- Email: [email protected]
In vitro O2-induced depression of T and B lymphocyte activation is reversed by diethyldithiocarbamate (DDC) treatment
Immunology Letters, 18 (1988)99-108
Elsevier IML 01054
In vitro O2-induced depression of T and B lymphocyte activation is reversed by diethyldithiocarbamate (DDC) treatment P h i l i p p e L a c o m b e , Isabelle Carre, Michel Fay a n d J e a n - J a c q u e s P o c i d a l o Inserm U.13, H6pital Claude Bernard, 75944 Paris, France
(Received25 February 1988; accepted 9 March 1988)
1. Summary
2. Introduction
In this study, we tried to establish a relationship between the immunopotentiating effects and the antioxidant activity of the immunostimulating compound, diethyldithiocarbamate (DDC). We studied the effects of DDC treatment on enriched T and B murine spleen lymphocytes in an in vivo - ex vivo model of O2-induced immune depression. Female C57B1/6 mice were injected subcutaneously with a single dose of DDC (125 mg. kg-1). Eight days after DDC injection, we evaluated, in vitro, the concanavalin A response o f the T cell fraction and the LPS response of the B cell fraction, under standard (air - 5% C O 2 ) and hyperoxic (600/0 0 2 -- 5% CO2) culture conditions. The results show that after a lag period, DDC is able [1] to enhance the mitogenic response of T and B murine lymphocytes under standard culture conditions [2] to restore the ConA response and to partially restore the LPS response under hyperoxic conditions. The results of this study suggest that the immunostimulatory effects of DDC could be related to the antioxidant activity of this compound on the lymphoid cellular metabolism. This activity apparently affects both T and B lymphocytes.
It was shown that, in vitro, the oxygen-derived metabolites which were produced by enzymatic systems suppressed the immune responses [1, 2]. Recently, we have demonstrated that in vivo hyperoxic exposure induced a depression of the murine immune responses [3 - 5]. Furthermore, we have developed a model of in vitro hyperoxic exposure which showed that oxygen and/or its metabolites directly depressed the mitogenic response of rat lymphoid cells [6, 7]. In these two latter models, the immune depression can be reversed by several antioxidant drugs such as 2-mercaptoethanol (2-ME) which is known to enhance the lymphocyte mitogenic response [8, 9]. Furthermore, one or more oxidation steps appear to be essential for the mitogenic response [10, 11]. Lymphocyte activation seems to depend on a dual effect of oxidant generation. On the other hand, it is well known that DDC is an immunostimulant [12, 13] and it has been suggested that it may shield against oxygen injury [14]. Recent pharmacological study in our laboratory demonstrated that a single injection of DDC (125 mg • kg -~ optimal dose) eight days before in vivo hyperoxic exposure, partially prevented the pulmonary and lymphoid oxygen toxicity [15]. But, in vivo, oxygen toxicity was firstly exerted on pulmonary cells [16], and the observed DDC protection of the lymphoid ceils against oxygen toxicity could be the result of the pulmonary DDC protection. At this stage, it was difficult to establish that DDCtreatment was able to induce a lymphoid protection against oxidative injury. In this work, we studied the antioxidant properties
Key words: Lymphocyteactivation;Diethyldithiocarbamate;Im-
munostimulation; Antioxidant activity Correspondence to: P. Lacombe,INSERMU.13,H6pital Claude Bernard, 10, Avenuede la Porte d'Aubervilliers,75944Paris, Cedex 19, France.
0165-2478 / 88 / $ 3.50 © 1988 ElsevierSciencePublishers B.V.(BiomedicalDivision)
99
of DDC on the lymphocyte mitogenic response. We tried to point out a relationship between the immunostimulating effects of this compound and the oxidant generation involved in the lymphocyte activation. We studied the mitogenic response of murine splenocytes in a model of in vitro Oz-induced immune depression. Several days after the injection of DDC or saline (control), we evaluated the ConA response of the T cell fraction and the LPS response of the B cell fraction, under standard (95% air - 5% CO2) or hyperoxic (60% Oz - 35% N 2 - 5 % C O 2 for 24 h) culture conditions. In parallel, the mitogenic responses of control cells were studied in presence of 2-ME, and served as positive controls. The results suggest (a) that DDC exerts an antioxidant activity on both T and B lymphocyte metabolism and Co) that the immunostimulating effects of DDC could be related, in part, to its antioxidant activity.
ded to one volume of 1.5 M NaCl to provide an isoosmotic stock solution of density 1.120 g/ml (100% Percoll). We prepared two solutions of density 1.096 g/ml (80% Percoll) and 1.079 g/ml (65% Percoll), by diluting 8 parts 100% Percoll to 2 parts 0.15 M NaC1 and 6.5 parts 100°70 Percoll to 3.5 parts 0.15 M NaC1, respectively. The cells (6 to 8 × 10 7 cells) were suspended in 2 m180% Percoll, in a 10-ml conical tube, and 4 m165% Percoll were layered over this initial layer. The tubes were centrifuged at 400 × g (1500 rpm) for 15 min. The first 4 ml at the top of the gradient were collected. The cells from this fraction were washed twice in Hank's balanced salt solution (HBSS). The recovery of spleen lymphocytes and monocytes was consistently 9 5 - 9 8 % and the viability determined by trypan blue dye exclusion was found to be greater than 97%. Cells in the treated group were then pooled randomly, as were cells in the control group, in order to obtain a sufficient number of cells for the cultures.
3. Materials and Methods
3.1. Reagents Anti-Thy-l.2 monoclonal antibody was purchased from Cedarlane Laboratories (Canada). Goat antimouse Igs antibodies (GAM) labelled with fluorescein (FITC) were purchased from Nordic Laboratories (Netherlands). Concanavalin A (ConA) and lipopolysaccharide from E. coli 055 : B5 (LPS) were purchased from Sigma Chemical Company (USA). 3.2. Animals Eight- to ten-week old female C57B1/6 mice (IFFA-CREDO, Lyon, France) were assigned randomly to experimental and control groups. 3.3. DDC treatment DDC (Institut M6rieux, France) was prepared as previously described [15]. Mice were given a single dose (125 mg. kg 1 body wt) of DDC by subcutaneous injection, eight days before ex vivo experimentation. Control mice were given an equivalent injection of sterile saline.
