, T., BEL1N, C., UZAN, A., RENAULT,C., DUBROEUCQ,M. C., GUEREMY,C. & LE FUR, G. (1984). Electrophysiological and pharmacological characterization of peripheral benzodiazepine receptors in a guinea-pig heart preparation. Life Sci., 35, 953 -962. PICK, E. & BROMBERG,Y. (1983). Regulation of macrophage function by lymphokines. Role of membrane phospholipids. In Advances in Immunopharmacology (ed. Hadden, J. W.) Vol. 2, pp. 243 - 249. Pergamon Press, New York. RUFF, M. R., PERT, C. B., WEBER, R. J., WAHL, L. M., WAHL, S. M. t~ PAUL, S. M. (1985). Benzodiazepine receptor mediated chemotaxis of human monocytes. Science, 229, 1281 - 1283. SCHOEMAKER, H., BLISS, M. & YAMAMURA,H. I. (1981). Specific high affinity saturable binding of tritium labeled Ro 54864 7-chloro-5-4-chlorophenyl-l-3-dihydro-l-methyl-2H-1,4benzodiazepine-2-one to benzodiazepine binding sites in the rat cerebral cortex. Eur. J. Pharmac., 71, 173 - 175. SCOTT, W. A., ZRIKE, J. M., HAM1LL,A. L., KEMPE,J. t~ COHN, Z. A. (1980). Regulation of arachidonic-acid metabolites in macrophages. J. exp. Med., 152, 324-335. SMITH, R. L. t~ WEIDMANN, M. J. (1980). Reactive oxygen production associated with arachidonic acid metabolism by peritoneal macrophages. Biochem. biophys. Res. Commun., 97, 973- 980. SUZUKI, T., SAITO-TAKI, T., SADAVISANR. & NITTA, T. (1982). Biochemical signal transmitted by Fcy receptors: Phospholipase A 2 activity of Fcy2b receptor of murine macrophage cell line P388D1. Proc. natn Acad. Sci. U.S.A., 79, 591 - 595. WANG, J. K. T., MORGAN, J. I. & SPECTOR, S. (1984). Benzodiazepines that bind at peripheral sites inhibit cell proliferation. Proc. natn Acad. Sci. U.S.A., 81, 753 - 756. ZAVALA, F., HAUMONT, J. & LENEANT, M. (1984). Interaction of Benzodiazepines with Mouse Macrophages. Eur. J. Pharmac., 106, 561- 566.
0192-0561/87 $3.00+ .00 International Society for Immunopharmacology.
PERIPHERAL BENZODIAZEPINES ENHANCE THE RESPIRATORY BURST OF MACROPHAGE-LIKE P388D1 CELLS STIMULATED BY ARACHIDONIC ACID FLORA ZAVALA and MARYSE LENFANT Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France (Received 29 July 1986 and in final form 23 October 1986).
Abstract - - P388D~, a murine cell with macrophage properties, responds to exogenous arachidonic acid with
superoxide anion production. This oxidative burst is enhanced by peripheral and mixed type benzodiazepines and this stimulation is specifically reversed by the peripheral antagonist PK 11195. In contrast, PK 11195 is unable to antagonize a stimulation caused by a non-benzodiazepine ligand such as the chemotactic peptide fMet-Leu-Phe. The optimal concentrations were close to 10 nM and corresponded to the affinities of the compounds for the peripheral benzodiazepine receptor detected on these cells. Compared to other tissues where peripheral benzodiazepines acted only at micromolar concentrations, the macrophage with its functional receptor appears as a privileged site of action for these molecules.
