Brain Research, 516 (1990) 345-348 Elsevier
345
BRES 24088
The nucleus basalis is involved in brain modulation of the immune system in rats J. Cherkaoui 1, W. Mayo 1, P.J. Neveu 1, K.W. Kelley 2, S. Vitiello 1, M. Le Moal 1 and H. Simon 1 ilNSERM, Unit~ 259, Universit~ de Bordeaux lI, Domaine de Carreire, 33077 Bordeaux (France) and 2Laboratory of Immunology, University of Illinois, Urbana, IL 61801 (U.S.A.)
(Accepted 30 January 1990) Key words: Nucleus basalis magnocellularis; Acetylcholine;Neuroimmunomodulation;Lymphoproliferation;Natural killer cell activity
Male rats were subjected to bilateral or unilateral excitotoxic lesions of the nucleus basalis magnocellulafis (NBM). Three weeks after surgery, mitogen-induced lymphoproliferation and natural killer (NK) cell activity were determined in the spleen. T-cell mitogenesis and NK cell activity were strongly enhanced after bilateral lesions but were not affected after fight or left unilateral lesions. B-cell mitogenesis and blood T-cell subset distribution remained unchanged after bilateral or unilateral lesions of the NBM. These results demonstrate that NBM cells are involved in the complex interrelations existing between the central nervous system and the immune system. Cerebral structures have been previously demonstrated to modulate the immune response. Lesion or stimulation of several hypothalamic nuclei modify the expression of various immunological indices 5. The cerebral neocortex has been shown to influence, in an asymmetrical way, the immune system. In rodents ablation of the left neocortex performed 6-10 weeks before testing, decreases both mitogen-induced responses of T-cells, IgG antibody synthesis and delays responses to alloantigens in the spleen cells. In contrast these parameters are enhanced after a symmetrical lesion of the right neocortex 16'19. Natural killer cell activity 1, activation of macrophages 15 and the synthesis or release of T-cell specific serum factors are also impaired by a left neocortical lesion 2°. Among the subcortical structures known to send projections to the neocortex, the nucleus basalis magnoceUularis (NBM) which provides the major source of cortical cholinergic innervation ~3 can modulate cortical activity2. In humans, degenerative processes involving the NBM are observed in some diseases such as senile dementia of the Alzheimer type (SDAT) which, apart classical histological alterations, is characterized by neurochemical abnormalities and cognitive dysfunctions4. In addition, modifications of immunological parameters have been described in S D A T 12'14'23'24. In animals, lesions of the NBM are known to induce biochemical and behavioral perturbations reproducing some of those
observed in S D A T 6's'9'11'22. Electrolytic lesions of the NBM decreased cellular immunity in rats ~8'2~, and in monkeys, excitotoxic lesions of the NBM were reported to depress natural killer cell activity 1°. In the present experiments, the possible role of NBM neurons in neuroimmunomodulation was assessed in rats after bilateral and unilateral lesions of the nucleus basalis 3 weeks before immunological testing. The results clearly show that after bilateral lesions, in contrast to unilateral ones, mitogen-induced T-cell proliferation as well as natural killer cell activity is strongly enhanced. Antibody production against xenogenic erythrocytes remained unchanged. The nucleus basalis is shown here to be endowed of immunoregulatory functions although its role in both the brain neocortex induced immunomodulation and the immunological perturbations observed in Alzheimer's disease remains to be elucidated. Sprague-Dawley rats weighing 300 + 50 g were used. The colony room was maintained on a constant light/dark cycle (lights on 06.00-20.00 h), at 22 °C and at a relative humidity of 60%. Food and water were provided at libitum. Surgery was conducted under chloral hydrate anesthesia (400 mg/kg i.p.). For unilateral or bilateral lesions, rats were placed in a stereotaxic apparatus (Kopf instruments) with the tooth bar 3.2 mm below the interaural line. The following coordinates for cannula placement were used: 0.8 mm posterior to the bregma, 2.3 mm
Correspondence: P.J. Neveu, INSERM, Unit6 259, Universit6 de Bordeaux II, Domaine de Carreire, rue Camille Saint-Sa~ns, 33077Bordeaux Cedex, France.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
