Journal of Neuroimmunology, 38 (1992) 19-26
19
© 1992 ElsevierSciencePublishers B.V. All rightsreserved 0165-5728/92/$05.00 JNI 02165
Immunomodulatory effect of peripheral benzodiazepine receptor ligands on human mononuclear cells H a n n a Bessler a, Ronit Weizman b, Moshe Gavish c, Ida Notti a and Meir Djaldetti a,d a Hematology Research Laboratory, Hasharon Hospital, Golda Medical Center, Petah Tiqva, b Hasharon Hospital, Petah Tiqva, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, c Department of Pharmacology, Faculty of Medicine and Rappaport Family Institute for Research in Medical Science, Technion, Institute of Technology, Haifa, and d Department of Medicine "B", Hasharon Hospital, Golda Medical Center, Petah Tiqva, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
(Received7 August,1991) (Revised, received12 December, 1991) (Accepted 12 December, 1991) Key words: Peripheral benzodiazepinereceptor; Mitogen-inducedproliferation;Interleukin-2;Interleukin-3-1ikeactivity
Summary Immunomodulatory effect of ligands active at the peripheral benzodiazepine receptor (PBR) was examined in human peripheral blood mononuclear cells (PBMC). Ro5-4864, PKll195 and diazepam suppressed phytohemagglutinin (PHA) and concanavalin A (ConA) induced proliferation of PBMC. All three ligands inhibited interleukin-3-1ike activity (IL-3-LA) secretion, while the production of interleukin-2 (IL-2) was inhibited by Ro5-4864 and diazepam only. The selective central benzodiazepine ligand clonazepam did not affect the cellular immune functions examined. Our results indicate an in-vitro immuno-suppressive activity of peripheral and mixed, but not central type benzodiazepine ligands.
Introduction Benzodiazepine (BZ) receptors have been identified and characterized in the synaptosomal fraction of the central nervous system (Mohler and Okada, 1977; Squires and Braestrup, 1977), as well as in a number of peripheral organs and non-neuronal brain tissue (Wang et al., 1980, 1981; Taniguchi et al., 1982; Schoemaker et al., 1981, 1983; De Souza et al., 1985). The central,
Correspondence to: M. Djaldetti, Department of Medicine 'B', HasharonHospital,GoldaMedicalCenter, P.O. Box 121, Petah Tiqva 49372, Israel.
but not the peripheral BZ receptors (PBR), are coupled to the 7-aminobutyric acid (GABA) receptors and the chlorid ion channels (Squires and Saederup, 1982). They mediate the anxiolytic, anticonvulsant and muscle relaxant activities of the BZs (Costa and Guidotti, 1979). It has been suggested that PBR ligands bind to ligand sites in the voltage-regulated calcium channel complex (Mestre et al., 1984). The high density of PBR in the cellular mitochondrial fraction implies a functional role for these receptors in intracellular metabolic events, rather than intercellular synaptic events (Anholt et al., 1986a, b; Snyder et al., 1987; Mukhin et al., 1989; Krueger and Papadopoulos, 1990; Papadopoulos et al., 1990).