3.4. Cell preparations Cells were obtained from the spleen as previously described [17], and isolated on Percoll density gradient to remove red blood cells and dead cells. Nine volumes of Percoll (Pharmacia France S.A.) were ad100
3.5. Spleen cell fractionation Splenocytes were fractionated by filtration through nylon wool columns. The barrels of 10-ml plastic syringes were packed to the 6-ml mark with 0.6 g of nylon wool (type 200, Fenwall Labs., USA) and sterilized before use. The columns were washed with 20 ml of RPMI 1640 (Gibco) - 5 % foetal calf serum (FCS, Gibco) (RPMI/FCS), incubated 1 h at 37 °C, for equilibration. After recovery from the Percoll density gradient, 2 × 108 spleen cells in 2 ml R P M I / F C S were loaded onto the nylon wool column, and incubated 45 rain at 37 °C. Effluent cells (T cell fraction) were collected by adding 25 ml of warm R P M I / F C S to the columns. The adherent cells (B cell fraction) were obtained by incubating the nylon wool in 25 ml of RPMI 1 6 4 0 - 4 % F C S - 0 . 2 % EDTA, 30 min, at 37 °C. The nylon wool was removed from the medium, pressed with a sterile syringe piston and the exudate was added to the medium. The cells were collected by centrifugation and washed three times with RPMI 1640. The lymphocyte sub-populations in the two fractions were identified by immunofluorescence. 3.6 Identification of lymphocyte sub-populations Anti-Thy-l.2 monoclonal antibody (anti-Thy-l.2 mAb) labelled all murine T lymphocytes. Immunofluorescent staining was performed using
1 - 2 x 1 0 6 cells in 50/A of HBSS (without phenol r e d ) - 4 % F C S - 2 % Hepes b u f f e r - 0 . 1 % sodium azide (fluorescence buffer, EB.) incubated with 25/zl of anti-Thy-l.2 mAb (1:20 dilution) for 30 min on ice. These cells were washed three times, incubated in 50 #1 EB. with 25 ixl F I T C - GAM conjugated for 30 min on ice, and washed again. B cells, surface Igbearing cells (SIg+), were identified by a direct staining with 25 ~1 F I T C - GAM conjugate. The percentage of positive cells was calculated as follows: % of positive cells = number of fluorescent cells x 100 total cell number The percentage o f T cells (Thy-l.2 +) was determined as follows: %Thy-l.2 ÷ = 070(GAM +Thy-l.2) ÷ - 070(GAM) +. A total of 100- 200 cells were counted for each determination. 3.7. 02 exposure Cultured cells were exposed to oxygen (60% 0 2 - 3 5 % N 2 - 5 % CO2) for 24 h, as previously described [7] after which incubation continued at 37°C in 5% CO2 enriched humidified air atmospher incubator (Heraus, France), for a total incubation time o f 48, 72 or 96 h (hyperoxic conditions). In parallel, cultures were grown in a i r - 5 % CO2 for the same incubation time (standard conditions). 3.8. Cultures Cells from both T cell and B cell fractions were cultured in triplicate as previously described [17] for 48, 72 and 96 h under hyperoxic or standard conditions. T cells were cultured with or without C o n A (2/~g/ml) and B cells were cultured with or without LPS (50 #g/ml). The cells from control animals cultured in presence of mitogen and 2-ME (5 x 10 -5 M) were used as positive control. 2-ME was added to the medium at the beginning of the culture. The mitogenic response was evaluated by [3H]thymidine incorporation ([3H]TdR uptake). Cells were pulsed with l t~Ci of [3H]TdR (1 Ci/mmole, C E A Saclay France) during the last 20 h of culture, and harvested using a multiple cell culture processor (Minimash, Dynatech). [3H]TdR
uptake was determined by counting in a/3-liquid scintillation spectrophotometer (LKB). Results were expressed as A counts (cpm of stimulated c e l l s - c p m of unstimulated cells) per minute per culture (A cpm/culture). 3.9. Statistical analysis The statistical analyses were made by using Student's t-test and analysis of variance. 4. Results
4.1. Identification of splenic lymphocyte sub-popu-
lations after fractionation on nylon wool column We observed no difference between control and DDC-treated animals, in the T cell and B cell fraction (Table 1). The percentages of T cells (Thy-l.2 ÷) in the T cell fraction and B cells (SIg +) in the B cell fraction were the same in both control and DDCtreated groups - 10070of B cells and 13-1407o of T cells contaminated the T cell and B cell fractions, respectively. 4.2. Effect of DDC treatment on the ConA response
of the T cell fraction Under standard conditions (9507o air-5070 CO2),
Table 1 Identification of splenic lymphocytesub-populations after fractionation on nylonwoolcolumn, from control and DDC-treated animals. T cell fraction
B cell fraction
SIg+ 070
Thy-l.2 + 070
SIg+ 07o
Thy-l.2 + %
Control
10.9 _+1.5
68.1 _+2.7
67.9 _+3.0
13.3 _+1.5
DDC
9.1 ___1.0
69.0 _+2.3
70.4 + 2.0
14.9 _+1.8
T and B cell fractions were obtained as described in Sec. 3. Lymphocyte sub-populations were identified as described in Sec. 3. B cells: Slg+; T cells: Thy-l.2 + . Results are expressed as the arithmetic mean of the percentageof fluorescentpositivecells + S.E.M. from twelve animals in both control and DDC-treated groups. No significant difference was observed between control and DDC-treated groups. 101
we observed after DDC injection a significant increase of the [3H]TdR uptake after 48 ( P < 0.05) and 72 h ( P < 0.05) of culture, but the peak [3H]TdR uptake was obtained after 48 h of culture (Fig. 1). Furthermore, no significant [3H]TdR incorporation was seen after 24 h of culture (data not shown). Concurrently, in presence of 2-ME (positive control) the ConA response of the control was significantly enhanced after 48 (P < 0.01) and 72 h (P < 0.001) of culture; the peak of [3H]TdR uptake was obtained after 72 h of culture. On the other hand, under hyperoxic culture conditions (60°70 0 2 - 35% N 2-5°70 CO2), DDC treatment also enhanced significantly the [3H]TdR uptake after 48 ( P < 0.01), 72 (P<0.01) and 96 h (P<0.001) of culture, as compared to the control culture, under the same conditions (Fig. 2). This enhancing effect of DDC treatment was significantly greater ( P < 0.01) than that of 2-ME (positive control), after 72 h of culture (Fig. 2). To determine the absolute effect of 2-ME and DDC treatment on the ConA response, we compared
the peaks of [3H]TdR uptake to the control peak (control cells in air). The results were expressed as the percentage of [3H]TdR uptake in control ceils in air (100°70) (Table 2). We observed that DDC treatment or 2-ME were able to enhance two-fold the ConA response, under standard conditions. On the other hand, under hyperoxic culture conditions, while control response was drastically inhibited, 2-ME restored and DDC treatment significantly increased ( P < 0.05) the ConA response, but the responses obtained with 2-ME or DDC were not significantly different. 4.3. Effect of DDC treatment on the L P S response of the B cell fraction As shown in Fig. 3, we observed that DDC treatment significantly increased the LPS response of the B cell fraction after 48 ( P < 0.001) and 72 h ( P < 0.05) of culture, under standard conditions. This enhancing effect of DDC treatment was the same order of magnitude as that of 2-ME. Furthermore, in all cases
150-•
eel
A t~
Ic) X
100--
E
O.
t.)
UJ
X4
n n-
50-
I--
co
O-
I 0
I }
I 48
I 72
I 96
CULTURE TIME ( HOURS )
Fig. 1. Effect of DDC treatment on the ConA response of the T cell fraction, under standard conditions. The cells were cultured under standard conditions (95 % a i r - 5 °7o CO2) and the ConA responses were evaluated, as described in Section 3. • . . . . . a , ConA response of the T cell fraction from DDC-treated animals. • - . - . - . • , ConA response in the presence of 2-ME of the T cell fraction from control animals (positive control). • ..... o, ConA response of the T cell fraction from control animals (control). Results are expressed as the arithmetic mean_+ S.E.M. (Acpm_+ S.E.M.) of 4 experiments from 3 pools of cells (3 animals/pool) in both control and DDC-treated groups. All results were compared to the mitogenic response of the control group. *P<0.05, **P<0.01, ***P<0.001.
