BRAIN RESEARCH

E L S E V I E R Brain Research 729 (1996) 285-288

Shor t c o m m u n i c a t i o n

Peripherally administered GM-CSF interferes with scopolamine-induced amnesia in mice: involvement of interleukin-1

M a u r o B i a n c h i *, P a o l a S a c e r d o t e , A l b e r t o E. Pane ra i

Department of Pharmacology, University ~!f'Milano, Via Vam,itelli 32, 20129 Milano, ltah'

Accepted 21 May 1996

Abstract

We studied the effects of granulocyte-macrophage colony stimulating factor (GM-CSF) on the classical behavioral test of scopolamine-induced amnesia lbr a passive avoidance response in the mouse. Pre-training intraperitoneal administration of this cytokine (1.25, 2.5, 5.0 or 10 p,g/mouse) partially, although significantly, reduced the amnesic action of the muscarinic receptor antagonist. The peripheral administration of a specific interleukin-1 receptor antagonist (IL-1 ra, 50 p~g/mouse i.p.) blocked the effect of GM-CSF. Our results suggest that GM-CSF is able to exert neuromodulatory actions and that it is involved (probably via IL-I) in the interactions between the immune system and the central nervous system.

Keywords: Interleukin-l: Passive avoidance; Scopolamine-induced anmesia; Recombinant granulocyte-macrophage colony-stimulating factor

Substancial evidence indicates that a bidirectional cir- cuit exists between the immune system and the brain. Consistently with such interaction, it has been reported that several mediators released from activated immune cells affect the brain function [5]. For instance, we previously demonstrated that, after peripheral administration, inter- leukin-1 (IL-I) and interleukin-2 (IL-2) differently inter- fere with scopolamine-induced amnesia in the mouse [1,3]. A large number of cytokines have been produced by recombinant DNA technology and are available for thera- peutic use; for many of them, included IL-I and IL-2, relevant central nervous system side effects have been reported [5]. A number of haemopoietic growth factors has been recently approved for the treatment of cytopenias induced by chemotherapy. Among them, granulocyte-mac- rophage colony-stimulating factor (GM-CSF) is currently available for clinical use in many countries. This cytokine stimulates the growth and differentation of monocyte, neu- trophil and eosinophil colonies. Moreover, it is a survival and activating factor for the mature cells, that enhances the biological activity of neutrophils, eosinophils and macrophages [10,12]. In human, it has been observed that the peripheral administration of GM-CSF can induce some

* Corresponding author. Fax: + 39 (2) 73-0470; E-mail: sacerdot@im- iucca.csi.unimi.it

neurological effects, including lethargy and mental contu- sion [10]. However, the possible interaction of this cy- tokine with brain functions has been taken into little consideration and has not been investigated yet. For all these reasons, we considered of interest to study the effects of GM-CSF on the central nervous system by using a behavioral model classically employed for the evaluation of drugs that interfere with cognitive functions in the experimental animal [2].

The following drugs were used: scopolamine hydrobro- mide (Sigma Chemical, St. Louis, MO, USA), human recombinant GM-CSF (Shering-Plough, Milano, Italy) and human IL-I receptor antagonist (IL-I ra, Synergen, Boul- der, CO, USA). All drugs were dissolved in sterile pyro- gen-free water and administered intraperitoneally (i.p.). We used male CD-I mice (Charles River, Calco, Italy), weighing 20-25 g and housed in air-conditioned rooms with a l ight/dark cycle of 12 h. The animals were acclima- tized to laboratory conditions for 7 days before the experi- ments. The effects of GM-CSF on scopolamine-induced amnesia for a passive avoidance response were assessed by a previously described method [2]. Briefly, the apparatus consists of two compartments, one light and one dark, connected via a sliding door. Each mouse was placed in the light compartment and allowed to enter the dark com- partment; the time (in seconds) taken to do so was recorded. Mice having a latencies greater than 40 s were eliminated.

0006-8993/96/$15.00 Copyright tC~ 1996 Elsevier Science B.V. All rights reserved. PII S 0 ( ) ( ) 6 - 8 9 9 3 ( 9 6 ) ( ) 0 5 9 1 - 4

286 M. Bianchi et al. / Brain Research 729 (1996) 285 288

200

180

~ 1 6 0 0

140 - o~

120 o

100 -

m 8 0 - g

6 0 -

4 0 -

2 0 -

1.25 2.50 5.00 10.00

Saline GM-CSF (pg / mouse)

GM-CSF Scopolamine (1 mg / kg) Scooolamine + GM-CSF

Fig. 1. Effects of the acute administration of GM-CSF alone or in combination with scopolamine on the passive avoidance response. Values are means _+ S.E.M. of 20 animals. * P < 0.05 vs. scopolamine.

Once the mouse was in the dark compar tment , the sl iding

door was c losed and an unavoidable electric shock (1 m A

for 1 s) de l ivered via the paws. The animal was then

p laced back in the h o m e cage until the retent ion trial. The

retent ion trial was carried out 24 h after the acquisi t ion

trial, by posi t ioning the mouse in the l ight compar tment

and recording the t ime taken to enter the dark compar t -

ment (retention latency). An increased retention latency

indicates that the animal has learned the associat ion be-

tween the shock and the dark compar tment . Dur ing the

retention trial, a cu t -of f t ime of 180 s was used. To

produce the amnesia for the avoidance task, mice were

treated with scopolamine (1.0 m g / k g i.p.) 15 min before

the acquisi t ion trial. Latency to re-enter the shock box 24 h

120

~ 1 0 0

8 ~ 80

= 60

g 4O

¢ 20

Scopolamine (1 mg / kg)

Scopolamine +GM-CSF (5 pg/mouse)

Scopolamine +lL-lra (50 pg/mouse)

Scopolamine +GM-CSF + IL-lra

0

Fig. 2. Blockade of the effect of GM-CSF (5.0 t~g/mouse) on scopolamine-induced amnesia induced by the simultaneous administration of IL-I ra (50 t~g/mouse). Values are means ___ S.E.M. of 20 animals. * P < 0.05 vs scopolamine; * * P < 0.05 vs. scopolamine + GM-CSF, and N.S. vs. scopolamine.