346 lateral to the midline, and 8.2 mm ventrally to the skull surface, according to the stereotaxic atlas of Paxinos and Watson 17. Quisqualic acid (Sigma) was dissolved in phosphate-buffered saline (pH 7.4) at a concentration of 23.3/~g//~l and was infused in a volume of 0.6/~1 over 4 min, through a 320 p m stainless steel cannula connected via polyethylene tubing to a 5/~1 Hamilton microsyringe. Bilateral lesions, but not unilateral lesions of the NBM induced a moderate syndrom of aphagia-adipsia in only 20% of the animals and this syndrom lasted not more than 3 days. Choline acetyltransferase (CAT) was assayed in both control and experimental groups. Three weeks after the lesion, rats were killed by decapitation. Then the brain was rapidly removed and chilled on an ice-cooled metal plate. CAT activity was measured in the neocortex by a modified method of Fonnum 7. Mitogen-induced lymphoproliferation. Spleen cells were dissociated in RPMI-1640 medium (GIBCO, Glasgow, UK) and after 3 washes adjusted to 2.106 cells/ml in medium containing 5% heat inactivated fetal calf serum and 1% antibiotic antimycotic solution (GIBCO). Triplicates of spleen cell suspensions (2.105 cells/well) were cultured, in microtest II plates (Falcon, Cockeysville, MD), for 4 days in the presence of diluted mitogens in a humidified atmosphere of 5% CO 2. Concanavalin A (ConA) were purchased from DIFCO (Detroit, MI) and lipopolysaccharide of Escherichia coli (LPS) from Sigma (St. Louis, MO). Concentrations of mitogens were expressed as final concentrations in the culture medium. Twenty-four h before the end of the culture, 2 pCi of 3H-thymidine (specific activity of 5 Ci/mM, CEA, France) was added in a volume of 50 ,ul. Cells were collected on fiber-glass strips using a multiple harvester (Mash II, Microbiological Associates, Bethesda, MD). The filters were dried and counted in 3 ml of Lipoluma (Lumac, Schaesberg, The Netherlands), in a liquid scintillation counter. The results were expressed as stimulation indexes calculated for each animal as follows:
TABLE I Choline acetyltransferase (CAT) activity (nmol. h ~.(mg protein) -1) in cerebral cortex of rats, 3 weeks after bilateral or unilateral quisqualic acid lesions of the nucleus basalis NB M lesion
CA T activity
%
Control (9) Bilateral (10) Left (10) Right (10)
27.08 + 14.38 + 14.50 + 12.80 +
-46.9 -39.7 -40.9
*P < 0.001 when compared to control group. < 0.001 when compared to the unlesioned side.
**P
were removed and counted in a counter. Spontaneous or total release was obtained by incubating labelled target cells in medium alone or with HCI respectively. NK activity was expressed as the percentage cytotoxicity as follows : % Cytotoxicity =
test cpm - spontaneous cpm total cpm - spontaneous cpm
Serum antibody levels. Five days before sacrifice, rats received an intra-peritoneal injection of 1.109 SRBC (sheep red blood cells). The sera were collected from trunk blood. Hemagglutination assays were performed according to standard procedures by doubling dilutions in microtiter trays. There was a significant decrease in CAT activity in the cortex of bilateral (t = 6.71, df = 17, P < 0.001, -46.9%) or unilateral (left: t = 6.14, df = 18, P < 0.001, -39.7%; right: t = 4.32, df = 18, P < 0.001, -40.9%) lesioned animals (Table I). T-lymphocyte proliferation induced by ConA was
2 ~g/ml
1 pg/ml
6G
X
SI=
1.5 1.1" 1.l** 1.0"*
50
mean cpm stimulated culture mean cpm unstimulated culture
Natural killer cell activity. Natural killer (NK) cell
activity in spleen was tested by a 51Cr release assay using YAC-1 cells as target cells (Flow Laboratories, Puteaux, France). Cultured target YAC-1 cells, a Moloney virus induced-lymphoma, were labelled by incubating 1.0.107 cells with 100/aCi Sodium 51Cr-chromate (NEN, Paris) 1 h at 37 °C and washed. Cytotoxicity was measured in triplicate samples at target to effector (E/T) ratios from 100:1 to 12.5:1 using 2.5.104 YAC target cells. After 4 h incubation at 37 °C, 100/~i aliquots of the supernatants
-Z
40
. j 30
m
20
o
SHAM
C
NBM
SH AM
C
NBM
Fig. 1. Con A-induced lymphoproliferation in normal control rats (C) or 3 weeks after sham-operation (S) or bilateral lesion of NBM (NBM). Results are expressed as stimulation index + S.E.M. The number of animals per group is indicated in brackets.
