Drug and Alcohol Dependence, 30 (1992) 181- 185
Elsevier Scientific Publishers
181
Ireland Ltd.
The possible mechanism
of action of ethanol on rat thymus
Mirela BudeC”, Olivera &ri@“, Vesna Koko”, Ruiica ASaninb aZn.stitute for Medical Research and bVeterinary Faculty, Department
of Microbiology, Beograd (Yugoslavia)
(Accepted January 23rd, 1992)
Alcohol is a known suppressant of the immune system and alcoholics frequently have impaired humoral and cell-mediated immunity. The purpose of this study was to investigate the effect of a single dose of ethanol on the thymus and the possible mechanism of its action. Adult female Wistar rats were divided into three groups which were treated with: (a) ethanol (4 g/kg i.p.), (b) naltrexone (5 mg/kg i.p.) and 45 min later with ethanol, (c) naltrexone alone. Untreated rats served as controls. The animals were killed 20 h after administration of alcohol. Thymuses were removed and fixed in Bouin’s solution. Paraffin sections were stained with hematoxylin-eosin and analysed using stereological measurements. Our results showed that a single dose of ethanol significantly decreased the volume of the thymus especially affecting the cortex. This effect was blocked by pretreatment with naltrexone. Therefore, it seems that the effect of ethanol on the thymus is mediated by an opioid-dependent mechanism. Key words:
Ethanol; Naltrexone;
Thymus; Female rats
Introduction Alcohol abuse has been associated with a increased susceptibility to infections and certain tumors [1,2]. The effects of ethanol on the immune system have been investigated in humans and animal models. In most studies chronic alcohol treatment has been used. Abundant evidence suggests that alcohol inhibits immune function leading to a loss of lymphoid cells from the thymus, spleen and peripheral blood [3,4] and alterations in cellular [5,6] and humoral immunity [7,8]. In vitro, it has been shown that ethanol is more immunosuppressive for Tlymphocytes than for B-lymphocytes [9]. Nonspecific aspects of immune mechanism including phagocyte cell function in the reticuloendothelial system and granulocyte mobility and function also are affected by alcohol 121. However, the mechanism of immunosuppressive action of alcohol has not been elucidated. Since nutrition can be an important variable in Correspondence
to: Mirela BudeC, Institute for Medical Research, P.O. Box 721, 11001 Beograd, Yugoslavia.
0376-8716/92/$05.00 0 1992 Elsevier Scientific Publishers Printed and Published in Ireland
immune modulation during chronic alcohol consumption, the present study was designed to investigate the ‘direct’ effect of a single-dose of ethanol on the thymus in rats and to determine whether this action is mediated through opiate receptors. Materials and Methods Twenty female Wistar rats (2 months of age and with body weights of 190 - 240 g) were housed in groups of five in a system of 12 h light/ 12 h dark (lights on 06:OO h); ambient temperature was 21- 23°C. The experimental animals were divided into three groups which were treated with: (a) ethanol (4 g/kg body wt.); (b) naltrexone (5 mg/kg body wt.) and 45 min later with ethanol (c) naltrexone alone. Untreated rats served as controls. On the injection day the rats were weighed and ethanol and naltrexone hydrochloride (ICN Biochemicals) solutions were prepared using sterile saline. The drugs were injected intraperitoneally between 12:00 h and 13:00 h. Twenty hours after ethanol administration the Ireland Ltd.
182
animals were killed by decapitation. In order to evaluate the possibility of contamination by intraperitoneal treatment, a bacteriological analysis was performed. Immediately after that the thymuses were removed, weighed and fixed in Bouin’s solution for paraffin sectioning. Sections 5 pm thick were stained with hematoxylineosin. Stereological analysis was performed on every 20th section of the thymus under a Galen III (Cambridge Instruments) microscope at an objective magnification of x 10. Volume density of the cortex and medulla was determined using Weibel’s multipurpose test system M-168 [lo]. The absolute volumes of the cortex and medulla were calculated from the formula V, = V++ x V, (Vf, absolute volume of fraction; Vvf, volume fraction; V,, absolute volume of organ). Statistical differences were determined by ANOVA and Student’s t-test.