3000 -

2500 -

~ 2000 -

1500 -

< 10o0 -

500

J

0

3500 12 A

10

o 8

6

~- 4 o

B

®

Saline

GM-CSF

Fig. 3. Effects of the acute administration of GM-CSF (10 i~g/mouse) on spontaneous locomotor activity (panel A), and nociceptive thresholds (panel B). Values are means + S.E.M. of ten animals.

M. Bianchi et al. / Brain Research 729 (I 996) 285 288 287

later was used as a mesure of memory retention. GM-CSF was administered at a dose of 0.625, 1.25, 2.5, 5.0 and 10 p,g/mouse 15 rain before the acquisition trial or simulta- neously with scopolamine. The IL-1 receptor antagonist was administered i.p. at a dose of 50 I~g/mouse, simulta- neously with GM-CSF or scopolamine. Twenty animals were used in each experimental group. Results obtained in the passive avoidance test are expressed as mean latency /group during the retention trial. For each experiment, control, scopolamine, and test groups were compared by the analysis of variance (ANOVA) followed by Tukey's test for multiple comparisons. Since a putative analgesic or sedative effect exerted by GM-CSF would have interfered with the acquisition trial, thus making difficult the interpre- tation of results, we evaluated spontaneous locomotor ac- tivity and nociceptive thresholds 15 rain after the adminis- tration of the highest dose (10 ~g /mouse ) of the cytokine. Locomotor activity was quantified using an Animex appa- ratus (L.K.B., Hagersten, Sweden). Upon the injection, the animals were returned to their cages. Fifteen minutes later, mice were introduced individually in a clean box of the same type as the home cage and the box was placed on the Animex apparatus. Activity counts were recorded for 30 rain. Ten animals were used in each experimental group. The results obtained in the study of locomotor activity were analysed by ANOVA. For the evaluation of nocicep- rive thresholds, mice were placed on a hot plate apparatus (Basile, Comerio, Italy) with a surface electrically heated to a temperature of 54 _+ 0.5°C. Reaction times were mea- sured betore and 15 min after GM-CSF administration. The endpoint used was the simultaneous licking of both the fore paws. Ten animals were used in each experimental group, and the results were analysed by ANOVA.

Fig. 1 shows that GM-CSF administered alone from a dose of 1.25 p~g/mouse to a dose of 10 p~g/mouse does not modify the passive avoidance response; however, it significantly reduces, although incompletely, the scopo- lamine-induced amnesia. A lower dose (e.g., 0.625 ~g /mouse ) was completely ineffective (data not shown). As we observed that GM-CSF decreased the scopolamine- induced amnesia in a way comparable to that previously described in the same experimental model after 1L-1 ad- ministration [1], and since it has been demonstrated that GM-CSF is able to induce IL-I production by immune cells [8,11], we evaluated whether the administration of a specific antagonist for IL-1 receptors was able to block the action of GM-CSF in our experimental conditions. After intraperitoneal administration, the IL-1 receptor antagonist, which is inactive by itself, blocks the action of GM-CSF (5.0 p,g/mouse) on scopolamine-induced amnesia (Fig. 2). The evaluation of spontaneous locomotor activity and no- ciceptive thresholds performed 15 rain after the administra- tion of the highest dose of GM-CSF indicates that this cytokine does not interfere with the acquisition trial; in fact, the cytokine does not induce sedative or analgesic effects (Fig. 3).

To our knowledge, this study gives the first demonstra- tion of a central action of GM-CSF after peripheral admin- istration in the experimental animal. In particular, our results suggest that GM-CSF might interact with the cholinergic system. In fact, this cytokine significantly re- duces the amnesic effect of scopolamine, a blocker of muscarinic receptors with central effects [3,9].

The finding that haemopoietic cytokines can also be produced by neural tissues has generated a great interest [6]; however, the presence of specific receptors for GM- CSF in the central nervous system has been never de- scribed. Therefore, we can suppose that the effects that we have observed are mediated by other cytokines: the block of GM-CSF effect on scopolamine-induced amnesia achieved by the administration of the IL-1 ra indicates that IL-I is involved. This hypothesis is consistent with the observations that GM-CSF increases the production ol 1L-l in human leukocytes [11], and with the behavioral and biochemical effects of peripheral administered IL-I de- scribed by us and others [1,4,7]. Systemic administration of any potent immunomodulator might be expected to elicit relevant side effect. Hopes for the use of agents like IL-I or IL-2 have been partially compromised by the obserwt- tion of a moderate to severe toxicity in humans [5]. Thus far, the preclinical investigations performed with GM-CSF have shown high safety [12]. Altogether, our results are in agreement with these observations: however, they, suggest that GM-CSF interferes, at least in the experimental ani- mal, with brain functions.

In conclusion, our results suggest that also GM-CSF might be involved in the immune system-central nervous system interactions. Further work is warranted in order to elucidate the possible role of this growth factor as neuro- modulator.

References

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