347 80
60
>I-x
o
I- 40 >.
20
I
100/1
I
50/1
I
2~/t
I
12.5/1
SPLENOCYTE / TARGET RATIO Fig. 2. Natural killer cell activity in spleen from groups of normal (O, N = 9) or in bilateral lesion of NBM ( 0 , N = 10) expressed as % cytotoxicity + S.E.M.
assessed in rats 3 weeks after lesioning the nucleus basalis. As seen in Fig. 1 which represents one of 3 independent experiments, lymphoproliferation induced by an optimal dose of ConA (2 mg/ml) was higher in animals after bilateral lesion of the NBM as compared to sham-operated rats (t = 2.56, df = 14, P < 0.05) or to normal controls (t = 1.93, df = 12, P < 0.10). No difference was observed between sham-operated and control animals. This enhancement of T-cell mitogenesis paralleled the increase of 11-2 production by Con-A stimulated spleen lymphocytes after lesioning the NBM. On the contrary, B-lymphocyte proliferation induced by LPS after bilateral lesions of the NBM was similar to that of controls (results not shown). In comparison, right or left unilateral lesions of NBM appeared not to modify lymphoproliferation induced by Con-A. Natural killer cell activity in spleen was higher after bilateral lesions of the NBM as compared to normal controls for each splenocyte-target ratio (100:1, t = 3.63, df = 17, P < 0.01; 50:1, t = 3.81, df = 17, P < 0.01; 25:1, t = 3.08, df = 17, P < 0.01; 12.5:1, t = 3.39, df = 15, P < 0.01) (Fig. 2). On the contrary, unilateral lesions of either side did not affect NK cell activity (results not shown). T-lymphocyte subsets in blood were similarly distributed whether the animals were bilaterally or unilaterally lesioned. The percentage of T-cells was 43.5 + 2.2 and
42.1 + 3.0; that of helper T-lymphocytes 34.0 + 1.3 and 32.7 + 1.9, and that of cytotoxic/suppressor cells was 15.1 + 1.5 and 15.5 + 1.4 for controls and bilateral lesioned animals, respectively. Serum antibody levels to sheep erythrocytes measured 5 days after immunization were similar in rats after bilateral lesions of the NBM and in controls (log 2 hemagglutination titers for lesioned and control animals were 5.6 + 0.5 and 6.3 + 0.2, respectively). The influence of the nucleus basalis on the immunological status was studied in rats 3 weeks after bilateral or unilateral lesions. After bilateral lesions, T cell responses, as indicated by Con A-induced mitogenesis and IL-2 production, were strongly enhanced by about 300% as compared to controls. These results may appear to be in opposition to these of Trobonj aca et al. 25 who reported a depression of cellular immunity after NBM lesions. However, they performed electrolytic lesions which destroy not only cell bodies but also fibers of passage, and also decrease catecholamine levels in the neocortex 6. In the present work, excitotoxic lesions involved only cell bodies in the NBM and spared fibers. This increase probably did not result from T-lymphocyte subset redistribution as the percentage of suppressor/cytotoxic and helper T-cells in the blood was unchanged after lesions of the NBM. The active signals required for the induction of T-cell activation remained to be explored. Likewise, the activity of NK cells which have been suggested to be derived from the T-cell lineage 21, were enhanced after bilateral lesions of the NBM. These results may also appear opposite to those of Kraus et al. 1° who reported a depression of NK cell activity after NBM lesions in monkeys. However the time interval between lesioning and testing was