20
However, other studies suggest that peripheral receptors can be found also in other subcellular compartments, such as nuclear (Schoemaker et al., 1983; Rago et al., 1990), sarcolemmic, and sarcoplasmic reticulum fractions (Doble et al., 1985). Several studies have demonstrated that the regulation of PBR in various tissues is under neural or hormonal control (for review see Saano, 1988; Verma and Snyder, 1989). Clonazepam and flumazenil are ligands specific for CBR, while PBR bind with high affinity the ligands Ro5-4864 (4-chlorodiazepam) and PKl1195 (an isoquinoline carboxamide derivative), which were suggested to be agonist and antagonist, respectively, to this receptor (Mestre et al., 1985). Diazepam is a mixed ligand which binds to both the central and peripheral receptors in nanomolar affinity (Wang et al., 1984). A line of evidence suggests a possible involvement of PBR in the regulation of immune function. PBR were identified in circulating mononuclear cells (Moingeon et al., 1983), macrophages (Zavala et al., 1984), thymocytes (Wang et al., 1981) and on a B-cell hybridoma (Pert et al., 1985). Autoradiographic study localized the anatomical distribution of PBR in organs of the rat immune system (Benavides et al., 1989). Ligands for PBR display immunomodulatory activity, such as enhancement of chemotaxis by human monocytes (Ruff et al., 1985). Peripheral and mixed type ligands but not central-type ligands potentiate the humoral response to sheep red blood cells (Zavala et al., 1984; Lenfant and Zavala, 1986). However, the role of PBR in the regulation of the immune function is as yet unknown. The purpose of the present study was to evaluate a possible in-vitro effect of specific PBR ligands on the immune system. Since interleukins play a major role in the regulation of immune function, we examined the effect of these ligands on the production of interleukin-2 (IL-2) and interleukin-3-1ike activity (IL-3-LA) by human peripheral blood mononuclear cells (PBMC). IL-3LA is a human growth factor similar to IL-3, released spontaneously by PBMC (Fishman et al., 1990). The effect of PBR ligands on mitogen-induced proliferation of human PBMC was also tested. The effects of the PBR ligands on the
immune functions were compared to those of clonazepam which is a selective CBR ligand.
Materials and methods
Materials Clonazepam, diazepam, flunitrazepam (FNZ) and Ro5-4868 (4'-chlorodiazepam) were supplied by Drs. H. Rutman and E. Kyburz (Hoffman La Roche, Basel, Switzerland). PKll195 (an isoquinoline carboxamide derivative) was donated by Dr. A. Bouvier, Rhone-Poulenc Sante (VitrySur-Seine, France). Ligand preparation All ligands were dissolved in absolute ethanol at 10 -3 M. Further dilutions were made in RPMI 1640 medium. The highest concentration of ligands used in our experiments was 10 -5 M revealing 1% ethanol in the incubation mixture. Cell preparation and incubation procedure Peripheral blood mononuclear ceils (PBMC) were isolated from the peripheral blood of healthy adult donors on Ficoll-Paque density gradient as described by Boyum (1968). Mononuclear cells (106/ml) were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% penicillin (10000 units/ml), 1% streptomycin sulphate (10 mg/ml) and amphotericin B (25 /zg/ml) (complete medium, CM). Aliquots of 0.18 ml were added to each well of a flat-bottomed microculture plate containing 0.02 ml of RPMI 1640 or various ligand concentrations. Cultures were stimulated with 1 ~ g / m l phytohemagglutinin (PHA-HA16, Wellcome-Research Lab. Bechman), or 5 ~zg/ml concanavalin A (ConA, Bio-Yeda, Israel). The cultures, set up in triplicate, were incubated for 72 h at 37°C in a humidified atmosphere containing 5% CO 2. 18 h prior to harvesting, the cells were pulsed with 0.5 ~Ci [3H]thymidine/well. The radioactivity was measured with an LKB liquid scintillation counter model 3380. The results are expressed as percent of control. IL-2 and IL-3-LA production For IL-2 production 1.5 × 10 6 PBMCs were suspended in CM containing 1 izg/ml PHA. 1-ml
21 aliquots of cell suspension were incubated for 24 h in 24-well plates (Nunc), in the absence or presence of various concentrations of the ligands. For IL-3-LA production, 1-ml aliquots of PBMCs suspension in CM (2 × 106 cells/ml) were incubated for 48 h in 24-well plates (Nunc) in the absence or presence of various concentrations of the ligands. At the end of the incubation period culture media were collected, cells were removed by centrifugation, and supernatants were kept at - 20°C until assayed.
