concomitant administration of fluoxetine and amantadine modulates the activity of peritoneal...

Concomitant administration of fluoxetine and

amantadine modulates the activity of peritoneal

macrophages of rats subjected to a forced

swimming test

Adam Roman1, Zofia Rogó¿2, Marta Kubera3, Dominika Nawrat1,

Irena Nalepa1

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Abstract:

Recent studies show that administration of a non-competitive NMDA receptor antagonist, amantadine (AMA), potentiates the action

of antidepressant drugs. Since antidepressants may modulate functioning of the immune system and activation of a pro-

inflammatory response in depressive disorders is frequently reported, the aim of the present study was to examine whether a com-

bined administration of AMA and the antidepressant, fluoxetine (FLU), to rats subsequently subjected to a forced swimming test

(FST) modifies the parameters of macrophage activity, directly related to their immunomodulatory functions, i.e., arginase (ARG)

activity and synthesis of nitric oxide (NO). We found that 10 mg/kg AMA and 10 mg/kg FLU, ineffective in FST for antidepressant-

like activity when administered alone, increased the ARG/NO ratio in macrophages when administered concomitantly. This effect

was accompanied by a decrease of cellular adherence. Concurrently, the basal metabolic activity of the cells measured with reduction

of resazurin, and intracellular host defense as assessed by a synthesis of superoxide anion, were not affected by such antidepressive

treatment. Our data indicate that co-administration of AMAand FLU decreases the pro-inflammatory properties of macrophages and

causes a redirection of immune response toward anti-inflammatory activity, as one can anticipate in the case of an effective antide-

pressive treatment.

Key words:

fluoxetine, amantadine, peritoneal macrophages, nitric oxide, arginase activity

Abbreviations: AMA – amantadine, ARG – arginase, FLU –

fluoxetine, FST – forced swimming test, iNOS – inductible ni-

tric oxide synthase, LPS – lipopolysaccharide, NBT – nitro-

tetrazolium blue chloride, PBS – phosphate buffered saline,

PMA – phorbol 12-myristate 13-acetate, Th – T helper lym-

phocytes

Introduction

A large majority of currently used antidepressants act

based on an enhancement of serotonergic and/or nora-

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drenergic neurotransmission. However, they are only

partially effective and more than 30% of depressed

patients fail to achieve remission despite multiple

treatment trials. Thus, there is an urgent need for de-

velopment of alternative, more effective pharmaco-

therapies. In recent years, the role of an increased glu-

tamatergic neurotransmission in the pathophysiology

of depressive disorders has been brought into focus.

Much attention has been paid to N-methyl-D-aspartic

acid (NMDA) receptor antagonists, in particular

amantadine (AMA). This compound was shown to

produce only mild antidepressive effects when admin-

istered alone, but was effective as an adjunctive ther-

apy in combination with other antidepressants in both

humans and in animal models [30, 31]. In addition,

AMA possesses immunomodulatory properties, as it

was shown to increase synthesis of interleukin (IL)-10

by splenocytes of rats subjected to a forced swimming

test (FST) [16].

Antidepressants may modulate functioning of the

immune system and many of them, as well as non-

pharmacological therapies, exert immunosuppressive

and/or anti-inflammatory action [17, 18, 32]. It has

been reported that fluoxetine (FLU), a selective sero-

tonin reuptake inhibitor, evinced anti-inflammatory

action by reducing paw edema in rats [1], decreasing

synthesis of pro-inflammatory cytokine tumor necro-

sis factor (TNF) in vivo and in vitro [34] and increas-

ing production of anti-inflammatory cytokine IL-10 in

depressed patients [17].

Involvement of immunological factors in the pa-

thophysiology of depressive disorders is widely ac-

cepted. Macrophages and their products, pro-

inflammatory cytokines, are now considered to be the

main agents involved in induction of depressive states

[21, 28]. On the basis of some functional criteria,

macrophages can be divided into at least two subsets

named M1 and M2 by association with their respec-

tive subpopulations of T helper (Th) lymphocytes,

Th1 and Th2 [22, 24]. The M1 macrophages support

the pro-inflammatory immune response of the Th1

type and show high production of nitric oxide (NO)

upon stimulation with a lipopolysaccharide (LPS).

