Psychological factors clearly influence immune responses in humans (Locke, 1982; Irwin, Daniels, Bloom, Smith, & Weiner, 1987; Temoshok, 1987) and other animals (Borysenko & Borysenko, 1982; Griffin, 1989). Relationships between the nervous system and the immune system may be mediated indirectly via the hypothalamic-hypophysial axis or directly through the autonomic nervous system. In addition, there are receptors for a variety of ligands associated with nervous system (neurotransmitters and hormones) located on cells that are components of the immune system (Jankovic, 1989; Rabin, Cohen, Ganguli, Lysle, & Cunnick, 1989; Khansari, Murgo, & Faith, 1990; Blalock, 1992; Jancovick & Radulovic, 1992).
It consequently seemed logical to examine the effects of psychoactive drugs on the immune system’s responses. A wide variety of neural opioid receptors are closely involved in behavioural changes (Rance, 1983). The relationship between endogenous morphine and stress suggests that endorphins and enkephalins influence emotionality (Rossier & Chapouthier, 1983). The most commonly-used opioid antagonist is naloxone, wich specifically blocks mu receptors at low doses but at higher doses it also influences kappa and delta receptors, as well as changing gabaergic activity (Dingledine, Iversen, & Breuker, 1978). This drug dose-dependently increases sensitivity to shock punishment on a food reinforced operant in the rat (Young, 1980). Under certain circumstances, morphine restores the punishment response in pigeons to a level similar to that seen after application of benzodiazepines. Moreover, those results mirror the anxiolytic effects of a morphine shown in humans (Iversen & Iversen, 1981). Lower doses of naloxone than those needed to increase the punishment effect, reverse the tolerance to punishment produced by diazepam administration (Soubrie, Jobert, & Thiebot, 1980).
Benzodiazepines have been characterized as efficient anxiolytics. Their therapeutic effect appears mainly mediated by interactions with receptors of the gabaergic system (Tollman, Skolnik, & Gallagher, 1980; Olsen, 1982; Paul, 1986; Doble & Martin, 1992). Many studies on the effects of benzodiazepines on anxiety have been carried out using experimental animals. Animals appear less fearful, and more ready to carry out activities for wich they have been punished, after treatment with these compounds (Treit, 1985; Iversen & Iversen, 1981). Chlordiazepoxide (CDP) is a classical benzodiazepine, widely-used in this type of research, wich has a strong anti-punishment effect (Iversen & Iversen, 1981).
The present study investigated the effects of naloxone and CDP on the primary immune response in male and female mice. Given the capacity of these drugs to affect emotional states, an influence on the immune response seemed likely. There are clear sex differences in emotionality, measured by behaviour in open field (defecation and ambulation) and, as response to novel situations such as stress and intense population density (Gray, 1971; Steenbergen, Heinsbroek, Van Hest, & Van de Poll, 1990; Johnston & File, 1991). The effects of sex on the immune response were consequently also examined.
Materials and methods
Sixty male and sixty female AP albino mice, from a stock obtained from ICI Pharmaceuticals, Macclesfield, U.K. were used in the study. The subjects were reared under highly controlled conditions at the animal facilities of University College of Swansea, U.K. (described in Brain, McAllister, & Walmsley 1989). All animals lived under a reversed lighting schedule (white lights on from 2200 to 1000 h local time).
Both naloxone hydrochloride and CDP were obtained from Sigma Chemical Company Ltd., Fancy Rd., Poole, Dorset, BH17 7NH, England. The materials were made up freshly in physiological (0,85%) saline.
All animals were injected subcutaneously (sc) each day for a 7 day period, two hours after the begining of the dark period. Categories of males and females (n=10) were given daily injections of either 1 or 2 mg/kg of naloxone or 5 or 10 mg/kg of CDP. Control categories received physiological saline. Five days after the end of this treatment, subjects received an antigenic challenge (see below).
Sheep red blood cells (SRBC) from Flow Laboratories (Irvine,
Scotland) were washed three times with physiological saline and made up to a
20% suspension in saline (there were approximately 109cells/ml).
The standard antigenic challenge consisted of a 0,1 ml intraperitoneal (ip)
injection of this suspension (108 cells). Blood was subsequently
collected from subjects by cardiac puncture 5 days after challenge and it was
allowed to clot at room temperature before being centrifuged at 3,000 rpm. The
resulting sera were collected and incubated for complement inactivation in a
water bath at 560C for 30 min. Serum titrations were carried out
in polystyrene trays (type 2-45128A, Nunc Gibco, Life Technologies, Paisley,
Scotland) using serial two-fold dilutions, with 0,85% saline and Tris/HCl buffer
solution at a pH of 7,4. After incubating the sera at 370C, dilutions containing
total hemagglutinins were recorded and immune response assessed in terms of
mean log2 titres for each category. Serum was also titrated after treatment
with 2-mercaptoethanol (Sigma Chemicals Company, LTD., Fancy Rd., Poole, Dorset,
BH17 7NH, England). MER-resistant antibody is the 7S (Sredberg units) variety,
whereas total antibody titre gives the 7S and 12S values (combined). The primary
immune response is largely in terms of 12S antibody (Brayton & Brain, 1974).