102
150--
oe ¢9 I o X
100--
E Q. tO
<1 v
UJ I-fl. ar 10 I-I "r ¢0
50--
,~mlmmD~ amml~ m mmLm gm ~ m ~
O--
I"-'1 0
I
nmbm mmmm m m m m ambumm mm~)
I
I
I
48
72
96
C U L T U R E TIME ( HOURS
)
Fig. 2. Effect of DDC treatment on the ConA response of the T cell fraction, under hyperoxicconditions. The cells were cultured under hyperoxic conditions (60% 02-35°70 N2-5% CO2) and the ConA responses were evaluated, as described in Section 3. • ..... •, • - . - . - m, • ..... e, Symbols are the same as in Fig. 1. Results are expressed and the comparisons were made, as described in Fig. I.
we observed no significant incorporation o f [3H]TdR after 24 h of culture (data not shown). Under hyperoxic conditions (Fig. 4), DDC treatment significantly enhanced the LPS response of the B cell fraction after 48 (P<0.01), 72 (P<0.01) and 96 h (P < 0.05) of culture, as compared to the control response under the same conditions of culture. This effect of DDC treatment was the same order of magnitude as that of 2-ME. We determined the absolute effect o f 2-ME or DDC treatment on the LPS response, by the same calculation as that in Table 2 for the C o n A response. We observed that 2-ME or DDC treatment enhanced two-fold the LPS response in air, but the corresponding effect under hyperoxic culture conditions was only a partial restoration of the LPS response (Table 3). 4.4. Cell viability As shown in Table 4, we observed no significant difference between the cell viabilities of DDCtreated group and control group during the C o n A
response o f the T cell fraction. Same results were observed for LPS response of the B cell fraction (data not shown).
5.
Discussion
In this study, using an in vitro model of O2-induced immune depression, we have shown that the DDC treatment completely restored the C o n A response and partially restored the LPS response of enriched T and B lymphocytes, respectively (Figs. 2 and 4). These results suggest that DDC exerts an antioxidant activity on the metabolism of both T and B murine splenocytes. This activity affected less B cells than T cells. This could be explained by the greater susceptibility of B lymphocytes than of T lymphocytes to O2 toxicity [18]. Concurrently, under standard culture conditions, DDC treatment enhances two-fold the C o n A and LPS responses of enriched T and B lymphocytes, respectively (Figs. 1 and 3). Although the C o n A re103
Table 2 Determination of the absolute effect of DDC treatment or 2-ME on the ConA response of the T cell fraction.
Table 3 Determination of the absolute effect of DDC treatment or 2-ME on the LPS response of the B cell fraction.
070 of the peak of the control response
Control a 2-ME b DDC c
Air
0 2
100_+ 13 188_+ 9*** 179 _+20**
24_+ 3** 122_+ 8 160 _+ 18'
°7o of the peak of the control response
Control 2-ME DDC
Air
0 2
100 _+20 196+ 14"* 172 + 13**
10 _+2"* * 33 _+ 1"* 39 _+3**
The cells were cultured as described in Table 2. The results are expressed as described in Table 2, except relating to LPS response instead of ConA response. Comparisons are made as described in Table 2. **, ***, are the same as Table 2.
The cells were cultured as described in Sec. 3 under standard (air) or hyperoxic (02) conditions. The results are expressed as follows: 070 of the peak of control response = cpm peak of [3H]TdR uptake of ConA response under both air or 0 2
sponse which was obtained with T cells from DDCtreated animals was shortened (Fig. 1), no significant [3H]TdR incorporation was observed after 24 h of culture under standard culture conditions (data not shown). Thus, the absolute effect of DDC treatment was a potentiation o f the ConA response as revealed by the comparison of the peaks of [3H]TdR uptake (Table 2). On the other hand, we studied the mitogenic responses of 70°70 enriched T and B cell subpopulations (Table 1). It was demonstrated that the ConA and LPS responses were specific to T [19] and B [20]
cpm peak of [3H]TdR uptake of ConA response of control cells in air The peak of [3H]TdR uptake obtained for the ConA response of control cells under standard culture conditions is considered as 100%. a ConA response of T cells from control animals (control cells). b ConA response of T cells from control animals, in presence of 2-ME (positive control). c ConA response of T cells from DDC-treated animals. Results are expressed as the arithmetic mean _+ S.E.M. of 4 experiments from 3 pools of cells (3 animals/pool) in both control and DDC-treated groups. All results were compared to 100070. * P < 0 . 0 5 , **P<0.01, ***P<0.001.
o o o
50-A I
0
o o o
x E
0
j
25--
........
iIll
IIIL
W
F¢r
pO--
I 0
I I
I 48
I 72
I 96
CULTURE TIME ( HOURS )
Fig. 3. Effect of DDC treatment on the LPS response of the B cell fraction, under standard conditions. The cells were cultured under standard conditions (95070 a i r - 5 % CO2) and the LPS responses were evaluated as described in Section 3. • . . . . . A, LPS response of the B cell fraction from DDC-treated animals, n - . - . - I, LPS response in presence of 2-ME of the B cell fraction from control animals (positive control). • ..... 0, LPS response of the B cell fraction from control animals (control). Results are expressed and the comparisons were made, as described in Fig. 1.
104
oe
q
A
10-I 0
I........ ...'"S',.:a:"....,
X
E
e~
o ,11 v
W
5--
I-Q. o n,-
I-I T O--
I
II
0
I
I
I
48
72
96
CULTURE ( HOURS
TIME )
Fig. 4. Effect of D D C treatment on the LPS response of the B cell fraction, under hyperoxic conditions. The cells were cultured under hyperoxic conditions (6007o 02-35°7o N 2 - 5 % CO2) and the LPS responses were evaluated as described in Section 3. • . . . . . • , • - . - . - i, • ..... • , Symbols are the same as in Fig. 3. Results are expressed and the comparisons were made, as described in Fig. 1.
Table 4 Cell viability during the C o n A response of the T cell fraction from control and DDC-treated groups. Culture time (h)
Control Air DDC Air Control O2 DDC 02
48
72
96
71 + 2
79_+4
60±5
75±2
76±3
56±6
63±3
64+3
61+6
61±2
65±3
59+4
The cell viability was determined by trypan blue dye exclusion after 48, 72 and 96 h of culture, under standard (air) or hyperoxic (02) conditions. The results are expressed as the percentage of viable cells: No. of viable cells ( Total no. of cells ) x 100) and as the arithmetic mean_+ S.E.M. of 4 experiments from 3 pools of cells (3 animals/pool) in both control and DDC-treated groups. No significant difference was observed between control and DDC-treated groups.