not indicated in their abstract and the number of lesioned animals was small. So it should be difficult to compare these results with ours which were additionally obtained in a different species. NBM lesions mainly modify T-cell function, on the contrary, B-cell response, as assessed by LPS-mitogenesis, were not affected by NBM lesioning. Similarly, antibody produc~ tion 5 days after immunization was similar to that of controls. Indeed, the early antibodies consist in IgM which production is known t o be independent of T-cell help 3. Surprisingly, unilateral NBM lesions had no effect of the immunological parameters tested so far. This could be due to the fact that after unilateral lesions, as compared to bilateral ones, the extent of the depletion in neurotransmitters was not sufficient to affect the immune system. NBM lesions were accompanied by a depletion of acetylcholine in several brain areas. However, other neurotransmitters may be affected by NBM lesions and it is difficult to make a link between depletion of acetyi-
348 choline and i m m u n e dysfunctions. N o correlation was found b e t w e e n C A T levels and Con A-stimulation index or N K cell activity. F u r t h e r m o r e , a high increase in lymphocyte reactivity has been observed in preliminary experiences whereas C A T was slightly depressed. The nature of the neurotransmitter(s) involved in immune p e r t u r b a t i o n s after N B M lesions are yet to be elucidated. The e n h a n c e m e n t of T-cell reactivity observed after bilateral N B M lesions is neither directly related to the effects of brain cortex ablation we have previously described 16 nor to the immune perturbations observed in A l z h e i m e r ' s disease. The brain neocortex and the N B M both mainly m o d u l a t e the activity of T-lymphocytes although the activity of the neocortex is lateralized whereas that of N B M appears not to be so. In Alzhei-
m e r ' s disease, the small i m m u n e dysfunctions already described 14'23'24 consist of decreases of some p a r a m e t e r s whereas after lesioning the N B M we observed a strong increase in T-cell functions. H o w e v e r in the present experiments, the effects of N B M lesions on immune status have been studied shortly after lesioning, the increase in lymphocyte reactivity may be only transient and followed by depression. F u r t h e r experiments performed at various time intervals are n e e d e d to answer this question. We d e m o n s t r a t e d for the first time that the N B M cells are involved in the complex interrelations existing between the central nervous system and the i m m u n e system but its precise role remains to be elucidated.
1 Bardos, P., Degenne, D., Lebranchu, Y., Bizi~re, K., and Renoux, G., Neocortical lateralization of NK activity in mice, Scand. J. lmmunol., 13 (1981) 609-611. 2 Buszaki, G., Bickford, R.G., Ponomareff, G., Thal, L.J., Mandel, R. and Gage, EH., Nucleus basalis and thalamic control of neocortical activity in the freely moving rat, J. Neurosci., 8 (1988) 4007-4026. 3 Cebra, J.J., Kamisar, J.L. and Schweitzer, P.A., CH isotype 'switching' during normal B-lymphocyte development, Annu. Rev. Immunol., 2 (1984) 493. 4 Collerton, D., Cholinergic function and intellectual decline in Alzheimer's disease, Neuroscience, 19 (1986) 1-28. 5 Cross, R.J., Brooks, W.H., Roszman, T.L. and Markesbery, W.R., Hypothalamic-immune interactions: effect of hypophysectomy on neuroimmuno-modulation, J. Neurol. Sci., 53 (1982) 557-566. 