Such an immune response is considered to be in-

volved in the pathomechanisms of depressive disor-

ders [21]. The M2 subset reinforces the humoral-

promoting Th2 type of immune response, showing

low NO production and high activity of arginase

(ARG). M1/Th1 and M2/Th2 cells promote their own

development and differentiation, as well as respective

immune responses. Thus, reduction of excessive pro-

inflammatory activity of macrophages (M1 type)

might be an important part of successful antidepres-

sive treatment.

Although macrophages play an important role in

the pathogenesis of depressive illness, studies on their

activity after antidepressive treatments are rare and

limited to a few papers. Recently, we have found that

a non-pharmacological antidepressive treatment, elec-

troconvulsive shock (ECS), modulates the immune

function of macrophages [33]. Furthermore, single

and prolonged administration of FLU has been shown

to decrease the cytotoxic activity of splenic macro-

phages [8]. The drug was also able to modulate the

phagocytic activity of macrophages derived from

stressed animals, while it did not affect the activity of

macrophages from non-stressed individuals [13]. In-

terestingly, FST, which is widely used for assessment

of the antidepressant activity of drugs in rodents and

is regarded as an animal model of depressive state

[27], may produce immunological alterations similar

to those observed in depressed patients, and some of

these alterations can be reversed by antidepressive

treatment [11].

In light of the lack of papers concerning effects of

antidepressants on macrophage activity and the data

suggesting that the combination of AMA and FLU in-

duces a more potent antidepressive effect [29], the

current study aimed to investigate whether co-

administration of these drugs to the FST-subjected

rats may influence parameters of macrophages’ activ-

ity related to their immunomodulatory function.

Materials and Methods

Animals

Male Wistar rats (230–250 g), obtained from a li-

censed animal breeder (T. Górzkowska, Warszawa,

Poland), were kept under standard animal house con-

ditions (at a room temperature of 23°C, on a 12/12 h

light/dark cycle, the light on at 07:00), with food and

water ad libitum. The rats were randomly divided into

five groups (six rats in each) and were acclimatized

for at least one week before the experiments. Experi-

ments were performed in accordance with the Euro-

1070 ��

pean Community Council Directive of 24 November

1986 (86/609 EEC). All of the experimental protocols

were approved by the Local Bioethics Commission

(for Animal Experiments) at the Institute of Pharma-

cology, Polish Academy of Sciences in Kraków.

Drug administration and FST

The animals were individually subjected to FST as

described in detail elsewhere [16]. FLU (hydrochlo-

ride; Farmacom, Kraków, Poland) and AMA (hydro-

chloride; Sigma-Aldrich, St. Louis, USA) were dis-

solved in distilled water and were administered at the

dose of 10 mg/kg. Either of the drugs or distilled wa-

ter (vehicle) were injected intraperitoneally (ip) three

times, at 24, 5 and 1 h before the test. FLU was also

co-injected with AMA in the doses and at the times

stated above. Control rats were injected with the vehi-

cle and were not subjected to the FST. These doses of

AMA or FLU given alone did not change the immo-

bility time of rats in the FST. However, combined ad-

ministration of FLU (10 mg/kg) and AMA (10 mg/kg)

significantly reduced the immobility time, indicating

an antidepressant-like effect [29]. The rats were sacri-

ficed by decapitation 2 h after the last vehicle/drug(s)

injection and 1 h after the FST.

Preparation of peritoneal macrophages

Peritoneal macrophages were prepared as described

previously [33]. The peritoneal cavity was washed out

with 6 ml ice-cold phosphate buffered saline (PBS;

Biomed, Lublin, Poland). The obtained cell suspen-

sions were centrifuged at 300 × g for 3 min. The cells

were then counted using Türk’s stain solution in

Bürker’s chamber, and resuspended in the culture me-

dium at a concentration of 1 × 106 cells/ml. The cul-

ture medium consisted of RPMI 1640 (Biomed,

Lublin, Poland), supplemented with 10% heat-

inactivated fetal bovine serum (Biochrom Seromed,

Berlin, Germany), 50 U/ml penicillin, 50 μg/ml strep-

tomycin, 2 mM L-glutamine, 8 mM HEPES and 50

μM 2-mercaptoethanol (all from Sigma-Aldrich, St.