murine lymphocytes, respectively. LPS response was known to be T cell-independent [21, 22]. Furthermore, we observed no significant difference between the percentages of T and B cell subpopulations (Table 1) and the cell viabilities (Table 4) of DDCtreated and control groups. These results showed that the effects of DDC treatment were independent of the cell viability, and were exerted on T and B lymphocyte metabolism. These results are partly in accordance with those of other authors [23]. These above observations suggest that DDC exerts its immunostimulating effect in part by antioxidative activity on the lymphoid cellular metabolism. Indeed, DDC has the same type of activity as the antioxidant drug, 2-ME, and the observed results with this latter compound were the same order of magnitude as those obtained with DDC (Tables 2 and 3). Although the primary mechanism of action of DDC and 2-ME may be different, these two compounds can act finally on the same metabolic target. Indeed, DDC is used in vivo and its effect can be observed only after a lag period, suggesting that DDC is active through indirect pathways. Furthermore, no effect is observed when DDC is used in vitro [24, 251. It has been demonstrated that DDC stimulates the synthesis of the enzymes of the glutathione (GSH) cycle I05
[26]. On the other hand, 2-ME was used in vitro. We have demonstrated that 2-ME enhanced the mitogenic response of murine lymphocytes via its action on the GSH metabolic pathways [27], and this action can be related to antioxidant properties of the GSH [6, 17]. It has been shown that the depletion of intracellular GSH involved an inhibition of the lymphocyte activation [28]. Thus, it seems that the antioxidant properties of DDC could be secondary to its action on the lymphocyte glutathione metabolic pathways. At this stage, there is a discrepancy between (a), the antioxidant properties of DDC related to its immunopotentiating effects and (b), the fact that oxidants must be generated if the events leading to lymphocyte proliferation are to be triggered [11]. The hypothesis that the DDC exerts its immunostimulatory effects by an antioxidant process, can be reconciled when we consider the duality of the effects of oxidant generation. While the 02 metabolites are necessary to trigger the early events of the lymphocyte activation, they are deleterious during the late phase of this activation [29]. Indeed, the early events which are triggered by the oxidant generation lead to the stimulation of arachidonate release [30, 31]. These processes are accompanied by the formation of and are modulated by lipoxygenase-leukotriene metabolites which may be the activation pathway during the lymphocyte proliferation [32]. This stimulation of 5-1ipoxygenase pathways increased the intracellular peroxide level during the lymphocyte activation. The increased peroxide level could lead to (a), autocatalytic lipid peroxidation which impairs cellular integrity [33], (b), inhibition of 5-1ipoxygenase pathways [34] and (c), stimulation of cyclooxygenase pathways [35] which are known to down-regulate the lymphocyte activation [36]. It appears that a limitation of the peroxide level can promote the lymphocyte activation. Such a limitation is performed by GSH via the GSH peroxidase [37]. This could explain the relationship between the immunostimulatory effects of DDC and its antioxidant activity which we point out in this study. This work strongly suggests that DDC exerts its immunostimulatory effects in part by antioxidative activity which could be related to an action on the lymphocyte glutathione metabolism. This activity of DDC is exerted on T and B lymphocytes. Our model of O2-induced immune depression in which 106
the oxidative processes involved in lymphocyte activation were amplified, appears to be a useful approach to study of these phenomena. Further studies are presently being performed to determine the relationship between the immunostimulating/antioxidant effects of DDC and lymphocyte glutathione metabolism, and to better understand the action of DDC on both T and B lymphocytes.
Acknowledgements The authors are grateful to Mrs. Annie-Christine Poidevin for typing the manuscript.
References [1] Hoffeld, J. T., Metzger, Z. and Oppenheim, J. J. (1981) in: Lymphokines (E. Pick, Ed.) pp. 63-86, Academic Press, London. [2] Kraut, E. H. and Sagone, L. (1981) J. Lab. Clin. Med. 98, 697. [3] Levacher-Place, M., Gougerot-Pocidalo, M. A., Rouveix, B., Kraus, L. and Pocidalo, J. J. (1983) Clin. Exp. Immunol. 54, 580. [4] Gougerot-Pocidalo, M. A., Fay, M. and Pocidalo, J. J. (1984) Clin. Exp. Immunol. 58, 428. [5] Gougerot-Pocidalo, M. A., Fay, M., Roche, Y., Lacombe, P. and Marquetty, C. (1985) J. Immunol. 135, 2045. [6] Kraus, L., Gougerot-Pocidalo, M. A., Lacombe, P. and Pocidalo, J. J. (1985) Int. J. Immunopharmacol. 7, 753. [7] Kraus, L., Lacombe, P., Fay, M. and Pocidalo, J. J. (1985) Immunol. Lett. ll, 51. [8] Broome, J. D. and Jeng, M. W. (1973) J. Exp. Med. 138, 574. [9] No,lie, R. J. and Lawrence, D. A. (1980) Cell. Immunol. 50, 416. [10] Coffey, R. G., Hadden, E. M. and Hadden, J. W. (1981) J. Biol. Chem. 256, 4418. [11] Mookerje, B. K., Wakerle, H. W., Sharon, N. and Fisher, H. (1984) J. Leukocyte Biol. 35, 427. [12] Renoux, G. and Renoux, M. (1979) J. Immunopharmacol. 1, 247. [13] Renoux, G. (1980) Drugs 19, 86. [14] Renoux, G. (1982) J. Pharmacol. (Paris) 13 (Suppl.), 95. [15] Mansour, H., Levacher-Place, M., Gougerot-Pocidalo, M. A., Rouveix, B. and Pocidalo, J. J. (1986) J. Pharmacol. Exp. Ther. 236, 476. [16] Kimball, R .E., Reddy, K., Pierce, T. H., Schwartz, L. W., Mustafa, M. G. and Cross, C. E. (1976) Am. J. Physiol. 230, 1425. [17] Lacombe, P., Kraus, L., Fay, M. and Pocidalo, J. J. (1986) Biochimie 68, 555. [18] Farber, C. M., Liebes, L. F., Kanganis, D. N. and Silber, R. (1984) J. Immunol. 132, 2543.
[19] Williams, M. and Benacerraf, B. (1972) J. Exp. Med. 135, 1279. [20] Peavy, D. L., Shands, J. W., Adler, W. H. and Smith, R. T. (1973) J. Immunol. Ill, 352. [21] Watson, J., Epstein, R., Nakoing, I. and Ralph, P. (1973) J. Immunol. ll0, 43. [22] Horan, M. A. and Fox, R. A. (1984) Mech. Age Dev. 26, 165. [23] Neveu, P. J., Perdoux, D. and Lafleur, L. (1982) Int. J. Immunopharmacol. 4, 9. [24] Hadden, J. W. (1977) in: Immunopharmacology (J. W. Hadden, R. G. Coffey and E Spreafico, Eds.) pp. 1- 28, Plenum Publishing Corp., New York. [25] Renoux, G. and Renoux, M. (1977) in: Control of Neoplasia by the Modulation of the Immune System (M. A. Chirigos, Ed.) pp. 67-80, Raven Press, New York. [261 Deneke, S. M. and Fanburg, B. L. (1980) Biochem. Pharmacol. 29, 1367. [27] Lacombe, P., Kraus, L., Fay, M. and Pocidalo, J. J. (1987) Biochimie 69, 37. [28] Fidelus, R. K., Ginouves, P., Lawrence, D. and Tsan, M. E (1987) Exp. Cell Res. 170, 269.
[29] Lacombe, P., Kraus, L., Fay, M. and Pocidalo, J. J. (1985) Febs Lett. 191,227. [30] Coffey, R. G. and Hadden, J. W. (1981) in: Advances in Immunopharmacology (J. W. Hadden, L. Chedid, P. Mullen and E Spreafico, Eds.) Vol. 1, pp. 365 - 373. Pergamon Press Ltd., Oxford. [31] Coffey, R. G., Hadden, E. M. and Hadden, J. W. (1981) J. Biol. Chem. 256, 4418. [32] Bailey, 3. M., Coffey, R., Merritt, W. D. and Hadden, J. W. (1985) in: Advances in Immunopharmacology (L. Chedid, J. W. Hadden, E Spreafico, P. Duker and D. Willoughby, Eds.) Vol. 3, pp. 177-188, Pergamon Press Ltd, Oxford. [33] Freeman, B. A. and Crapo, J. D. 0982) Lab. Invest. 47, 412. [34] German, J. B. and Kinsella, J. E. (1986) Biochim. Biophys. Acta 879, 378. [35] Hemler, M. E., Cook, H. W. and Lands, W. E. M. (1979) Arch. Biochem. Biophys. 193, 340. [36] Bray, M. A. (1987) ISI Atlas of Science: Pharmacology 1, 101. [37] Meister, A. and Anderson, M. E. (1983) Rev. Biochem. 52, 711.