6 Dubois, B., Mayo W., Agid Y., Le Moal, M. and Simon, H., Profound disturbances of spontaneous and learned behaviors following lesions of the nucleus basalis magnocellularis in the rat, Brain Research, 338 (1985) 249-258. 7 Fonnum, E, A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1969) 407-409. 8 Hepler, D.J., Wenk, G.L., Cribbs, B.L., Olton, D.S. and Coyle, J.T., Memory impairments following basal forebrain lesions, Brain Research, 346 (1985) 8-14. 9 Kesner, R.P., Adelstein, T. and Crutcl~er, K.A., Rats with nucleus basalis lesions mimic mnemonic symptomatology observed in patients with dementia of Alzheimer type, Behav. Neurosci., 101 (1987) 451-456. 10 Kraus, L.J., Moss, M.B. and Rosene, D.L., Decreased natural killer cell activity in young adult basal forebrain damaged and normal aged, rhesus monkeys, Soc. Neurosci. Abstr., vol. 11, part. 2 (1985) 252.8. 11 Mayo, W., Kharouby, M., Le Moal, M. and Simon, H., Memory disturbances following ibotenic acid injections in the nucleus basalis magnocellularis of the rat, Brain Research, 455 (1988) 213-222. 12 McDonald, S.M., Goldstone, A.H., Morris, J.E., Exton-Smith, A.N. and Callard, R.E., Immunological parameters in the aged and in Alzheimer's disease, Clin. Exp. Immunol., 49 (1982) 123-128.
13 Mesulam, M.M., Mufson, E.J., Wainer, B.H. and Levey, A.I., Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Chl-Ch6), Neuroscience, 10 (1983) 1185-1201. 14 Miller, A.E., Neighbour, P.A., Katzman, R., Aronson, M. and Lipkowitz, R., Immunological studies in senile dementia of the Alzheimer type: evidence for enhanced suppressor cell activity, Ann. Neurol., 10 (1981) 506-510. 15 Neveu, P.J., Barn6oud, P., Georgiades, O., Vitiello, S., Vincendeau, P. and Le Moal, M., Brain neocortex influence on the mononuclear phagocytic system. J. Neurol. Sci., 22 (1989) 188-193. 16 Neveu, P.J., Cerebral neocortex modulation of immune functions, Life Sci., 42 (1988) 1917-1923. 17 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 18 Radosevic-Stasic, B., Cuk, M., Markovcic-Sutic, I., Trobonjaca, Z., Salamon, R., Stojanov, L. and Rukavina D., Immunological consequences of lesions of nucleus basalis in rats, Int. J. Neurosci., in press. 19 Renoux, G., Bizi6re, K., Renoux, M. and Guillaumin, J.M., The production of T-cell-inducing factors in mice is controlled by the brain neocortex, Scand. J. lmmunol., 17 (1983) 45-50. 20 Renoux, G., Bizi6re, K., Renoux, M., Guillaumin, J.M. and Degenne, D.A., A balanced brain asymmetry modulates T-cell mediated events, J. Neuroimmunol., 5 (1983) 227-238. 21 Robertson, M., T-cell receptor: the present state of recognition, Nature (Lond.), 317 (1985) 768-771. 22 Salamone, J.D., B6art, P.M., Alpert, J.E. and Iversen, S.D., Impairment in T-maze reinforced alternation performance following nucleus basalis magnocellularis lesions in rats, Behav. Brain Res.,, 13 (1984) 63-70. 23 Singh, V.K., Fudenberg, H.H. and Brown, ER., Immunologic dysfunction simultaneous study of Alzheimer's and older down's patients, Mech. Aging Devel., 37 (1987) 257-264. 24 Tavolato, B. and Argentiero, V., Immunological indices in presenile Alzheimer's disease, J. Neurol. Sci., 46 (1980) 325331. 25 Trobonjaca, Z., Salamon, R., Radosevi-Stasic, B., Stojanov, L., Cuk, M., Zupan, G., Simonic, A. and Rukavina, D., Suppression of cell-mediated immunity after bilateral nucleus basalis lesions in rats, Yugosl. Physiol. Pharmacol. Acta, in press.