Mean log2 antibody titres for total and mercaptoethanol-resistant (MER-resistant) hemagglutinins
together with ANOVA comparisons between categories are given in Tables 1 and
2, respectively. Figures 1 to 4 represent the effects of naloxone and CDP doses
on total and MER-resistant antibody production.
The total hemagglutinin measure showed a significant interaction
between drug and sex (p< 0.04). The 10 mg/kg dose affected males and females
in different ways.
In males there was a drastic reduction of antibody titres,
but females showed an increased response. MER-resistant antibody showed a significant
increase after treatment with naloxone (p< 0.0001). Both doses (1 mg/kg and
10 mg/kg) differed significantly from the control group (p< 0.001 and p<
0.009). There were also significant effects of sex (p< 0.02) on this measure,
males having higher antibody titres than females in the different groups.
A significant effect on the total hemagglutinin titre of mice
was observed when subjects were treated with CDP ( p< 0.03). The 10 mg/kg
dose produced a significant reduction in antibody titre compared with controls
(p< 0.01). There were no sex differences in total hemagglutinin production.
CDP tended to reduce the MER-resistant antibody titre (p< 0.07). Nevertheless,
when median values for the groups were compared, there was a significant difference
between the 5 and 10 mg/kg doses (p< 0.0001) and between the 5 mg/kg and
control (p< 0.0001). A reduction in the MER-resistant antibody concentration
was observed with the 5 mg/kg dose but not with the 10 mg/kg dose. There was
a significant difference between sexes in the MER-resistant hemagglutinin response
(p< 0.05), with males producing more antibody than females.
Treatment with naloxone at both doses (1 and 10 mg/kg) increased
antibody titre in response to SRBC inoculation on the MER-resistant hemagglutinin
test. This effect on immunopotential is reminiscent of that reported by Inostroza,
Teschemacher, & Mueller-Eckhardt, (1987), Jankovic & Maric (1987) and
Mediratta, Das, Gupta, & Sen, (1988), wich all suggest that naloxone antagonizes
the immunosuppressive effects of endorphins. This immunosuppression has also
been observed both on cellular (Shavit. Lewis, Terman, Gale, & Liebeskind,
1986; Maric & Jankovic, 1987; Gabrilovac, Antica & Osmak, 1992; Freier
& Fuchs, 1994) and humoral (Johnson, Smith, Torres, & Blaloc, 1982;
Mediratta et al., 1988) immune responses.
Naloxone´s effects on MER-resistant hemagglutinin differ from
those on total hemagglutinin, suggesting that this drug affect those two types
of hemagglutinins in different ways. This difference was sex-dependent. In the
case of the MER-resistant hemagglutinin a quantitative difference on the antibody
response was observed, while in the total hemagglutinin titre a sex-linked qualitative
difference in response to naloxone was found, with a significant sex-drug interaction.
Sex differences in the immune system have also been reported
by Globe & Konopka (1973) and Rabin et al. (1988) and it is generally accepted
that males are more vulnerable to the effects of stress than females (Strausser,
Fiore, & Belisle, 1984; McFarland & Bigley, 1989), although existing
results are not conclusive.
In the present experiment, a higher production of MER-resistant
hemagglutinin was evident in males, independently of the drug used. This dimorphism
was also observed in the control groups.
The present results also show that CDP application affects
the immune system. This drug depressed the antibody response in both tests,
although the overall effect observed on MER-resistant hemagglutinin did not
quite reach significance. In this test, the 5 mg/kg dose produced a significant
reduction on the antibody production compared to both control group and 10 mg/kg
group. Pericic, Manev, Boranic, Poljak-Blazi, & Lehic (1987), reported that
another benzodiazepine (Diazepam) also has an immunosuppressive effect on spleen
plaque-forming cell production in rats. It has also been observed the sex differences
in MER-resistant hemagglutinin (but not total hemagglutinin) titre were evident
in this study. Relationships between the immune response and benzodiazepine
receptors have been suggested by Arora, Hanna, Paul, & Skolnick (1987).
Receptors for these drugs are located in regions of the cerebral cortex which
are concerned with both anxiety mechanisms and the immune response.