107
Elsevier IML 01054
In vitro O2-induced depression of T and B lymphocyte activation is reversed by diethyldithiocarbamate (DDC) treatment P h i l i p p e L a c o m b e , Isabelle Carre, Michel Fay a n d J e a n - J a c q u e s P o c i d a l o Inserm U.13, H6pital Claude Bernard, 75944 Paris, France
(Received25 February 1988; accepted 9 March 1988)
1. Summary
2. Introduction
In this study, we tried to establish a relationship between the immunopotentiating effects and the antioxidant activity of the immunostimulating compound, diethyldithiocarbamate (DDC). We studied the effects of DDC treatment on enriched T and B murine spleen lymphocytes in an in vivo - ex vivo model of O2-induced immune depression. Female C57B1/6 mice were injected subcutaneously with a single dose of DDC (125 mg. kg-1). Eight days after DDC injection, we evaluated, in vitro, the concanavalin A response o f the T cell fraction and the LPS response of the B cell fraction, under standard (air - 5% C O 2 ) and hyperoxic (600/0 0 2 -- 5% CO2) culture conditions. The results show that after a lag period, DDC is able [1] to enhance the mitogenic response of T and B murine lymphocytes under standard culture conditions [2] to restore the ConA response and to partially restore the LPS response under hyperoxic conditions. The results of this study suggest that the immunostimulatory effects of DDC could be related to the antioxidant activity of this compound on the lymphoid cellular metabolism. This activity apparently affects both T and B lymphocytes.
It was shown that, in vitro, the oxygen-derived metabolites which were produced by enzymatic systems suppressed the immune responses [1, 2]. Recently, we have demonstrated that in vivo hyperoxic exposure induced a depression of the murine immune responses [3 - 5]. Furthermore, we have developed a model of in vitro hyperoxic exposure which showed that oxygen and/or its metabolites directly depressed the mitogenic response of rat lymphoid cells [6, 7]. In these two latter models, the immune depression can be reversed by several antioxidant drugs such as 2-mercaptoethanol (2-ME) which is known to enhance the lymphocyte mitogenic response [8, 9]. Furthermore, one or more oxidation steps appear to be essential for the mitogenic response [10, 11]. Lymphocyte activation seems to depend on a dual effect of oxidant generation. On the other hand, it is well known that DDC is an immunostimulant [12, 13] and it has been suggested that it may shield against oxygen injury [14]. Recent pharmacological study in our laboratory demonstrated that a single injection of DDC (125 mg • kg -~ optimal dose) eight days before in vivo hyperoxic exposure, partially prevented the pulmonary and lymphoid oxygen toxicity [15]. But, in vivo, oxygen toxicity was firstly exerted on pulmonary cells [16], and the observed DDC protection of the lymphoid ceils against oxygen toxicity could be the result of the pulmonary DDC protection. At this stage, it was difficult to establish that DDCtreatment was able to induce a lymphoid protection against oxidative injury. In this work, we studied the antioxidant properties
Key words: Lymphocyteactivation;Diethyldithiocarbamate;Im-
munostimulation; Antioxidant activity Correspondence to: P. Lacombe,INSERMU.13,H6pital Claude Bernard, 10, Avenuede la Porte d'Aubervilliers,75944Paris, Cedex 19, France.
0165-2478 / 88 / $ 3.50 © 1988 ElsevierSciencePublishers B.V.(BiomedicalDivision)
99
of DDC on the lymphocyte mitogenic response. We tried to point out a relationship between the immunostimulating effects of this compound and the oxidant generation involved in the lymphocyte activation. We studied the mitogenic response of murine splenocytes in a model of in vitro Oz-induced immune depression. Several days after the injection of DDC or saline (control), we evaluated the ConA response of the T cell fraction and the LPS response of the B cell fraction, under standard (95% air - 5% CO2) or hyperoxic (60% Oz - 35% N 2 - 5 % C O 2 for 24 h) culture conditions. In parallel, the mitogenic responses of control cells were studied in presence of 2-ME, and served as positive controls. The results suggest (a) that DDC exerts an antioxidant activity on both T and B lymphocyte metabolism and Co) that the immunostimulating effects of DDC could be related, in part, to its antioxidant activity.
ded to one volume of 1.5 M NaCl to provide an isoosmotic stock solution of density 1.120 g/ml (100% Percoll). We prepared two solutions of density 1.096 g/ml (80% Percoll) and 1.079 g/ml (65% Percoll), by diluting 8 parts 100% Percoll to 2 parts 0.15 M NaC1 and 6.5 parts 100°70 Percoll to 3.5 parts 0.15 M NaC1, respectively. The cells (6 to 8 × 10 7 cells) were suspended in 2 m180% Percoll, in a 10-ml conical tube, and 4 m165% Percoll were layered over this initial layer. The tubes were centrifuged at 400 × g (1500 rpm) for 15 min. The first 4 ml at the top of the gradient were collected. The cells from this fraction were washed twice in Hank's balanced salt solution (HBSS). The recovery of spleen lymphocytes and monocytes was consistently 9 5 - 9 8 % and the viability determined by trypan blue dye exclusion was found to be greater than 97%. Cells in the treated group were then pooled randomly, as were cells in the control group, in order to obtain a sufficient number of cells for the cultures.
3. Materials and Methods
3.1. Reagents Anti-Thy-l.2 monoclonal antibody was purchased from Cedarlane Laboratories (Canada). Goat antimouse Igs antibodies (GAM) labelled with fluorescein (FITC) were purchased from Nordic Laboratories (Netherlands). Concanavalin A (ConA) and lipopolysaccharide from E. coli 055 : B5 (LPS) were purchased from Sigma Chemical Company (USA). 3.2. Animals Eight- to ten-week old female C57B1/6 mice (IFFA-CREDO, Lyon, France) were assigned randomly to experimental and control groups. 3.3. DDC treatment DDC (Institut M6rieux, France) was prepared as previously described [15]. Mice were given a single dose (125 mg. kg 1 body wt) of DDC by subcutaneous injection, eight days before ex vivo experimentation. Control mice were given an equivalent injection of sterile saline.