IL-2 and IL-3-LA bioassays IL-2 activity in the supernatants was determined using the CTLL-2 cell line as reported previously (Serrate et al., 1987). IL-3-LA was evaluated by using the 32-D-cl-23 cell line as described by Fishman et al. (1990). The results were calculated as u n i t s / m l as compared to standard IL-2- or IL-3-containing preparations (supernatants of ConA-stimulated rat splenocytes) which was arbitrarily assigned as a value of 1000 units/ml. The results are expressed as percent of control (cells incubated with alcohol at concentrations similar to those carried over with the drug, in the absence of ligands).
Results
Effect of ethanol on the immune function Since the ligands were dissolved in ethanol, the effect of ethanol at concentrations carried out with the ligands (1%; 0.1%; 0.01%) was examined on the parameters tested in the present study. Ethanol at these concentrations had no significant effect on PHA- and ConA-induced proliferation of PBMC or IL-2 and IL-3-LA production by these ceils ( 4 - 6 experiments).
Effect of BZ receptor ligands on ConA-induced proliferation of PBMC A dose-dependent inhibitory effect on ConAinduced proliferation of PBMCs was observed when Ro5-4864, diazepam and P K l l 1 9 5 were added at concentrations between 10 -6 M and 10 -5 M (Fig. 1). No effect was found when clonazepam was added at the same concentrations. A highly significant reduction in ConA-induced proliferation was achieved by Ro5-4864 (54%) at 10 -5 M ( P < 0.01), while diazepam and P K l l 1 9 5 at the same concentration inhibited the proliferation by 26% and 20% only ( P < 0.05; Fig. 1). Ro5-4864 was the only drug that exhibited signifi-
Effect of the ligands on IL-2 and IL-3-LA activity The cells used in the present study for determination of IL-2 and IL-3-LA (CTLL 2 and 32D-cl-23) are of lymphoid origin and may express PBR. Experiments were designed to examine the possible direct effect of the ligands on the IL-2 and IL-3-LA activities in the assay systems. CM was incubated in the absence or presence of 10 -5 M of the various ligands for 24 h (IL-2) and 48 h (IL-3-LA), then media were collected and tested for their effect on the lymphokine activities in the assay systems. Briefly, 0.1 ml of CTLL-2 or 32-Dcl-23 cells (105/ml) suspended in GM, containing IL-2 or IL-3-LA (100 u n i t s / m l ) respectively, were added to 0.1 ml of the various preincubated media, and assayed for IL-2 and IL-3-LA activities as described above.
Statistical evaluation Statistical analysis was performed using Student's t-test. All results are expressed as mean + SEM.
Effect of BZ receptor ligands on Con-A induced proliferation of PBMC
2
110 tO 0 , ~ 100 E
90.
•
,~. P,
~,*
'*
DIAZEPAM
~ 700e~
PK 11195 RO5-4684
50-
CLONAZEPAM ---A--10-9
110-8 - - -
0 1 -7
I /J I 10 4 2.5X10 -6
I 5Xl0q~
I 7.5}(10 ~
Ligand concentration [M]
Fig. 1. The effect of benzodiazepine receptor ligands on ConA induced proliferation of human PBMC. PBMC were incubated for 72 h with the indicated concentrations of the various ligands and ConA (5/zg/ml). [3H]TdR was added for the last 18 h. Each experimental point represents the mean+ SEM of 4-8 experiments. Asterisk indicates statisticallysignificant differences from control cultures (incubated without any ligand) (* P < 0.05; ** P < 001).
22
Effect of BZ receptor ligands on PHA induced proliferation of PBMC g
110
0
100 I..
0
'~::::::'-"'~i- .........._.-.?
" ~ I
DIAZEPAM A
o 0 .~
.
.
! ......... % I ..... =
"--..
PK..I~I195 70
"-j, *
. I
.......
""-.., j
CLONAZEPAM
"0
~
10 .9
~-A-I 10.8
•
"mr.....