Essentially the present results confirm that naloxone and CDP
have opposite effects on antibody production. Several data show these drugs
alter lymphocytes T population (Arora, Hanna, Paul & Skonick, 1987; Inostroza,
Teschemacher & Mueller-Eckhardt, 1987; Manfredi, Sacerdote, Vianchi, Locatelli,
Veljic-Radulovic & Panerai, 1993). A subset of lymphocytes cells, helper
- T cells, influence antibody production; the B cells’ response to antigens
is totally dependent on these cells. This mechanism could be the responsible
of the effects of both sustances on immune primary response. There is evidence
that endogenous opiates decrease antibody response to T dependent antigens (SRBC)
and this effect was reversed by naloxone (Inostroza, Teschemacher & Mueller-Eckhardt,
1987; Manfredy, Sacerdote, Bianchi, Locatelli & Veljic-Radulovic, 1993).
In the other hand, as naloxone and CDP affect anxiety in opposite
ways (naloxone is anxiogenic whereas CDP is anxiolytic) the opposite effects
on antibody production seems a logical finding. The different anxiety states
generated by these drugs (with a possible gabaergic involvement and productions
of endogenous opioids), could be the responsible for the varied effects of these
psychoactive compounds on the immune response. The regions of the cerebral cortex
that influence anxiety mechanisms also alter the immune system. The data suggest
that emotional states are likely to influence immune responsiveness and that
there are likely to be immunological sequelae of clinical treatment with psychoactive
compounds. The potential impact of psychoactive compounds on disease resistance
must, for example, be of great interest to AIDS researchers, as AIDS sufferens
are frequently treated for depression. It is notable that males and females
appear to show differing responses (in terms of immune capacity) to these compounds.
Arora, P. K., Hanna, E. E., Paul, S. M. & Skolnick, P. (1987). Suppression of the immune response by benzodiazepine receptor inverse agonists. Journal of Neuroimmunology, 15, 1-9.
Blalock, J.E. (1992). Neuroimmunoendocrinology. Basel: S. Karger.
Borysenko, M. & Borysenko, J. (1982). Stress, behavior, and immunity: Animal models and mediating mechanisms. General Hospital Psychiatry, 4, 59-67.
Brain, P.F., McAllister, K.H. & Walmsley, S.V. (1989). Drug effects on social behaviour. In A.A. Boulton, G.B. Baker & A.J. Greenshaw (Eds.), Neuromethods: Vol. 13. Psychopharmacology (pp. 689-739). Clifton: The Humana Press Inc..
Brayton, A.R. & Brain, P.F. (1974). Effects of differential housing and glucocorticoid administration on immune responses to sheep red blood cells in albino "TO" strain mice". Journal of Endocrinology, 64, 4-5.
Dingledine, R., Iversen, L. L. & Breuker, E. (1978). Naloxone as a Gaba antagonist: Evidence from iontophoretic receptor binding and convulsant studies. European Journal of Pharmacology, 47, 19-27.
Doble, A. & Martin, I. (1992). Multiple benzodiazepine receptors. Trends in Pharmacological Science, 13, 76-81.
Freier, B. O. & Fuchs, B. A. (1994). A mechanism of action for morphine-induced immunosuppression: corticosterone mediates morphine-induced suppression of natural killer cell activity. Journal of Pharmacology and Experimental Therapeutics, 270, 1127-1133.
Gabrilovac, J., Antica, M. & Osmak, M. (1992). In vivo bidirectional regulation of mouse natural killer cell cytotoxic activities by Leu-enkephalin: reversibility by naloxone. Life Sciences, 50, 29-37.
Globe, F. C. & Konopka, E. A. (1973). Sex as a factor in infectious disease. Transactions of the New York Academy of Science, 35, 326.
Gray, J. (1971). The psychology of fear and stress. San Francisco: McGraw-Hill.
Griffin, J. F. T. (1989). Stress and immunity: A unifying concept. Veterinary Immunology and Immunopathology, 20, 263-312.
Inostroza, J., Teschemacher, H. J. & Mueller-Eckhardt, C. (1987). Péptidos opioides y sistema inmunitario. Inmunología, 6, 93-99.
Irwin, M., Daniels, M., Bloom, E. T., Smith, T. L. & Weiener, H. (1987). Life events depressive symptoms and immune function. American Journal of Psychiatry, 144, 437-441.
Iversen, S. D. & Iversen, L. L. (1981). Behavioral Pharmacology. New York: Oxford University Press.
Jankovic, B. D. (1989). Neuroimmunomodulation: Facts and dilemmas. Immunology Letters, 21, 101-118.
Jankovic, B. D. & Maríc, D. (1987). Enkephalins and immunity. I: In vivo suppression and potentiation of humoral immune response. Annals of the New York Academy of Sciences, 496, 115-125.