3.4. Cell preparations Cells were obtained from the spleen as previously described [17], and isolated on Percoll density gradient to remove red blood cells and dead cells. Nine volumes of Percoll (Pharmacia France S.A.) were ad100
3.5. Spleen cell fractionation Splenocytes were fractionated by filtration through nylon wool columns. The barrels of 10-ml plastic syringes were packed to the 6-ml mark with 0.6 g of nylon wool (type 200, Fenwall Labs., USA) and sterilized before use. The columns were washed with 20 ml of RPMI 1640 (Gibco) - 5 % foetal calf serum (FCS, Gibco) (RPMI/FCS), incubated 1 h at 37 °C, for equilibration. After recovery from the Percoll density gradient, 2 × 108 spleen cells in 2 ml R P M I / F C S were loaded onto the nylon wool column, and incubated 45 rain at 37 °C. Effluent cells (T cell fraction) were collected by adding 25 ml of warm R P M I / F C S to the columns. The adherent cells (B cell fraction) were obtained by incubating the nylon wool in 25 ml of RPMI 1 6 4 0 - 4 % F C S - 0 . 2 % EDTA, 30 min, at 37 °C. The nylon wool was removed from the medium, pressed with a sterile syringe piston and the exudate was added to the medium. The cells were collected by centrifugation and washed three times with RPMI 1640. The lymphocyte sub-populations in the two fractions were identified by immunofluorescence. 3.6 Identification of lymphocyte sub-populations Anti-Thy-l.2 monoclonal antibody (anti-Thy-l.2 mAb) labelled all murine T lymphocytes. Immunofluorescent staining was performed using
1 - 2 x 1 0 6 cells in 50/A of HBSS (without phenol r e d ) - 4 % F C S - 2 % Hepes b u f f e r - 0 . 1 % sodium azide (fluorescence buffer, EB.) incubated with 25/zl of anti-Thy-l.2 mAb (1:20 dilution) for 30 min on ice. These cells were washed three times, incubated in 50 #1 EB. with 25 ixl F I T C - GAM conjugated for 30 min on ice, and washed again. B cells, surface Igbearing cells (SIg+), were identified by a direct staining with 25 ~1 F I T C - GAM conjugate. The percentage of positive cells was calculated as follows: % of positive cells = number of fluorescent cells x 100 total cell number The percentage o f T cells (Thy-l.2 +) was determined as follows: %Thy-l.2 ÷ = 070(GAM +Thy-l.2) ÷ - 070(GAM) +. A total of 100- 200 cells were counted for each determination. 3.7. 02 exposure Cultured cells were exposed to oxygen (60% 0 2 - 3 5 % N 2 - 5 % CO2) for 24 h, as previously described [7] after which incubation continued at 37°C in 5% CO2 enriched humidified air atmospher incubator (Heraus, France), for a total incubation time o f 48, 72 or 96 h (hyperoxic conditions). In parallel, cultures were grown in a i r - 5 % CO2 for the same incubation time (standard conditions). 3.8. Cultures Cells from both T cell and B cell fractions were cultured in triplicate as previously described [17] for 48, 72 and 96 h under hyperoxic or standard conditions. T cells were cultured with or without C o n A (2/~g/ml) and B cells were cultured with or without LPS (50 #g/ml). The cells from control animals cultured in presence of mitogen and 2-ME (5 x 10 -5 M) were used as positive control. 2-ME was added to the medium at the beginning of the culture. The mitogenic response was evaluated by [3H]thymidine incorporation ([3H]TdR uptake). Cells were pulsed with l t~Ci of [3H]TdR (1 Ci/mmole, C E A Saclay France) during the last 20 h of culture, and harvested using a multiple cell culture processor (Minimash, Dynatech). [3H]TdR
uptake was determined by counting in a/3-liquid scintillation spectrophotometer (LKB). Results were expressed as A counts (cpm of stimulated c e l l s - c p m of unstimulated cells) per minute per culture (A cpm/culture). 3.9. Statistical analysis The statistical analyses were made by using Student's t-test and analysis of variance. 4. Results
4.1. Identification of splenic lymphocyte sub-popu-
lations after fractionation on nylon wool column We observed no difference between control and DDC-treated animals, in the T cell and B cell fraction (Table 1). The percentages of T cells (Thy-l.2 ÷) in the T cell fraction and B cells (SIg +) in the B cell fraction were the same in both control and DDCtreated groups - 10070of B cells and 13-1407o of T cells contaminated the T cell and B cell fractions, respectively. 4.2. Effect of DDC treatment on the ConA response
of the T cell fraction Under standard conditions (9507o air-5070 CO2),
Table 1 Identification of splenic lymphocytesub-populations after fractionation on nylonwoolcolumn, from control and DDC-treated animals. T cell fraction
B cell fraction
SIg+ 070
Thy-l.2 + 070
SIg+ 07o
Thy-l.2 + %
Control
10.9 _+1.5
68.1 _+2.7
67.9 _+3.0
13.3 _+1.5
DDC
9.1 ___1.0
69.0 _+2.3
70.4 + 2.0
14.9 _+1.8
T and B cell fractions were obtained as described in Sec. 3. Lymphocyte sub-populations were identified as described in Sec. 3. B cells: Slg+; T cells: Thy-l.2 + . Results are expressed as the arithmetic mean of the percentageof fluorescentpositivecells + S.E.M. from twelve animals in both control and DDC-treated groups. No significant difference was observed between control and DDC-treated groups. 101
we observed after DDC injection a significant increase of the [3H]TdR uptake after 48 ( P < 0.05) and 72 h ( P < 0.05) of culture, but the peak [3H]TdR uptake was obtained after 48 h of culture (Fig. 1). Furthermore, no significant [3H]TdR incorporation was seen after 24 h of culture (data not shown). Concurrently, in presence of 2-ME (positive control) the ConA response of the control was significantly enhanced after 48 (P < 0.01) and 72 h (P < 0.001) of culture; the peak of [3H]TdR uptake was obtained after 72 h of culture. On the other hand, under hyperoxic culture conditions (60°70 0 2 - 35% N 2-5°70 CO2), DDC treatment also enhanced significantly the [3H]TdR uptake after 48 ( P < 0.01), 72 (P<0.01) and 96 h (P<0.001) of culture, as compared to the control culture, under the same conditions (Fig. 2). This enhancing effect of DDC treatment was significantly greater ( P < 0.01) than that of 2-ME (positive control), after 72 h of culture (Fig. 2). To determine the absolute effect of 2-ME and DDC treatment on the ConA response, we compared
the peaks of [3H]TdR uptake to the control peak (control cells in air). The results were expressed as the percentage of [3H]TdR uptake in control ceils in air (100°70) (Table 2). We observed that DDC treatment or 2-ME were able to enhance two-fold the ConA response, under standard conditions. On the other hand, under hyperoxic culture conditions, while control response was drastically inhibited, 2-ME restored and DDC treatment significantly increased ( P < 0.05) the ConA response, but the responses obtained with 2-ME or DDC were not significantly different. 4.3. Effect of DDC treatment on the L P S response of the B cell fraction As shown in Fig. 3, we observed that DDC treatment significantly increased the LPS response of the B cell fraction after 48 ( P < 0.001) and 72 h ( P < 0.05) of culture, under standard conditions. This enhancing effect of DDC treatment was the same order of magnitude as that of 2-ME. Furthermore, in all cases
150-•
eel
A t~
Ic) X
100--
E
O.
t.)
UJ
X4
n n-
50-
I--
co
O-
I 0
I }
I 48
I 72
I 96
CULTURE TIME ( HOURS )
Fig. 1. Effect of DDC treatment on the ConA response of the T cell fraction, under standard conditions. The cells were cultured under standard conditions (95 % a i r - 5 °7o CO2) and the ConA responses were evaluated, as described in Section 3. • . . . . . a , ConA response of the T cell fraction from DDC-treated animals. • - . - . - . • , ConA response in the presence of 2-ME of the T cell fraction from control animals (positive control). • ..... o, ConA response of the T cell fraction from control animals (control). Results are expressed as the arithmetic mean_+ S.E.M. (Acpm_+ S.E.M.) of 4 experiments from 3 pools of cells (3 animals/pool) in both control and DDC-treated groups. All results were compared to the mitogenic response of the control group. *P<0.05, **P<0.01, ***P<0.001.