R05-4864 ..... ~11.....
n--
,60
.
**
"'I i'
i
1(77
10 4
2.5x10 -8
5X10 ~
7.5x10 -6
10.8
Ligand concentration [M] Fig. 2. T h e e f f e c t o f b e n z o d i a z e p i n e r e c e p t o r l i g a n d s o n PHA-induced proliferation of human PBMC. PBMC were
incubated for 72 h with the indicated concentrations of the various ligands and PHA (1 ;zg/ml). [3H]TdR was added for the last 18 h. Each experimental point represents the mean + SEM of 4-8 experiments. Asterisk indicates statistically significant differences from control cultures (incubated in the absence of any ligand) (* P < 0.05; * * P < 0.01). cant inhibitory effect (12%, P < 0.025) at a concentration as low as 10 -8 M.
Effect of BZ receptor ligands on PHA induced proliferation of PBMC A dose-dependent inhibitory effect of Ro54864 on PHA-induced PBMC proliferation was found when the ligand was added at concentrations ranging from 10 -7 M to 10 -5 M, reaching 32% inhibition at 10 -5 M ( P < 0.01; Fig. 2). At 10 - s M Ro5-4864 had no effect on PHA-induced PBMC proliferation. Incubation of PBMC with P K l l 1 9 5 at 10 -7 M resulted in a slight, yet significant, inhibition of 15% ( P < 0 . 0 5 ) . Diazepam significantly inhibited ( P < 0.05) PHA-induced proliferation of PBMC at concentrations ranging from 2.5 X 10 -6 M to 10 -5 M. The addition of clonazepam to PBMC did not reveal any inhibitory effect.
reduction of less than 10% was obtained. The addition of Ro5-4864 and diazepam to the IL-3L A assay system revealed 18% and 13% reduction in IL-3-LA, respectively, whereas PKl1195 and clonazepam induced an inhibition of 8% only. This direct effect of the ligands on the IL-2 and IL-3-LA assay systems was substracted from the activities detected in the supernatants, and the results represent the net effect of the ligands on the production of these lymphokines. A significant inhibition of IL-2 production by PBMC was obtained when diazepam or Ro5-4864 were added at 10 -5 M (32% and 26%, respectively, P < 0.05; Fig. 3). Clonazepam and PK11195 did not exert any significant effect on IL-2 production. The addition of diazepam, Ro5-4864 or PKl1195 at 10 -2 M to PBMC revealed a significant reduction of 18-19% in IL-3-LA production, ( P < 0.05; Fig. 4). In contrast, clonazepam did not affect IL-3-LA production by PBMC at this concentration.
Effect of flunitrazepam (FNZ) on the immune function The action of ligands acting on mitochondrial BZ receptors is inhibited by flunitrazepam (Papadopoulos et al., 1991). In order to test the Effect of BZ receptor ligands at 105M on IL-2 production by human PBMC 12o.
~ CONTROL
loo. ~ o O
'~
80-
~
~
~
To evaluate a possible direct effect of the ligands on the assay systems for IL-2 and IL-3-LA, we tested their effect at 10 -5 M on these systems. IL-2 activity was inhibited by 15% when Ro5-4864 was added, whereas for the other ligands a slight
i/
\,\\ ,,\\\
[ ] DIAZEPAM [ ] Ro 5-4864 [ ] PK 11195 E~CLONAZEPAM
\\\\ 40.
¢d
--
"
20.
\ \ \ \
"
"
"
"
.
.
.
.
• . .