Benzodiazepine molecules (BZD) have been divided into three main classes (central, peripheral and mixed types) according to their pharmacological properties as well as to the localization of their receptors. Central type molecules exhibit at low doses anticonvulsant, anxiolytic and sedative effects, which are well correlated with their binding to receptors in the central nervous system (CNS), mostly localized at synapses. Peripheral type molecules bind in the CNS to non-neuronal sites (Schoemaker, Bliss & Yamamura, 1981) and to several peripheral organs and cell types (Del Zompo, Bocchetta, Corsini, Tallman & Gessa, 1983; Martin, 1984; Haefely, Kyburz, Gerecke & Mohler, 1985). The pharmacological relevance of the peripheral binding sites is not clearly established since only large doses ( 5 - 2 0 mg/kg) of Ro 5-4864, a specific peripheral ligand, induce in vivo an anxiogenic and convulsant response (File, Green, Nutt & Vincent, 1984). Mixed type benzodiazepines share the properties of both central and peripheral type molecules, and bind to both types of receptors. In a previous study (Zavala, Haumont & Lenfant, 1984), we disclosed a hitherto unknown in vivo pharmacological activity which is restricted to peripheral and mixed type benzodiazepines, namely their ability to stimulate in mice the humoral antibody response to sheep red blood cells. This
immunomodulating activity was observed when the compounds were injected at doses of 1 mg/kg i.p. one day after immunization, i.e. around the time of presentation of the antigen by the macrophage to immunocompetent cells. Furthermore, the respective degrees of this in vivo activity of the tested compounds correlated well with their in vitro binding affinities for mouse thioglycollate-elicited peritoneal macrophages. It was, therefore, suggested that the immunomodulation observed in vivo could be exerted through the binding of these molecules to their receptors on macrophages, which might result in a modulation of physiologicaI functions of these important ceils in the immune system. In the present study, we used P388Dt a murine cell line which exhibits many macrophage properties (Koren, Handwerger & Wunderlich, 1975); it is both adherent and phagocytic, it possesses surface receptors for IgG and C3b, whereas it bears neither immunoglobulin nor Thy-I antigen; it functions as an effector cell in ADCC (antibody-dependent cellmediated cytotoxicity), possesses non-specific esterase (a cytochemical marker for monocytes) and secretes lysozyme. We report here that P388D~ cells are able to produce superoxide anion when triggered with arachidonic acid. The activities of peripheral, mixed and central type benzodiazepines on this cellular
269
270
F. ZAVALA and M. LENFANT
response are studied and the implication in this process of the high affinity BZD binding site, which we found to be present on these cells, is evaluated. EXPERIMENTAL PROCEDURES
8
oo 150 x tq
I00 P388D~ cells were a gift from Dr. A. Adam (Orsay, France). They were maintained in ascitic form in DBA2/J female mice (Iffa Credo, France) by weekly i.p. transfer of 107 cells. 02 production was measured by the reduction of cytochrome c (Cyt c) (Cohen & Chovaniec, 1978): P388D~ cells were suspended at 10 6 cells/ml in Hanks-balanced salt solution (HBSS) supplemented with Hepes 20 mM and containing 80 /aM Cyt c. The assay was conducted at 37°C in plastic cuvettes in a total volume of 1 ml. Cells were preincubated 15 min with the test compounds before stimulation with arachidonate (AA). At given time intervals, the absorbance at 550 nm was measured; 20,000/M/cm was used as the extinction coefficient for reducedminus-oxidized Cyt c at 550 nm. The reference cuvette received concomitantly an equivalent volume of AA solvent (EtOH/H20, 60/40). Controls also included Cyt c and the test compounds in the absence of cells. The maximal concentration of EtOH used in the experiments (0.1°70) had no effect on the reduction of Cyt c and did not affect cell viability. The influence of superoxide dismutase (SOD) and catalase are presented under Results. The ICs0values for displacement of [3H] Ro 5-4864 specifically bound to P388D~ cells by Ro 5-4864, PK 11195, diazepam, and clonazepam, were assessed as follows: P388Dl cells (106 cells/ml) were incubated in HBSS with 2 nM [3H] Ro 5-4864 and various concentrations of unlabeled drugs. After a 30 min incubation at 0 ° - 4 ° C , the reactions were terminated by rapid vacuum filtration over Whatman GF/B glass fiber filters, previously moistened with 3 ml ice cold HBSS. The filters were further rinsed twice with the same amount of buffer and then counted for radioactivity in a/3-scintillation counter. Non-specific binding was defined as the binding observed in the presence of 10/aM unlabeled Ro 5-4864. Specific binding was defined as total binding minus non-specific binding and represented 84°70 of total binding. All experiments were performed three times in triplicate.
Materials Ro 5-4864, diazepam and clonazepam were obtained from the Research Division of HoffmannLa Roche (Basel, Switzerland). PK 11195 was from
| 50
o
o
2o
40
60
80
Incubation time (mln)
Fig. 1. Kinetic curves of the response of P388D, cells to various concentrations of arachidonic acid. To P388Dt cells (106/ml) suspended at 37°C in 80/aM Cyt c in Hanks were added at to 100 /aM (A), 150 /aM ((3) or 200 /aM (o) arachidonic acid. The reduction of Cyt c was measured by the increase of absorbance (AA) (1 munit = 0.001 AA) at 550 nm at different time intervals. Results are expressed as the mean of triplicate measurements in a representative experiment. Pharmuka, Rh6ne-Poulenc Sant6 (Gennevilliers, France). Fmet-Leu-Phe (FMLP) was purchased from Bachem AG (Switzerland), catalase from Boehringer Mannheim (France), arachidonate, cytochrome c, superoxide dismutase, indomethacin from Sigma, [3HI RO 5-4864 (76.6 Ci/ml) from Amersham, France. Nafazatrom and BW 755C were a gift from Dr. J. L. Robin, RhOne-Poulenc Sant6 (Vitry, France).