various treatments is shown in Fig. la-d. Alcohol significantly altered the ratio between the cortex and medulla in the thymus (P < 0.001) (Fig. 2). This effect was completely blocked by pretreatment with naltrexone. Naltrexone also was found to have no effect on the cortex/medulla ratio. To determine which tissue is mainly affected by ethanol, absolute volumes of the cortex and medulla were calculated. (Fig. 3) Administration of ethanol resulted in a significant reduction of the cortex (P < 0.001) and a less significant reduction of the medulla (P < 0.05). The effect of alcohol on the cortex is prevented by naltrexone (P < 0.05). There was no significant difference in volume of the medulla between the alcohol and naltrexone plus alcohol treated groups (P > 0.05). Discussion
Results Table I shows the effect of a single dose of ethanol on the thymic weight. The ethanoltreated group had a significantly reduced thymic weight compared with controls (P c 0.001). The naltrexone pretreated group showed less decreased thymic weight, but this difference was not significant (P > 0.05). Using thymus/body weight ratio similar differences were observed. The relative thymic weight was also significantly reduced in etahnol-treated rats (P < 0.001). The histological picture of the thymuses under
Table
1.
Thymic weight in different groups.
Thymus (g) 5 S.E.M. Thymus (g)body +z S.E.M. ‘(P
Alcohol is known to suppress the immune response but the underlying mechanism accounting for the immunosuppression is still not clearly elucidated. Of particular interest is the association of ethanol ingestion and impairment of the cell-mediated immune response [3,5,11,6]. It has been shown that ethanol treatment resulted in a marked loss of spleen and thymus cells in rats [3] and mice [4]. The thymuses of fetal mice exposed prenatally to ethanol also were reduced in cell number [12] and thymus immaturity is one of the accompanying features of fetal alcohol syndrome [13]. Our results showing
wt. @g)
Ethanol (N = 5)
Naltrexone + ethanol (N = 5)
Naltrexone (N = 5)
Controls (N = 5)
0.346’ 0.036 1.79* 0.178
0.471 0.036 2.25 0.174
0.489 0.040 2.12 0.138
0.596 0.031 2.65 0.076
< 0.001) compared with controls.
183
184 RELATIVE PROPORTIONS “/, IOOj~
-~
_________?
methanol (Lg/kg) analtrexone (5 mg/kg)
IZZ naltrexcne +ethanol m controls
Fig. 2. The relative proportion of the cortex (C) and the medulla (M) in different groups. **Significantly different (P < 0.001) compared with controls. *#Significantly different (P < 0.001) compared with naltrexone + ethanol-treated group.
a profound effect of alcohol on rat thymus are consistent with the previous findings. After intraperitoneal administration of ethanol no bacteriological invasion in abdominal cavity was ABSOLUTE VOLUMES
CM
mm3
CM
CM
methanol (Lg/kg) ardtrexone (5mg/kg) Fig. 3. medulla
The absolute
volumes
CM
~Z?Znaltrexone+ethanol m controls of the cortex
(C) and
the
(M) in different groups. **Significantly different (P < 0.001) compared with controls. *Significantly different (P < 0.05) compared with controls. #Significantly different (P < 0.05) compared with naltrexone + ethanol-treated group.
found. It is clear from the data presented in this report that a single hypnotic dose (4 g/kg) of ethanol significantly reduced the volume of the thymus especially affecting the cortex. Although it is possible that ethanol directly affects lymphocyte function in vivo, there are studies suggesting that this is not the major mechanism of lymphocyte impairment. This is supported by findings that the most profound impairment in lymphocyte proliferation occurs during the period after cessation of ethanol administration when withdrawal symptoms are evident [3]. Jerrells et al. [ll] have established that corticosteroids, produced most abundantly after withdrawal of ethanol, are at least partially responsible for the defect in lymphocyte proliferation. A number of reports have shown that acute and chronic ethanol treatment in vivo and in vitro alters the activity of the endogenous opioid system [14- 161, Ethanol may interact with the endogenous opioid system by: (a) production of certain ethanol metabolites, for example the isoquinolines which bind to opiate receptors, (b) altering the binding properties of opiate receptors and (c) altering the release, synthesis and post-translational processing of endogenous opioid peptides [17]. It has been suggested that ethanol may exert some of its effects like the reinforcing [18] and epileptogenic [19] effects via interaction with the endogenous opioid system. Since the cells of the immune system possess opioid receptors that are biochemically, physicochemically and functionally related to receptors of neuroendocrine origin [20], we assumed that the effects of ethanol on the thymus may be mediated by an opioid dependent mechanism. In order to test this hypothesis we used the opioid antagonist naltrexone. Naltrexone pretreatment prevented ethanol-induced changes in thymus morphology, whereas naltrexone alone was found to have no effect. These results indicate that ethanol exerts its effect on the thymus by an opioid dependent mechanism. Having in mind that naltrexone is a p-preferring antagonist and that functional ptype opioid receptors are present on cells of the immune system [21], one may assume that the
185
effect of ethanol is mediated by p-opiate receptors. Considering that the neuroendocrine system can influence immune function [22,23] it seems logical that brain opiate receptors may be involved in this phenomenon.