Effect of BZ receptor ligands on IL-2 and IL-3-LA production
*
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1 I ×y
Fig. 3. The effect o f benzodiazepine receptor ligands on IL-2 production by human PBMC. P B M C were treated with 10 -5 M of the varius ligands as indicated. Culture supernatants were collected after 24 h and the IL-2 content determined as described in Materials and Methods. Results are expressed as mean percent o f control _+S E M o f 3 - 8 experiments. Asterisk indicates statistically significant difference from control ( P
19
© 1992 ElsevierSciencePublishers B.V. All rightsreserved 0165-5728/92/$05.00 JNI 02165
Immunomodulatory effect of peripheral benzodiazepine receptor ligands on human mononuclear cells H a n n a Bessler a, Ronit Weizman b, Moshe Gavish c, Ida Notti a and Meir Djaldetti a,d a Hematology Research Laboratory, Hasharon Hospital, Golda Medical Center, Petah Tiqva, b Hasharon Hospital, Petah Tiqva, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, c Department of Pharmacology, Faculty of Medicine and Rappaport Family Institute for Research in Medical Science, Technion, Institute of Technology, Haifa, and d Department of Medicine "B", Hasharon Hospital, Golda Medical Center, Petah Tiqva, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
(Received7 August,1991) (Revised, received12 December, 1991) (Accepted 12 December, 1991) Key words: Peripheral benzodiazepinereceptor; Mitogen-inducedproliferation;Interleukin-2;Interleukin-3-1ikeactivity
Summary Immunomodulatory effect of ligands active at the peripheral benzodiazepine receptor (PBR) was examined in human peripheral blood mononuclear cells (PBMC). Ro5-4864, PKll195 and diazepam suppressed phytohemagglutinin (PHA) and concanavalin A (ConA) induced proliferation of PBMC. All three ligands inhibited interleukin-3-1ike activity (IL-3-LA) secretion, while the production of interleukin-2 (IL-2) was inhibited by Ro5-4864 and diazepam only. The selective central benzodiazepine ligand clonazepam did not affect the cellular immune functions examined. Our results indicate an in-vitro immuno-suppressive activity of peripheral and mixed, but not central type benzodiazepine ligands.
Introduction Benzodiazepine (BZ) receptors have been identified and characterized in the synaptosomal fraction of the central nervous system (Mohler and Okada, 1977; Squires and Braestrup, 1977), as well as in a number of peripheral organs and non-neuronal brain tissue (Wang et al., 1980, 1981; Taniguchi et al., 1982; Schoemaker et al., 1981, 1983; De Souza et al., 1985). The central,
Correspondence to: M. Djaldetti, Department of Medicine 'B', HasharonHospital,GoldaMedicalCenter, P.O. Box 121, Petah Tiqva 49372, Israel.
but not the peripheral BZ receptors (PBR), are coupled to the 7-aminobutyric acid (GABA) receptors and the chlorid ion channels (Squires and Saederup, 1982). They mediate the anxiolytic, anticonvulsant and muscle relaxant activities of the BZs (Costa and Guidotti, 1979). It has been suggested that PBR ligands bind to ligand sites in the voltage-regulated calcium channel complex (Mestre et al., 1984). The high density of PBR in the cellular mitochondrial fraction implies a functional role for these receptors in intracellular metabolic events, rather than intercellular synaptic events (Anholt et al., 1986a, b; Snyder et al., 1987; Mukhin et al., 1989; Krueger and Papadopoulos, 1990; Papadopoulos et al., 1990).