102
150--
oe ¢9 I o X
100--
E Q. tO
<1 v
UJ I-fl. ar 10 I-I "r ¢0
50--
,~mlmmD~ amml~ m mmLm gm ~ m ~
O--
I"-'1 0
I
nmbm mmmm m m m m ambumm mm~)
I
I
I
48
72
96
C U L T U R E TIME ( HOURS
)
Fig. 2. Effect of DDC treatment on the ConA response of the T cell fraction, under hyperoxicconditions. The cells were cultured under hyperoxic conditions (60% 02-35°70 N2-5% CO2) and the ConA responses were evaluated, as described in Section 3. • ..... •, • - . - . - m, • ..... e, Symbols are the same as in Fig. 1. Results are expressed and the comparisons were made, as described in Fig. I.
we observed no significant incorporation o f [3H]TdR after 24 h of culture (data not shown). Under hyperoxic conditions (Fig. 4), DDC treatment significantly enhanced the LPS response of the B cell fraction after 48 (P<0.01), 72 (P<0.01) and 96 h (P < 0.05) of culture, as compared to the control response under the same conditions of culture. This effect of DDC treatment was the same order of magnitude as that of 2-ME. We determined the absolute effect o f 2-ME or DDC treatment on the LPS response, by the same calculation as that in Table 2 for the C o n A response. We observed that 2-ME or DDC treatment enhanced two-fold the LPS response in air, but the corresponding effect under hyperoxic culture conditions was only a partial restoration of the LPS response (Table 3). 4.4. Cell viability As shown in Table 4, we observed no significant difference between the cell viabilities of DDCtreated group and control group during the C o n A
response o f the T cell fraction. Same results were observed for LPS response of the B cell fraction (data not shown).
5.
Discussion
In this study, using an in vitro model of O2-induced immune depression, we have shown that the DDC treatment completely restored the C o n A response and partially restored the LPS response of enriched T and B lymphocytes, respectively (Figs. 2 and 4). These results suggest that DDC exerts an antioxidant activity on the metabolism of both T and B murine splenocytes. This activity affected less B cells than T cells. This could be explained by the greater susceptibility of B lymphocytes than of T lymphocytes to O2 toxicity [18]. Concurrently, under standard culture conditions, DDC treatment enhances two-fold the C o n A and LPS responses of enriched T and B lymphocytes, respectively (Figs. 1 and 3). Although the C o n A re103
Table 2 Determination of the absolute effect of DDC treatment or 2-ME on the ConA response of the T cell fraction.
Table 3 Determination of the absolute effect of DDC treatment or 2-ME on the LPS response of the B cell fraction.
070 of the peak of the control response
Control a 2-ME b DDC c
Air
0 2
100_+ 13 188_+ 9*** 179 _+20**
24_+ 3** 122_+ 8 160 _+ 18'
°7o of the peak of the control response
Control 2-ME DDC
Air
0 2
100 _+20 196+ 14"* 172 + 13**
10 _+2"* * 33 _+ 1"* 39 _+3**
The cells were cultured as described in Table 2. The results are expressed as described in Table 2, except relating to LPS response instead of ConA response. Comparisons are made as described in Table 2. **, ***, are the same as Table 2.
The cells were cultured as described in Sec. 3 under standard (air) or hyperoxic (02) conditions. The results are expressed as follows: 070 of the peak of control response = cpm peak of [3H]TdR uptake of ConA response under both air or 0 2
sponse which was obtained with T cells from DDCtreated animals was shortened (Fig. 1), no significant [3H]TdR incorporation was observed after 24 h of culture under standard culture conditions (data not shown). Thus, the absolute effect of DDC treatment was a potentiation o f the ConA response as revealed by the comparison of the peaks of [3H]TdR uptake (Table 2). On the other hand, we studied the mitogenic responses of 70°70 enriched T and B cell subpopulations (Table 1). It was demonstrated that the ConA and LPS responses were specific to T [19] and B [20]
cpm peak of [3H]TdR uptake of ConA response of control cells in air The peak of [3H]TdR uptake obtained for the ConA response of control cells under standard culture conditions is considered as 100%. a ConA response of T cells from control animals (control cells). b ConA response of T cells from control animals, in presence of 2-ME (positive control). c ConA response of T cells from DDC-treated animals. Results are expressed as the arithmetic mean _+ S.E.M. of 4 experiments from 3 pools of cells (3 animals/pool) in both control and DDC-treated groups. All results were compared to 100070. * P < 0 . 0 5 , **P<0.01, ***P<0.001.
o o o
50-A I
0
o o o
x E
0
j
25--
........
iIll
IIIL
W
F¢r
pO--
I 0
I I
I 48
I 72
I 96
CULTURE TIME ( HOURS )
Fig. 3. Effect of DDC treatment on the LPS response of the B cell fraction, under standard conditions. The cells were cultured under standard conditions (95070 a i r - 5 % CO2) and the LPS responses were evaluated as described in Section 3. • . . . . . A, LPS response of the B cell fraction from DDC-treated animals, n - . - . - I, LPS response in presence of 2-ME of the B cell fraction from control animals (positive control). • ..... 0, LPS response of the B cell fraction from control animals (control). Results are expressed and the comparisons were made, as described in Fig. 1.
104
oe
q
A
10-I 0
I........ ...'"S',.:a:"....,
X
E
e~
o ,11 v
W
5--
I-Q. o n,-
I-I T O--
I
II
0
I
I
I
48
72
96
CULTURE ( HOURS
TIME )
Fig. 4. Effect of D D C treatment on the LPS response of the B cell fraction, under hyperoxic conditions. The cells were cultured under hyperoxic conditions (6007o 02-35°7o N 2 - 5 % CO2) and the LPS responses were evaluated as described in Section 3. • . . . . . • , • - . - . - i, • ..... • , Symbols are the same as in Fig. 3. Results are expressed and the comparisons were made, as described in Fig. 1.
Table 4 Cell viability during the C o n A response of the T cell fraction from control and DDC-treated groups. Culture time (h)
Control Air DDC Air Control O2 DDC 02
48
72
96
71 + 2
79_+4
60±5
75±2
76±3
56±6
63±3
64+3
61+6
61±2
65±3
59+4
The cell viability was determined by trypan blue dye exclusion after 48, 72 and 96 h of culture, under standard (air) or hyperoxic (02) conditions. The results are expressed as the percentage of viable cells: No. of viable cells ( Total no. of cells ) x 100) and as the arithmetic mean_+ S.E.M. of 4 experiments from 3 pools of cells (3 animals/pool) in both control and DDC-treated groups. No significant difference was observed between control and DDC-treated groups.