11
12
Acknowledgement This work was supported by the Republic of Serbia Research Fund. The technical help of Mrs. Sneiana Markovic is highly appreciated. References C.S. Lieber et al. (1979) Alcohol-related diseases and carcinogenesis. Cancer Res. 39, 2863. R.R. Mac Gregor (1986) Alcohol and immune defense. J. Am. Med. Assoc. 256, 1474. T.R. Jerrells et al. (1986) Effects of ethanol administration on parameters of immunocompetency in rats. J. Leukocyte Biol. 39, 499. 4 T.R. Jerrelis, W. Smith, M. J. Eckardt (1990) Murine model of ethanol-induced immunosuppression. Alcoholism: Clin. Exp. Res. 14, 546. 5 G.A. Roselle, L. Mendenhall (1984) Ethanol-induced alterations in lymphocyte function in the guinea pig. Alcoholism: Clin. EXD. Res. 8. 62. 6 C.J. Grossman, C.L.‘Mendenhall, G.A. Roselle (1988) Alcohol and immune regulation. In vivo effects of ethanol on concanavalin A sensitive thymic lymphocyte function. Int. J. Immunopharmacol. 10, 187. 7 W.J. Smith, Jr. et al. (1980) Altered immunity in male patients with alcoholic liver disease: evidence for defective immune regulation. Alcoholism: Clin. Exp. Res. 4, 199. 8 M. Aldo-Benson (1989) Mechanisms of alcohol-induced suppression of B-cell response. Alcoholism: Clin. Exp. Res., 13, 469. 9 C. Levallois et al. (1989) Effects of ethanol in vitro on some parameters of the immune response. Drug Alcohol Depend. 24, 239. 10 E.R. Weibel (1979) Stereological Methods, Vol. I,
13
14
15
16
17
18
19
20
21
22 23
Practical methods for biological morphometry. Academic Press, London-New York-Toronto-SidneySan Francisco. T.R. Jerrells et al. (1989) Mechanisms of suppression of cellular immunity induced by ethanol. Alcoholism: Clin. Exp. Res. 13, 490. S.J. Ewald, W.W. Frost (1987) Effect of prenatal exposure to ethanol on development of the thymus. Thymus 9, 211. S.J. Ewald, S.M. Walden (1988) Flow cytometric and histological analysis of mouse thymus in fetal alcohol syndrome. J. Leukocyte Biol. 44, 434. C. Gianoulakis, A. Barcomb (1987) Effect of acute ethanol in vivo and in vitro on the P-endorphin system in the rat. Life Sci. 40, 19. C. Gianoulakis (1990) Characterization of the effects of acute ethanol administration on the release of flendorphin peptides by the rat hypothalamus. Eur. J. Pharmacol. 180, 21. B.R. Seizinger (1984) Enhanced activity of the fland endorphinergic system in the anterior neurointermediate lobe after chronic treatment with ethanol liquid diet. J. Pharmacol. Exp. Ther. 230, 455. C. Gianoulakis (1989) The effect of ethanol on the biosynthesis and regulation of opioid peptides. Experientia 45, 4.28. H.L. Altshuler, P.E. Philips, D.E. Feinhaudler (1980) Alteration of ethanol self-administration by naltrexone. Life Sci. 26, 679. E. Triana, J.F. Richard, P.E. Strokes (1980) The relationship between endorphins and alcohol induced subcortical activity. Am. J. Psychiat. 127, 491. D.J.J. Carr (1988) Opioid receptors on cells of the immune systems. Prog. Neurol. Endocrinol. Immunol. 1, 8. R.T. Radulescu et al. (1991) Biochemical and functional characterization of p-opioid receptor binding site on cells of the immune system. Prog. Neurol. Endocrinol. Immunol. 4, 166. J.E. Blalock (1984) The immune system as a sensory organ. J. Immunol. 132, 1067. P.J. Neveu, M. Le Moal. (1990) Physiological basis for neuroimmunomodulation. Fund. Clin. Pharmacol. 4, 281.