20
However, other studies suggest that peripheral receptors can be found also in other subcellular compartments, such as nuclear (Schoemaker et al., 1983; Rago et al., 1990), sarcolemmic, and sarcoplasmic reticulum fractions (Doble et al., 1985). Several studies have demonstrated that the regulation of PBR in various tissues is under neural or hormonal control (for review see Saano, 1988; Verma and Snyder, 1989). Clonazepam and flumazenil are ligands specific for CBR, while PBR bind with high affinity the ligands Ro5-4864 (4-chlorodiazepam) and PKl1195 (an isoquinoline carboxamide derivative), which were suggested to be agonist and antagonist, respectively, to this receptor (Mestre et al., 1985). Diazepam is a mixed ligand which binds to both the central and peripheral receptors in nanomolar affinity (Wang et al., 1984). A line of evidence suggests a possible involvement of PBR in the regulation of immune function. PBR were identified in circulating mononuclear cells (Moingeon et al., 1983), macrophages (Zavala et al., 1984), thymocytes (Wang et al., 1981) and on a B-cell hybridoma (Pert et al., 1985). Autoradiographic study localized the anatomical distribution of PBR in organs of the rat immune system (Benavides et al., 1989). Ligands for PBR display immunomodulatory activity, such as enhancement of chemotaxis by human monocytes (Ruff et al., 1985). Peripheral and mixed type ligands but not central-type ligands potentiate the humoral response to sheep red blood cells (Zavala et al., 1984; Lenfant and Zavala, 1986). However, the role of PBR in the regulation of the immune function is as yet unknown. The purpose of the present study was to evaluate a possible in-vitro effect of specific PBR ligands on the immune system. Since interleukins play a major role in the regulation of immune function, we examined the effect of these ligands on the production of interleukin-2 (IL-2) and interleukin-3-1ike activity (IL-3-LA) by human peripheral blood mononuclear cells (PBMC). IL-3LA is a human growth factor similar to IL-3, released spontaneously by PBMC (Fishman et al., 1990). The effect of PBR ligands on mitogen-induced proliferation of human PBMC was also tested. The effects of the PBR ligands on the
immune functions were compared to those of clonazepam which is a selective CBR ligand.
Materials and methods
Materials Clonazepam, diazepam, flunitrazepam (FNZ) and Ro5-4868 (4'-chlorodiazepam) were supplied by Drs. H. Rutman and E. Kyburz (Hoffman La Roche, Basel, Switzerland). PKll195 (an isoquinoline carboxamide derivative) was donated by Dr. A. Bouvier, Rhone-Poulenc Sante (VitrySur-Seine, France). Ligand preparation All ligands were dissolved in absolute ethanol at 10 -3 M. Further dilutions were made in RPMI 1640 medium. The highest concentration of ligands used in our experiments was 10 -5 M revealing 1% ethanol in the incubation mixture. Cell preparation and incubation procedure Peripheral blood mononuclear ceils (PBMC) were isolated from the peripheral blood of healthy adult donors on Ficoll-Paque density gradient as described by Boyum (1968). Mononuclear cells (106/ml) were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% penicillin (10000 units/ml), 1% streptomycin sulphate (10 mg/ml) and amphotericin B (25 /zg/ml) (complete medium, CM). Aliquots of 0.18 ml were added to each well of a flat-bottomed microculture plate containing 0.02 ml of RPMI 1640 or various ligand concentrations. Cultures were stimulated with 1 ~ g / m l phytohemagglutinin (PHA-HA16, Wellcome-Research Lab. Bechman), or 5 ~zg/ml concanavalin A (ConA, Bio-Yeda, Israel). The cultures, set up in triplicate, were incubated for 72 h at 37°C in a humidified atmosphere containing 5% CO 2. 18 h prior to harvesting, the cells were pulsed with 0.5 ~Ci [3H]thymidine/well. The radioactivity was measured with an LKB liquid scintillation counter model 3380. The results are expressed as percent of control. IL-2 and IL-3-LA production For IL-2 production 1.5 × 10 6 PBMCs were suspended in CM containing 1 izg/ml PHA. 1-ml
21 aliquots of cell suspension were incubated for 24 h in 24-well plates (Nunc), in the absence or presence of various concentrations of the ligands. For IL-3-LA production, 1-ml aliquots of PBMCs suspension in CM (2 × 106 cells/ml) were incubated for 48 h in 24-well plates (Nunc) in the absence or presence of various concentrations of the ligands. At the end of the incubation period culture media were collected, cells were removed by centrifugation, and supernatants were kept at - 20°C until assayed.