Louis, USA). The cells were placed on 96-well, flat-

bottomed culture plates (TPP, Trasadingen, Switzer-

land) in a volume of 100 μl per well and cultured un-

der standard conditions (37°C; 5% CO2; 95% humid-

ity) for 1 h. The viability of cells always exceeded

95%, as assessed by a standard trypan blue dye

method. The whole peritoneal cell population was

used for assays. Because the majority of these cells

are macrophages [12, 23] the cells will be referred to

as peritoneal macrophages. Regardless of treatment, a

similar number of cells were obtained from each rat

(about 17×106 cells per rat).

Resazurin reduction assay

Assessment of the metabolic activity of macrophages

was carried out using the resazurin assay as described

previously [33]. In brief, after a 1-hour preincubation

period, 10 μl resazurin (Sigma-Aldrich, St. Louis,

USA) solution (0.44 mM in PBS) was added to each

well and then the plates were incubated for 1 h under

standard conditions. Afterwards, absorbance was

measured at 570 nm using a Multiskan Spectrum mi-

croplate spectrophotometer (Thermo Labsystems,

Helsinki, Finland) under the control of Multiskan

Spectrum Software v. 1.1 (Thermo Labsystems, Hel-

sinki, Finland), run on a PC-compatible machine. The

data are expressed in absorbance units as the optical

density (OD570).

Nitrotetrazolium blue chloride (NBT) reduction

test

The capacity for the spontaneous and induced synthe-

sis of superoxide anion (O2–) was assessed by a NBT

reduction test according to the method described pre-

viously [33]. In brief, after preincubation (for 1 h) of

macrophages under standard conditions, some cul-

tures were stimulated with 2 μg/ml (final concentra-

tion) phorbol 12-myristate 13-acetate (PMA; Sigma-

Aldrich, St. Louis, USA), dissolved in dimethyl sul-

foxide (DMSO; POCh, Gliwice, Poland). Unstimu-

lated cultures were supplemented with 10 μl PBS. Im-

mediately after stimulation with PMA, 10 μl per well

1% aqueous NBT (Sigma-Aldrich, St. Louis, USA)

solution was added, and the incubation was continued

for 2 h. Afterwards, the supernatants were gently re-

moved and the wells were washed three times with

100% methanol. The accumulated formazan was dis-

solved by adding 120 μl 2 M potassium hydroxide

(POCh, Gliwice, Poland) per well and 140 μl DMSO

per well, and the mixtures were vigorously mixed

with a multipipette. Absorbance was measured at 630

nm with the Multiskan Spectrum apparatus. The data

are expressed in absorbance units as OD630.

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Co-administration of fluoxetine and amantadine affects macrophages��

Crystal violet staining

Adhered macrophages were stained with crystal violet

as described by Roman et al. [33]. Briefly, after

macrophage preincubation (for 1 h) under standard

conditions, 100 μl 2% formaldehyde (POCh, Gliwice,

Poland) were added to each well (yielding a 1% final

concentration) and the cells were fixed for 2 h at

a room temperature. Afterwards, supernatants were

removed and the wells were washed with 50 μl

deionized water. Cells were stained with 50 μl 0.5%

crystal violet (Fluka, Basel, Switzerland) and dis-

solved in 20% methanol (POCh, Gliwice, Poland) for

5 min. The plates were then washed three times with

deionized water and absorbed dye was extracted with

100 μl 100% methanol per well. Absorbance was im-

mediately measured at 570 nm with the Multiskan

Spectrum spectrophotometer. The data are expressed

in absorbance units as OD570.

NO synthesis assay

The basal and lipopolysaccharide (LPS)-induced NO

synthesis in macrophages was assessed as accumula-

tion of nitrites in a culture medium during a 24-h in-

cubation period using Griess’s reaction as described

previously [33]. Briefly, the macrophages were stimu-

lated with LPS (E. coli, serotype 0111:B4; Sigma-

Aldrich, St. Louis, USA) at a final concentration of

1 μg/ml. After 24 h of incubation, 50 μl of super-

natants were mixed with 30 μl 1% sulfanilamide

(Sigma-Aldrich, St. Louis, USA) and dissolved in 5%

phosphoric acid (POCh, Gliwice, Poland) and 30 μl

0.1% N-(1-Naphthyl)ethylenediamine dihydrochloride

(Sigma-Aldrich, St. Louis, USA), dissolved in water.