Elsevier Scientific Publishers
181
Ireland Ltd.
The possible mechanism
of action of ethanol on rat thymus
Mirela BudeC”, Olivera &ri@“, Vesna Koko”, Ruiica ASaninb aZn.stitute for Medical Research and bVeterinary Faculty, Department
of Microbiology, Beograd (Yugoslavia)
(Accepted January 23rd, 1992)
Alcohol is a known suppressant of the immune system and alcoholics frequently have impaired humoral and cell-mediated immunity. The purpose of this study was to investigate the effect of a single dose of ethanol on the thymus and the possible mechanism of its action. Adult female Wistar rats were divided into three groups which were treated with: (a) ethanol (4 g/kg i.p.), (b) naltrexone (5 mg/kg i.p.) and 45 min later with ethanol, (c) naltrexone alone. Untreated rats served as controls. The animals were killed 20 h after administration of alcohol. Thymuses were removed and fixed in Bouin’s solution. Paraffin sections were stained with hematoxylin-eosin and analysed using stereological measurements. Our results showed that a single dose of ethanol significantly decreased the volume of the thymus especially affecting the cortex. This effect was blocked by pretreatment with naltrexone. Therefore, it seems that the effect of ethanol on the thymus is mediated by an opioid-dependent mechanism. Key words:
Ethanol; Naltrexone;
Thymus; Female rats
Introduction Alcohol abuse has been associated with a increased susceptibility to infections and certain tumors [1,2]. The effects of ethanol on the immune system have been investigated in humans and animal models. In most studies chronic alcohol treatment has been used. Abundant evidence suggests that alcohol inhibits immune function leading to a loss of lymphoid cells from the thymus, spleen and peripheral blood [3,4] and alterations in cellular [5,6] and humoral immunity [7,8]. In vitro, it has been shown that ethanol is more immunosuppressive for Tlymphocytes than for B-lymphocytes [9]. Nonspecific aspects of immune mechanism including phagocyte cell function in the reticuloendothelial system and granulocyte mobility and function also are affected by alcohol 121. However, the mechanism of immunosuppressive action of alcohol has not been elucidated. Since nutrition can be an important variable in Correspondence
to: Mirela BudeC, Institute for Medical Research, P.O. Box 721, 11001 Beograd, Yugoslavia.
0376-8716/92/$05.00 0 1992 Elsevier Scientific Publishers Printed and Published in Ireland
immune modulation during chronic alcohol consumption, the present study was designed to investigate the ‘direct’ effect of a single-dose of ethanol on the thymus in rats and to determine whether this action is mediated through opiate receptors. Materials and Methods Twenty female Wistar rats (2 months of age and with body weights of 190 - 240 g) were housed in groups of five in a system of 12 h light/ 12 h dark (lights on 06:OO h); ambient temperature was 21- 23°C. The experimental animals were divided into three groups which were treated with: (a) ethanol (4 g/kg body wt.); (b) naltrexone (5 mg/kg body wt.) and 45 min later with ethanol (c) naltrexone alone. Untreated rats served as controls. On the injection day the rats were weighed and ethanol and naltrexone hydrochloride (ICN Biochemicals) solutions were prepared using sterile saline. The drugs were injected intraperitoneally between 12:00 h and 13:00 h. Twenty hours after ethanol administration the Ireland Ltd.
182
animals were killed by decapitation. In order to evaluate the possibility of contamination by intraperitoneal treatment, a bacteriological analysis was performed. Immediately after that the thymuses were removed, weighed and fixed in Bouin’s solution for paraffin sectioning. Sections 5 pm thick were stained with hematoxylineosin. Stereological analysis was performed on every 20th section of the thymus under a Galen III (Cambridge Instruments) microscope at an objective magnification of x 10. Volume density of the cortex and medulla was determined using Weibel’s multipurpose test system M-168 [lo]. The absolute volumes of the cortex and medulla were calculated from the formula V, = V++ x V, (Vf, absolute volume of fraction; Vvf, volume fraction; V,, absolute volume of organ). Statistical differences were determined by ANOVA and Student’s t-test.