RESULTS
Oxidative burst o f P388DI cells P388D~ cells responded only poorly to phorbol myristate acetate (PMA) stimulation. In contrast, like human neutrophils (Curnutte, Badwey, Robinson, Karnovsky & Karnovsky, 1984) and guinea pig peritoneal macrophages (Pick & Bromberg, 1983), P388D~ cells (after 7 days in ascites in the mice) reproducibly responded in vitro to arachidonate stimulation with superoxide anion production as monitored by Cyt c reduction. A kinetic curve at three concentrations is given in Fig. 1, showing that, in those cells, the response was obtained only at an A A concentration about twice as high as that necessary for the former cells. At an intermediary AA concentration (150 /aM), 5 - 6 nmoles of Cyt c were reduced in the first 5 min. This reduction of Cyt c was inhibited by 8007o when superoxide dismutase (SOD) 30/ag/ml and catalase
Peripheral Benzodiazepines Enhance the Respiratory Burst I
I
g
I
I
I
1
271
I
la
¢)
150
"~
% g
IOO
150
I00
S
E
[ 0
50
<]
0
0
0
I0 20
40
60
0
80
Fig. 2. Kinetics of O~ production by P388D, cells. Cells (106/ml) were preincubated for 15 min with control solvent (0), SOD 30 ug/ml (A), catalase 40/ag/ml (IS]), SOD 30tag/ ml + catalase 40/ag/ml (&), or Ro 5-4864 10 nM ( o ) . At to, 150/aM AA were added and the reduction of Cyt c was measured at 550 nm. Results are expressed as the mean of triplicate measurements in a representative experiment.
Incubation
Indomethacin BW 755 C Nafazatrom
Concentration OaM)
070Modulation of control Cyt c reduction
0.25 25 1 10 1 10
+ 36% + 67 % + 54% + 141% - 42% +25%
The enzyme inhibitors were preincubated during 15 min at 37°C with P388D, cells (106/ml 80/aM Cyt c in Hanks) prior to stimulation by 150/aM arachidonate at to; the resultant Cyt c reductions were followed at different time intervals. Results are expressed as °70 of modulation of control response after 5 min of AA stimulation of P388D~ cells. 40 /~/gl were added. SOD a l o n e suppressed the r e d u c t i o n o f Cyt c by only 20°70 (Fig. 2). D u r i n g the c o n s u m p t i o n o f O 2 - , S O D produces H202 which in turn participates in the h y d r o p e r o x i d a t i o n o f the highly reactive A A leading to H P E T E metabolites. T h e latter A A derivatives are k n o w n to be p r o d u c e d in m a c r o p h a g e s (Scott, Znike, Hamill, K e m p e & C o h n , 1980) a n d to f u r t h e r stimulate 0 2 - p r o d u c t i o n in phagocytes (Smith & W e i d m a n n , 1980). Catalase a l o n e is inactive o n the reduction o f Cyt c by A A stimulated cells. However, w h e n a d d e d together with SOD, it c o n s u m e s H202
60
80
time (rain)
Fig. 3. Effect of Mn z÷ on the biphasic response of P388Dl cells to 150/aM arachidonate. Hanks buffer (©) or MnSO4 50/ag/ml in Hanks (A) were added to P388D~ cells prior to the addition of 150/aM AA. The reduction of Cyt c was measured at 550 nm at different time intervals. Results are expressed as the mean of triplicate measurements in a representative experiment.