IL-2 and IL-3-LA bioassays IL-2 activity in the supernatants was determined using the CTLL-2 cell line as reported previously (Serrate et al., 1987). IL-3-LA was evaluated by using the 32-D-cl-23 cell line as described by Fishman et al. (1990). The results were calculated as u n i t s / m l as compared to standard IL-2- or IL-3-containing preparations (supernatants of ConA-stimulated rat splenocytes) which was arbitrarily assigned as a value of 1000 units/ml. The results are expressed as percent of control (cells incubated with alcohol at concentrations similar to those carried over with the drug, in the absence of ligands).
Results
Effect of ethanol on the immune function Since the ligands were dissolved in ethanol, the effect of ethanol at concentrations carried out with the ligands (1%; 0.1%; 0.01%) was examined on the parameters tested in the present study. Ethanol at these concentrations had no significant effect on PHA- and ConA-induced proliferation of PBMC or IL-2 and IL-3-LA production by these ceils ( 4 - 6 experiments).
Effect of BZ receptor ligands on ConA-induced proliferation of PBMC A dose-dependent inhibitory effect on ConAinduced proliferation of PBMCs was observed when Ro5-4864, diazepam and P K l l 1 9 5 were added at concentrations between 10 -6 M and 10 -5 M (Fig. 1). No effect was found when clonazepam was added at the same concentrations. A highly significant reduction in ConA-induced proliferation was achieved by Ro5-4864 (54%) at 10 -5 M ( P < 0.01), while diazepam and P K l l 1 9 5 at the same concentration inhibited the proliferation by 26% and 20% only ( P < 0.05; Fig. 1). Ro5-4864 was the only drug that exhibited signifi-
Effect of the ligands on IL-2 and IL-3-LA activity The cells used in the present study for determination of IL-2 and IL-3-LA (CTLL 2 and 32D-cl-23) are of lymphoid origin and may express PBR. Experiments were designed to examine the possible direct effect of the ligands on the IL-2 and IL-3-LA activities in the assay systems. CM was incubated in the absence or presence of 10 -5 M of the various ligands for 24 h (IL-2) and 48 h (IL-3-LA), then media were collected and tested for their effect on the lymphokine activities in the assay systems. Briefly, 0.1 ml of CTLL-2 or 32-Dcl-23 cells (105/ml) suspended in GM, containing IL-2 or IL-3-LA (100 u n i t s / m l ) respectively, were added to 0.1 ml of the various preincubated media, and assayed for IL-2 and IL-3-LA activities as described above.
Statistical evaluation Statistical analysis was performed using Student's t-test. All results are expressed as mean + SEM.
Effect of BZ receptor ligands on Con-A induced proliferation of PBMC
2
110 tO 0 , ~ 100 E
90.
•
,~. P,
~,*
'*
DIAZEPAM
~ 700e~
PK 11195 RO5-4684
50-
CLONAZEPAM ---A--10-9
110-8 - - -
0 1 -7
I /J I 10 4 2.5X10 -6
I 5Xl0q~
I 7.5}(10 ~
Ligand concentration [M]
Fig. 1. The effect of benzodiazepine receptor ligands on ConA induced proliferation of human PBMC. PBMC were incubated for 72 h with the indicated concentrations of the various ligands and ConA (5/zg/ml). [3H]TdR was added for the last 18 h. Each experimental point represents the mean+ SEM of 4-8 experiments. Asterisk indicates statisticallysignificant differences from control cultures (incubated without any ligand) (* P < 0.05; ** P < 001).
22
Effect of BZ receptor ligands on PHA induced proliferation of PBMC g
110
0
100 I..
0
'~::::::'-"'~i- .........._.-.?
" ~ I
DIAZEPAM A
o 0 .~
.
.
! ......... % I ..... =
"--..
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"-j, *
. I
.......
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"0
~
10 .9
~-A-I 10.8
•
"mr.....
R05-4864 ..... ~11.....
n--
,60
.