various treatments is shown in Fig. la-d. Alcohol significantly altered the ratio between the cortex and medulla in the thymus (P < 0.001) (Fig. 2). This effect was completely blocked by pretreatment with naltrexone. Naltrexone also was found to have no effect on the cortex/medulla ratio. To determine which tissue is mainly affected by ethanol, absolute volumes of the cortex and medulla were calculated. (Fig. 3) Administration of ethanol resulted in a significant reduction of the cortex (P < 0.001) and a less significant reduction of the medulla (P < 0.05). The effect of alcohol on the cortex is prevented by naltrexone (P < 0.05). There was no significant difference in volume of the medulla between the alcohol and naltrexone plus alcohol treated groups (P > 0.05). Discussion
Results Table I shows the effect of a single dose of ethanol on the thymic weight. The ethanoltreated group had a significantly reduced thymic weight compared with controls (P c 0.001). The naltrexone pretreated group showed less decreased thymic weight, but this difference was not significant (P > 0.05). Using thymus/body weight ratio similar differences were observed. The relative thymic weight was also significantly reduced in etahnol-treated rats (P < 0.001). The histological picture of the thymuses under
Table
1.
Thymic weight in different groups.
Thymus (g) 5 S.E.M. Thymus (g)body +z S.E.M. ‘(P
Alcohol is known to suppress the immune response but the underlying mechanism accounting for the immunosuppression is still not clearly elucidated. Of particular interest is the association of ethanol ingestion and impairment of the cell-mediated immune response [3,5,11,6]. It has been shown that ethanol treatment resulted in a marked loss of spleen and thymus cells in rats [3] and mice [4]. The thymuses of fetal mice exposed prenatally to ethanol also were reduced in cell number [12] and thymus immaturity is one of the accompanying features of fetal alcohol syndrome [13]. Our results showing
wt. @g)
Ethanol (N = 5)
Naltrexone + ethanol (N = 5)
Naltrexone (N = 5)
Controls (N = 5)
0.346’ 0.036 1.79* 0.178
0.471 0.036 2.25 0.174
0.489 0.040 2.12 0.138
0.596 0.031 2.65 0.076
< 0.001) compared with controls.
183
184 RELATIVE PROPORTIONS “/, IOOj~
-~
_________?
methanol (Lg/kg) analtrexone (5 mg/kg)
IZZ naltrexcne +ethanol m controls
Fig. 2. The relative proportion of the cortex (C) and the medulla (M) in different groups. **Significantly different (P < 0.001) compared with controls. *#Significantly different (P < 0.001) compared with naltrexone + ethanol-treated group.
a profound effect of alcohol on rat thymus are consistent with the previous findings. After intraperitoneal administration of ethanol no bacteriological invasion in abdominal cavity was ABSOLUTE VOLUMES
CM
mm3
CM
CM
methanol (Lg/kg) ardtrexone (5mg/kg) Fig. 3. medulla
The absolute
volumes
CM
~Z?Znaltrexone+ethanol m controls of the cortex
(C) and
the
(M) in different groups. **Significantly different (P < 0.001) compared with controls. *Significantly different (P < 0.05) compared with controls. #Significantly different (P < 0.05) compared with naltrexone + ethanol-treated group.
found. It is clear from the data presented in this report that a single hypnotic dose (4 g/kg) of ethanol significantly reduced the volume of the thymus especially affecting the cortex. Although it is possible that ethanol directly affects lymphocyte function in vivo, there are studies suggesting that this is not the major mechanism of lymphocyte impairment. This is supported by findings that the most profound impairment in lymphocyte proliferation occurs during the period after cessation of ethanol administration when withdrawal symptoms are evident [3]. Jerrells et al. [ll] have established that corticosteroids, produced most abundantly after withdrawal of ethanol, are at least partially responsible for the defect in lymphocyte proliferation. A number of reports have shown that acute and chronic ethanol treatment in vivo and in vitro alters the activity of the endogenous opioid system [14- 161, Ethanol may interact with the endogenous opioid system by: (a) production of certain ethanol metabolites, for example the isoquinolines which bind to opiate receptors, (b) altering the binding properties of opiate receptors and (c) altering the release, synthesis and post-translational processing of endogenous opioid peptides [17]. It has been suggested that ethanol may exert some of its effects like the reinforcing [18] and epileptogenic [19] effects via interaction with the endogenous opioid system. Since the cells of the immune system possess opioid receptors that are biochemically, physicochemically and functionally related to receptors of neuroendocrine origin [20], we assumed that the effects of ethanol on the thymus may be mediated by an opioid dependent mechanism. In order to test this hypothesis we used the opioid antagonist naltrexone. Naltrexone pretreatment prevented ethanol-induced changes in thymus morphology, whereas naltrexone alone was found to have no effect. These results indicate that ethanol exerts its effect on the thymus by an opioid dependent mechanism. Having in mind that naltrexone is a p-preferring antagonist and that functional ptype opioid receptors are present on cells of the immune system [21], one may assume that the
185
effect of ethanol is mediated by p-opiate receptors. Considering that the neuroendocrine system can influence immune function [22,23] it seems logical that brain opiate receptors may be involved in this phenomenon.