Table 1. Modulation of the respiratory burst of AA stimulated P388D~ cells by cyclooxygenase and lipoxygenase inhibitors. Inhibitor
40
20
Incubation time (rain)
8O
g 60
o
2
0
40
20
¢3 T
[0
9
8
7
6
e
- log r concentration (M]]
Fig. 4. Modulation of O2 production of P388D, cells by BDZ binding site ligands. P388D~ cells (l&/ml) were preincubated for 15 min with increasing concentrations of Ro 5-4864 ( e ) , PK 11195 (ll), diazepam (©), clonazepam (V) or solvent. At to, 150 /aM AA were added and the reduction of Cyt c at 550 nm was compared after 5 min of stimulation. Results are expressed as the mean _+ S.E. of triplicate measurements in a representative experiment. p r o d u c e d by SOD reaction a n d thus prevents the f o r m a t i o n o f H P E T E derivatives (otherwise f a v o r e d by the r a t h e r high A A c o n c e n t r a t i o n used) a n d the subsequent restimulation of cellular 02 p r o d u c t i o n . This explains why SOD a n d catalase together effectively inhibit the reduction of c y t o c h r o m e c which is t h e r e f o r e a t t r i b u t a b l e to O z initially p r o d u c e d by P 388D1 cells stimulated with AA.
272
F. ZAVALAand M. LENFANT
A
7a
B
+
-F
+
C
0
E
28
N RoS-4e64. PK II igO
RO5-4864 + PK 1119~
O~2epam PKIII96
Olazepam 4PK 11196
FMLP
PK I I I ~
F'MLP ÷ I=KIII95
Fig. 5. Specific antagonism by PK 11195 of the BDZs induced stimulation of AA triggered 02- production. P388Dj cells (10~/ml) were preincubated for 15 min with 10 nM concentrations of: (A) Ro 5-4864, PK 11195, Ro 5-4864 + PK 11195; (B) diazepam, PK 11195, diazepam + PK 11195; (C) FMLP, PK 11195, FMLP + PK 11195. At to, 150 gM AA were added and the 02 - productions were compared to control after 5 min of stimulation. Results are expressed as the mean _+S.E. of triplicate measurements in representative experiments.
i
IOO
.
.
.
.
.
Nitta, 1982) resulted in the specific inhibition of the second phase of the response to AA (Fig. 3). The reoxidation of Cyt c occurring in the last minutes of the kinetics is apparently specific of P388D~ cells since it did not occur with thioglycollate-elicited mouse peritoneal macrophages nor BCG-elicited rabbit alveolar macrophages, stimulated with AA (data not shown). It possibly reflects the production of some oxidant factor by the stimulated tumoral cells.
~""~
~ i so
o
~ ~
,
,
I0
9 -
~
8
,
7
log Econcentration
6
5
(M)-1
Fig. 6. Inhibition of [3H] Ro 5-4864 (2 nM) specific binding to P388D, ceils (106/ml) by increasing concentrations of PK 11195 (ll), Ro 5-4864 (e), diazepam (O) and clonazepam (V).
The biphasic kinetics of the curve suggested a sequential activation of enzymatic systems resulting in an activation NADPH-oxidase. Indeed, preincubation of P388D~ cells with cyclooxygenase and lipoxygenase inhibitors such as indomethacin, BW 755C and nafazatrom resulted, as previously observed on guinea pig peritoneal macrophages (Pick & Bromberg, 1983), in a modulation of the response to AA from the first minutes of the kinetics (Table 1). Preincubation with Mn 2÷ 50 /ag/ml, a concentration known to uncouple adenylate cyclase in P388D~ cells (Suzuki, Saito-Taki, Sadavisan and
I n f l u e n c e o f b e n z o d i a z e p i n e s on 0 2 P388D~ cells
p r o d u c t i o n by
A 15 min incubation of the cells with various concentrations of Ro 5-4864, PK 11195, diazepam and clonazepam did not induce production of superoxide. However, when this 15 min period was followed by addition of 150 /~M AA, the resulting 0 2 production was significantly enhanced in the presence of Ro 5-4864, a specific peripheral ligand of BZD sites (Fig. 2). This stimulating effect was apparent from the first minutes of 02 production and did not occur at optimal AA concentration (200 /aM). The addition of SOD and catalase to the reaction containing the BZD receptor ligands suppressed the Cyt c reduction to the same level as in the absence of ligands. Preincubation with a concentration range of Ro 5-4864 resulted in a bellshaped curve (Fig. 4) where maximal stimulation ( + 6 5 % ) was obtained at 10 nM. 1 nM and 100 nM concentrations produced intermediate stimulation whereas 0.1 nM as well as 1/aM did not significantly modulate the response to AA. A 53% stimulation