**
"'I i'
i
1(77
10 4
2.5x10 -8
5X10 ~
7.5x10 -6
10.8
Ligand concentration [M] Fig. 2. T h e e f f e c t o f b e n z o d i a z e p i n e r e c e p t o r l i g a n d s o n PHA-induced proliferation of human PBMC. PBMC were
incubated for 72 h with the indicated concentrations of the various ligands and PHA (1 ;zg/ml). [3H]TdR was added for the last 18 h. Each experimental point represents the mean + SEM of 4-8 experiments. Asterisk indicates statistically significant differences from control cultures (incubated in the absence of any ligand) (* P < 0.05; * * P < 0.01). cant inhibitory effect (12%, P < 0.025) at a concentration as low as 10 -8 M.
Effect of BZ receptor ligands on PHA induced proliferation of PBMC A dose-dependent inhibitory effect of Ro54864 on PHA-induced PBMC proliferation was found when the ligand was added at concentrations ranging from 10 -7 M to 10 -5 M, reaching 32% inhibition at 10 -5 M ( P < 0.01; Fig. 2). At 10 - s M Ro5-4864 had no effect on PHA-induced PBMC proliferation. Incubation of PBMC with P K l l 1 9 5 at 10 -7 M resulted in a slight, yet significant, inhibition of 15% ( P < 0 . 0 5 ) . Diazepam significantly inhibited ( P < 0.05) PHA-induced proliferation of PBMC at concentrations ranging from 2.5 X 10 -6 M to 10 -5 M. The addition of clonazepam to PBMC did not reveal any inhibitory effect.
reduction of less than 10% was obtained. The addition of Ro5-4864 and diazepam to the IL-3L A assay system revealed 18% and 13% reduction in IL-3-LA, respectively, whereas PKl1195 and clonazepam induced an inhibition of 8% only. This direct effect of the ligands on the IL-2 and IL-3-LA assay systems was substracted from the activities detected in the supernatants, and the results represent the net effect of the ligands on the production of these lymphokines. A significant inhibition of IL-2 production by PBMC was obtained when diazepam or Ro5-4864 were added at 10 -5 M (32% and 26%, respectively, P < 0.05; Fig. 3). Clonazepam and PK11195 did not exert any significant effect on IL-2 production. The addition of diazepam, Ro5-4864 or PKl1195 at 10 -2 M to PBMC revealed a significant reduction of 18-19% in IL-3-LA production, ( P < 0.05; Fig. 4). In contrast, clonazepam did not affect IL-3-LA production by PBMC at this concentration.
Effect of flunitrazepam (FNZ) on the immune function The action of ligands acting on mitochondrial BZ receptors is inhibited by flunitrazepam (Papadopoulos et al., 1991). In order to test the Effect of BZ receptor ligands at 105M on IL-2 production by human PBMC 12o.
~ CONTROL
loo. ~ o O
'~
80-
~
~
~
To evaluate a possible direct effect of the ligands on the assay systems for IL-2 and IL-3-LA, we tested their effect at 10 -5 M on these systems. IL-2 activity was inhibited by 15% when Ro5-4864 was added, whereas for the other ligands a slight
i/
\,\\ ,,\\\
[ ] DIAZEPAM [ ] Ro 5-4864 [ ] PK 11195 E~CLONAZEPAM
\\\\ 40.
¢d
--
"
20.
\ \ \ \
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.
.
.
.
• . .
Effect of BZ receptor ligands on IL-2 and IL-3-LA production
*
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1 I ×y
Fig. 3. The effect o f benzodiazepine receptor ligands on IL-2 production by human PBMC. P B M C were treated with 10 -5 M of the varius ligands as indicated. Culture supernatants were collected after 24 h and the IL-2 content determined as described in Materials and Methods. Results are expressed as mean percent o f control _+S E M o f 3 - 8 experiments. Asterisk indicates statistically significant difference from control ( P