murine lymphocytes, respectively. LPS response was known to be T cell-independent [21, 22]. Furthermore, we observed no significant difference between the percentages of T and B cell subpopulations (Table 1) and the cell viabilities (Table 4) of DDCtreated and control groups. These results showed that the effects of DDC treatment were independent of the cell viability, and were exerted on T and B lymphocyte metabolism. These results are partly in accordance with those of other authors [23]. These above observations suggest that DDC exerts its immunostimulating effect in part by antioxidative activity on the lymphoid cellular metabolism. Indeed, DDC has the same type of activity as the antioxidant drug, 2-ME, and the observed results with this latter compound were the same order of magnitude as those obtained with DDC (Tables 2 and 3). Although the primary mechanism of action of DDC and 2-ME may be different, these two compounds can act finally on the same metabolic target. Indeed, DDC is used in vivo and its effect can be observed only after a lag period, suggesting that DDC is active through indirect pathways. Furthermore, no effect is observed when DDC is used in vitro [24, 251. It has been demonstrated that DDC stimulates the synthesis of the enzymes of the glutathione (GSH) cycle I05
[26]. On the other hand, 2-ME was used in vitro. We have demonstrated that 2-ME enhanced the mitogenic response of murine lymphocytes via its action on the GSH metabolic pathways [27], and this action can be related to antioxidant properties of the GSH [6, 17]. It has been shown that the depletion of intracellular GSH involved an inhibition of the lymphocyte activation [28]. Thus, it seems that the antioxidant properties of DDC could be secondary to its action on the lymphocyte glutathione metabolic pathways. At this stage, there is a discrepancy between (a), the antioxidant properties of DDC related to its immunopotentiating effects and (b), the fact that oxidants must be generated if the events leading to lymphocyte proliferation are to be triggered [11]. The hypothesis that the DDC exerts its immunostimulatory effects by an antioxidant process, can be reconciled when we consider the duality of the effects of oxidant generation. While the 02 metabolites are necessary to trigger the early events of the lymphocyte activation, they are deleterious during the late phase of this activation [29]. Indeed, the early events which are triggered by the oxidant generation lead to the stimulation of arachidonate release [30, 31]. These processes are accompanied by the formation of and are modulated by lipoxygenase-leukotriene metabolites which may be the activation pathway during the lymphocyte proliferation [32]. This stimulation of 5-1ipoxygenase pathways increased the intracellular peroxide level during the lymphocyte activation. The increased peroxide level could lead to (a), autocatalytic lipid peroxidation which impairs cellular integrity [33], (b), inhibition of 5-1ipoxygenase pathways [34] and (c), stimulation of cyclooxygenase pathways [35] which are known to down-regulate the lymphocyte activation [36]. It appears that a limitation of the peroxide level can promote the lymphocyte activation. Such a limitation is performed by GSH via the GSH peroxidase [37]. This could explain the relationship between the immunostimulatory effects of DDC and its antioxidant activity which we point out in this study. This work strongly suggests that DDC exerts its immunostimulatory effects in part by antioxidative activity which could be related to an action on the lymphocyte glutathione metabolism. This activity of DDC is exerted on T and B lymphocytes. Our model of O2-induced immune depression in which 106
the oxidative processes involved in lymphocyte activation were amplified, appears to be a useful approach to study of these phenomena. Further studies are presently being performed to determine the relationship between the immunostimulating/antioxidant effects of DDC and lymphocyte glutathione metabolism, and to better understand the action of DDC on both T and B lymphocytes.
Acknowledgements The authors are grateful to Mrs. Annie-Christine Poidevin for typing the manuscript.
References [1] Hoffeld, J. T., Metzger, Z. and Oppenheim, J. J. (1981) in: Lymphokines (E. Pick, Ed.) pp. 63-86, Academic Press, London. [2] Kraut, E. H. and Sagone, L. (1981) J. Lab. Clin. Med. 98, 697. [3] Levacher-Place, M., Gougerot-Pocidalo, M. A., Rouveix, B., Kraus, L. and Pocidalo, J. J. (1983) Clin. Exp. Immunol. 54, 580. [4] Gougerot-Pocidalo, M. A., Fay, M. and Pocidalo, J. J. (1984) Clin. Exp. Immunol. 58, 428. [5] Gougerot-Pocidalo, M. A., Fay, M., Roche, Y., Lacombe, P. and Marquetty, C. (1985) J. Immunol. 135, 2045. [6] Kraus, L., Gougerot-Pocidalo, M. A., Lacombe, P. and Pocidalo, J. J. (1985) Int. J. Immunopharmacol. 7, 753. [7] Kraus, L., Lacombe, P., Fay, M. and Pocidalo, J. J. (1985) Immunol. Lett. ll, 51. [8] Broome, J. D. and Jeng, M. W. (1973) J. Exp. Med. 138, 574. [9] No,lie, R. J. and Lawrence, D. A. (1980) Cell. Immunol. 50, 416. [10] Coffey, R. G., Hadden, E. M. and Hadden, J. W. (1981) J. Biol. Chem. 256, 4418. [11] Mookerje, B. K., Wakerle, H. W., Sharon, N. and Fisher, H. (1984) J. Leukocyte Biol. 35, 427. [12] Renoux, G. and Renoux, M. (1979) J. Immunopharmacol. 1, 247. [13] Renoux, G. (1980) Drugs 19, 86. [14] Renoux, G. (1982) J. Pharmacol. (Paris) 13 (Suppl.), 95. [15] Mansour, H., Levacher-Place, M., Gougerot-Pocidalo, M. A., Rouveix, B. and Pocidalo, J. J. (1986) J. Pharmacol. Exp. Ther. 236, 476. [16] Kimball, R .E., Reddy, K., Pierce, T. H., Schwartz, L. W., Mustafa, M. G. and Cross, C. E. (1976) Am. J. Physiol. 230, 1425. [17] Lacombe, P., Kraus, L., Fay, M. and Pocidalo, J. J. (1986) Biochimie 68, 555. [18] Farber, C. M., Liebes, L. F., Kanganis, D. N. and Silber, R. (1984) J. Immunol. 132, 2543.
[19] Williams, M. and Benacerraf, B. (1972) J. Exp. Med. 135, 1279. [20] Peavy, D. L., Shands, J. W., Adler, W. H. and Smith, R. T. (1973) J. Immunol. Ill, 352. [21] Watson, J., Epstein, R., Nakoing, I. and Ralph, P. (1973) J. Immunol. ll0, 43. [22] Horan, M. A. and Fox, R. A. (1984) Mech. Age Dev. 26, 165. [23] Neveu, P. J., Perdoux, D. and Lafleur, L. (1982) Int. J. Immunopharmacol. 4, 9. [24] Hadden, J. W. (1977) in: Immunopharmacology (J. W. Hadden, R. G. Coffey and E Spreafico, Eds.) pp. 1- 28, Plenum Publishing Corp., New York. [25] Renoux, G. and Renoux, M. (1977) in: Control of Neoplasia by the Modulation of the Immune System (M. A. Chirigos, Ed.) pp. 67-80, Raven Press, New York. [261 Deneke, S. M. and Fanburg, B. L. (1980) Biochem. Pharmacol. 29, 1367. [27] Lacombe, P., Kraus, L., Fay, M. and Pocidalo, J. J. (1987) Biochimie 69, 37. [28] Fidelus, R. K., Ginouves, P., Lawrence, D. and Tsan, M. E (1987) Exp. Cell Res. 170, 269.
[29] Lacombe, P., Kraus, L., Fay, M. and Pocidalo, J. J. (1985) Febs Lett. 191,227. [30] Coffey, R. G. and Hadden, J. W. (1981) in: Advances in Immunopharmacology (J. W. Hadden, L. Chedid, P. Mullen and E Spreafico, Eds.) Vol. 1, pp. 365 - 373. Pergamon Press Ltd., Oxford. [31] Coffey, R. G., Hadden, E. M. and Hadden, J. W. (1981) J. Biol. Chem. 256, 4418. [32] Bailey, 3. M., Coffey, R., Merritt, W. D. and Hadden, J. W. (1985) in: Advances in Immunopharmacology (L. Chedid, J. W. Hadden, E Spreafico, P. Duker and D. Willoughby, Eds.) Vol. 3, pp. 177-188, Pergamon Press Ltd, Oxford. [33] Freeman, B. A. and Crapo, J. D. 0982) Lab. Invest. 47, 412. [34] German, J. B. and Kinsella, J. E. (1986) Biochim. Biophys. Acta 879, 378. [35] Hemler, M. E., Cook, H. W. and Lands, W. E. M. (1979) Arch. Biochem. Biophys. 193, 340. [36] Bray, M. A. (1987) ISI Atlas of Science: Pharmacology 1, 101. [37] Meister, A. and Anderson, M. E. (1983) Rev. Biochem. 52, 711.
107