Jankovic, B. D. & Radulovic, J. (1992). Enkephalins, brain and immunity: modulation of immune responses by methionine-enkephalin injected into the cerebral cavity. International Journal of Neuroscience, 67, 241-270.
Johnson, H. M., Smith, E. M., Torres, B. A. & Blaloc, J. E. (1982). Neuroendocrine hormone regulation of in vitro antibody production. Proceedings of the National Academy of Sciences of the United States of America, 79, 4171-4174.
Johnston, A. L. & File, S. E. (1991). Sex differences in animal tests of anxiety. Physiology and Behavior, 49, 245-250.
Khansari, D., Murgo, A. & Faith, R. E. (1990). Effects of stress on the immune system. Immunology Today, 11, 170-175.
Locke, S. E. (1982). Stress, adaptation, and immunity: Studies in humans. General Hospital Psychiatry, 4, 49-58.
Manfredi, B., Sacerdote, P., Bianchi, M., Locatelli, L., Veljic-Radulovic, J. & Panerai, A. E. (1993). Evidence for an opioid inhibitory effect on T cell proliferation. Journal of Neuroimmunology, 44, 43-48.
Maríc, D. & Jankovic, B. D. (1987). Enkephalins and immunity. II: In vivo modulation of cell-mediated immunity. Annals New York Academy of Sciences, 496, 126-136.
McFarland, H. I. & Bigley, N. J. (1989). Sex-dependent, early cytokine production by NK-like spleen cells following infection with the D variant of encephalomyocarditis virus (EMCV-D). Viral Immunology, 2, 205-214.
Mediratta, P. K., Das, N., Gupta, V. S. & Sen, P. (1988). Endogenous opioids and immune responses: An experimental study. NIDA Research Monography, 87, 209-216.
Olsen, R. W. (1982). Drug interactions at the GABA receptor-Ionophore complex. Annual Review of Pharmacological Toxicology, 22, 245-277.
Paul, S. M., Crawley, J. N. & Skolnick, P. (1986). The neurobiology of anxiety: the role of the GABA-benzodiazepine receptor complex. In P. A. Berger & K. H. Brodie (Eds.), American Handbook of Psychyatry, Vol. 8. (pp. 581-596). New York: Basic Books.
Pericic, D., Manev, H., Boranic, M., Poljak-Blazi, M. & Lekic, N. (1987). Effect of diazepam on brain neurotransmitters, plasma corticosterone and the immune system of stressed rats. Annals New York Academy of Sciences, 496, 450-458.
Rabin, B. S., Cohen, S., Ganguli, R., Lysle, D. T. & Cunnick, J. E. (1989). Bidirectional interaction between the central nervous system and the immune system. Critical Reviews in Immunology, 9, 279-312.
Rabin, B. S., Cunnick, J. E. & Lysle, D. T. (1988). Alteration of the immune system by housing. Advances, 5, 15-25.
Rance, M. J. (1983). Multiple opiate receptors-their ocurrence and significance. Clinics in Anaesthesiology, 1, 183-199.
Rossier, J. and Chapouthier, G., 1983. Encefalinas y endorfinas. Mundo Científico, 3, 68-79.
Shavit, Y., Lewis, J. W., Terman, G. W., Gale, R. P. & Liebeskind, J. C. (1986). Stress, opiod peptides and immune function. In R.C.A. Frederickson, H. Hendire and J. Hingtgen (Eds.), Neuroregulation of autonomic, endocrine and immune systems (pp. 343-366). Boston: Martinus Nijhoff Publishing.
Soubrie, P., Jobert, A. & Thiebot, M. H. (1980). Differential effects of naloxone against the diazepam-induced release of behavior in rats in three aversive sutuations. Psychopharmacolgy, 69, 101-105.
Steenbergen, H. L., Heinsbroek, R. P., Van Hest, A. &Van de Poll, N. E. (1990). Sex-dependent effects of inescapable shock administration on shuttlebox-escape performance and elevated plus-maze behaviour. Physiology and Behavior, 48, 571-576.
Strausser, H. R., Fiore, R. P. & Belisle, E. H. (1984). Alterations in immune function with age, sex, hormones and stress. In E.L. Cooper (Ed.), Stress Immunity and Aging. New York: Marcel Dekker.
Temoshok, L. (1987). Personality, coping style, emotion and cancer: Towards and integrative model. Cancer Survey, 6, 545-567.
Tollman, J. F., Skolnik, P. & Gallagher, D. W. (1980). Receptors for the age of anxiety: Pharmacology of the benzodiazepines. Science, 207, 274-281.
Treit, D. (1985). Animal models for the study of anti-anxiety agents: A review. Neuroscience and Biobehavioral Reviews, 9, 203-222.
Young, G. A. (1980). Naloxone enhancement of punishment in the rat. Life Science, 26, 1787-1792.
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