Peripheral Benzodiazepines Enhance the Respiratory Burst was obtained with 10 nM diazepam, a mixed type ligand, while the central type ligand clonazepam and the peripheral antagonist PK 11195, in a concentration range varying from 0.1 nM to 10/aM, showed no significant modulation of the production of 0 2 - following A A stimulation. Interestingly, the stimulatory effect of l0 nM Ro 5-4864 or diazepam was completely antagonized by addition of l0 nM PK 11195 during the 15 min preincubation with the cells (Fig. 5A, B); the resulting O2 production was not different from that of control cells triggered by A A alone. The specificity of this antagonism was established by the lack of activity of PK I 1195 on a non-BZD ligand induced stimulation: FMLP, a chemotactic peptide, did not trigger 0 2 production when incubated at 10 nM or 100 nM for 15 min with P388Dj cells, but at 10 nM, it was able to enhance 02' production ( + 61o70) triggered by A A in a way similar to that observed with the BZDs; however, equimolar or higher concentrations of PK 11195 were unable to antagonize the effect of FMLP (Fig. 5C). This observation clearly establishes the specificity of the antagonism by PK 11195, which is restricted to the actions mediated through the BZD receptor. Binding affinities o f benzodiazepines and P K 11195 on P388DI cells
The IC50values for the specific binding of [3HI Ro 5-4864 on P388Dj cells were evaluated for these compounds (Fig. 6). PK 11195 was the most effective ligand with an ICso value of 7.5 nM; Ro 5-4864 followed with an Ics0 of 45 nM; diazepam had an Ics0 of 200 nM; clonazepam did not reach 50% inhibition at concentrations up to 10 laM.
DISCUSSION Our results show that P388D~ cells are able to respond by an oxidative burst to the stimulation by A A , thus sharing this property with human neutrophils (Curnutte et aL, 1984), and guinea pig peritoneal macrophages in which Pick & Bromberg (1983) have stressed the central role of A A in the regulation of the oxidative metabolism. As suggested by the latter authors, the resulting inhibition or stimulation of the cellular response to A A after preincubation with lipoxygenase and/or cyclooxygenase inhibitors possibly reflects the accumulation of either inhibiting PGEs or stimulating leukotrienes and thromboxanes. Further study is in progress to ascertain this point. We have
273
obtained similar results with BCG-elicited rabbit alveolar macrophages whose response to A A was also stimulated with peripheral BZD Ro 5-4864 (data not shown). The easy availability of large amounts of P388D~ cells and the reproducibility of their response, make these cells an interesting tool. Peripheral and mixed type BDZ did not stimulate the respiratory burst by themselves but specifically increased the response to AA. This stimulation was clearly shown to be mediated through the binding of these compounds to their specific high affinity receptor on P388D~ cells, since the Ic50 values of the BZD were close to the active concentrations of these compounds on the respiratory burst. The specific antagonism by PK 11195 further demonstrated the implication of this site in the observed activity. Interestingly, the affinity values measured on P388D~ cells were quite similar to those measured by Ruff et aL on human monocytes (Ruff, Pert, Weber, Wahl, Wahl & Paul, 1985) who reported that peripheral and mixed type molecules stimulated the chemotaxis of these cells. In the latter case, the chemotactic concentrations (1, 10 pM) were lower than I¢50 values. In the hitherto described in vitro activities of peripheral BZDs, such as inhibition of the in vitro growth of thymoma cells (Wang, Morgan & Spector, 1984), decrease of cardiac muscle contractility (Mestre, Carriot, Belin, Uzan, Renault, Dubroeucq, Gueremy & Le Fur, 1984), induction of melanogenesis in B 16/C3 melanoma cells (Matthews, Laskin, Zimmerman, Weinstein, Hsu & Englehardt, 1981), there was a discrepancy between the affinities of the molecules for their receptors on the corresponding cells, which were nanomolar, and their effective concentrations which were micromolar. In contrast, macrophages are affected by nearly nanomolar concentrations of peripheral BZDs, and appear thus as a privileged site of action for these molecules. Since O2'- production is an important biochemical correlate of the activity of macrophages in host's defense against infection and malignant cells (Cohn, 1978), the potential use of peripheral and mixed type BZDs and PK 11195 in the modulation of these functions as well as their pro- or anti-inflammatory properties, respectively, certainly deserve to be investigated. Acknowledgements -- We would like to thank Dr. G. H.
Werner, Dr. J. F. Petit and Dr. P. Potier for stimulating discussions and pertinent advice. We thank S. Guengard for typing the manuscript. This work was supported by Centre National de la Recherche Scientifique-Programme Interdisciplinairede Recherche sur les bases scientifiques du M6dicament (C.N.R.S.-P.I.R.M.E.D).
274
F. ZAVALAand M. LENFANT
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