11
12
Acknowledgement This work was supported by the Republic of Serbia Research Fund. The technical help of Mrs. Sneiana Markovic is highly appreciated. References C.S. Lieber et al. (1979) Alcohol-related diseases and carcinogenesis. Cancer Res. 39, 2863. R.R. Mac Gregor (1986) Alcohol and immune defense. J. Am. Med. Assoc. 256, 1474. T.R. Jerrells et al. (1986) Effects of ethanol administration on parameters of immunocompetency in rats. J. Leukocyte Biol. 39, 499. 4 T.R. Jerrelis, W. Smith, M. J. Eckardt (1990) Murine model of ethanol-induced immunosuppression. Alcoholism: Clin. Exp. Res. 14, 546. 5 G.A. Roselle, L. Mendenhall (1984) Ethanol-induced alterations in lymphocyte function in the guinea pig. Alcoholism: Clin. EXD. Res. 8. 62. 6 C.J. Grossman, C.L.‘Mendenhall, G.A. Roselle (1988) Alcohol and immune regulation. In vivo effects of ethanol on concanavalin A sensitive thymic lymphocyte function. Int. J. Immunopharmacol. 10, 187. 7 W.J. Smith, Jr. et al. (1980) Altered immunity in male patients with alcoholic liver disease: evidence for defective immune regulation. Alcoholism: Clin. Exp. Res. 4, 199. 8 M. Aldo-Benson (1989) Mechanisms of alcohol-induced suppression of B-cell response. Alcoholism: Clin. Exp. Res., 13, 469. 9 C. Levallois et al. (1989) Effects of ethanol in vitro on some parameters of the immune response. Drug Alcohol Depend. 24, 239. 10 E.R. Weibel (1979) Stereological Methods, Vol. I,
13
14
15
16
17
18
19
20
21
22 23
Practical methods for biological morphometry. Academic Press, London-New York-Toronto-SidneySan Francisco. T.R. Jerrells et al. (1989) Mechanisms of suppression of cellular immunity induced by ethanol. Alcoholism: Clin. Exp. Res. 13, 490. S.J. Ewald, W.W. Frost (1987) Effect of prenatal exposure to ethanol on development of the thymus. Thymus 9, 211. S.J. Ewald, S.M. Walden (1988) Flow cytometric and histological analysis of mouse thymus in fetal alcohol syndrome. J. Leukocyte Biol. 44, 434. C. Gianoulakis, A. Barcomb (1987) Effect of acute ethanol in vivo and in vitro on the P-endorphin system in the rat. Life Sci. 40, 19. C. Gianoulakis (1990) Characterization of the effects of acute ethanol administration on the release of flendorphin peptides by the rat hypothalamus. Eur. J. Pharmacol. 180, 21. B.R. Seizinger (1984) Enhanced activity of the fland endorphinergic system in the anterior neurointermediate lobe after chronic treatment with ethanol liquid diet. J. Pharmacol. Exp. Ther. 230, 455. C. Gianoulakis (1989) The effect of ethanol on the biosynthesis and regulation of opioid peptides. Experientia 45, 4.28. H.L. Altshuler, P.E. Philips, D.E. Feinhaudler (1980) Alteration of ethanol self-administration by naltrexone. Life Sci. 26, 679. E. Triana, J.F. Richard, P.E. Strokes (1980) The relationship between endorphins and alcohol induced subcortical activity. Am. J. Psychiat. 127, 491. D.J.J. Carr (1988) Opioid receptors on cells of the immune systems. Prog. Neurol. Endocrinol. Immunol. 1, 8. R.T. Radulescu et al. (1991) Biochemical and functional characterization of p-opioid receptor binding site on cells of the immune system. Prog. Neurol. Endocrinol. Immunol. 4, 166. J.E. Blalock (1984) The immune system as a sensory organ. J. Immunol. 132, 1067. P.J. Neveu, M. Le Moal. (1990) Physiological basis for neuroimmunomodulation. Fund. Clin. Pharmacol. 4, 281.