Absorbance was measured at 540 nm with the Multis-

kan Spectrum apparatus. The data are expressed in ab-

sorbance units as OD540.

Measurement of ARG activity

ARG activity was evaluated after a 24-hour incuba-

tion of macrophages, which were non-stimulated or

stimulated with LPS (E. coli, serotype 0111:B4) at

a final concentration of 1 μg/ml, as described previ-

ously [33]. Briefly, the previously washed macro-

phages (with 50 μl PBS) were lysed with 50 μl per

well of a mixture containing 0.1% Triton X-100 (MP

Biomedicals, Illkirch, France), 5 μg/ml pepstatin, 5 μg/ml

aprotinin and 5 μg/ml antipain (all AppliChem GmbH,

Darmstadt, Germany) as protease inhibitors, and the

plates were shaken for 30 min at a room temperature.

Afterwards, 50 μl per well 10 mM manganese chlo-

ride tetrahydrate (MP Biomedicals, Illkirch, France),

dissolved in 50 mM Tris-HCl (ICN Pharmaceuticals,

Irvine, USA), pH 7.5, were added and the enzyme

was activated by shaking for 10 min at 55°C. Then,

100 μl 0.5 M arginine (Fluka, Basel, Switzerland)

were added, and hydrolysis was carried out at 37°C

for 1 h. Triplicates were collected and 25 μl of the

mixture were transferred to Eppendorf tubes. The re-

action was terminated by adding of 400 μl of an acid

mixture containing sulfuric acid (POCh, Gliwice, Po-

land), phosphoric acid and deionized water (mixed in

a ratio of 1:3:7). The urea was quantified by adding

25 μl 9% 1-phenyl-1,2-propanedione-2-oxime (Sigma-

Aldrich, St. Louis, USA), dissolved in 100% ethanol

(POCh, Gliwice, Poland), and by heating at 100°C for

45 min. After cooling, 200 μl of the mixture were

transferred to 96-well flat-bottomed plates and absor-

bance was measured at 540 nm with the Multiskan

Spectrum apparatus. The data are expressed in absor-

bance units as OD540.

Statistical analysis

For a statistical analysis of the results, Statistica 8.0

software (Statsoft, Tulsa, USA), run on a PC-compati-

ble computer, was used. The data were evaluated by

a two-way analysis of variance (ANOVA) (vehicle or

AMA × vehicle or FLU administration) with vehicle-

injected and not subjected to the FST group of rats as

an additional control group followed by planned com-

parisons. Three types of contrasts were tested: un-

treated control vs vehicle, vehicle vs AMA, or FLU or

joint AMA + FLU, and AMA + FLU vs AMA or FLU

alone. Normality of the distribution of variables and the

homogeneity of variances were checked by Shapiro-

Wilk’s and Levene’s tests, respectively. All of the assays

were conducted in sextuples. The data are given as the

means of six rats ± SEM. The p values lower than 0.05

were regarded as statistically significant.

Results

Metabolic activity of macrophages

The basal metabolic activity of the cells was assessed

with the resazurin reduction assay. The mean absor-

1072 ��

bance ± SEM of the measurement was 0.580 ± 0.008.

There were no significant differences between groups

in this parameter of cell activity (data not shown).

Synthesis of O2–

Synthesis of O2– was assessed using the NBT reduc-

tion test both in basal conditions and after stimulation

with PMA. The mean absorbance ± SEM of basal and

PMA-induced reduction of NBT were 0.141 ± 0.007

and 0.380 ± 0.020, respectively. No significant differ-

ences between groups were observed (data not

shown).

Cellular adherence

The ability of macrophages to adhere to plastic was

assessed by crystal violet staining method. There was

no difference between FST-subjected control and

non-stressed control rats (Fig. 1). In groups treated

with FLU (alone and in combination with AMA)

a statistically significant decrease of adherence was

observed (main effect of FLU: F(1,25) = 40.13;

p < 0.001). Treatment with FLU alone resulted in

a highly significant decrease of macrophages’ adherence

in comparison to FST-treated control group (p < 0.001).

A similar effect was observed in the group co-injected

with AMA + FLU (p < 0.001). Administration of

AMA alone did not alter this parameter of cellular ac-

tivity. Combined administration of AMA + FLU re-

sulted in a statistically highly significant (p < 0.001)

decrease of cellular adherence in comparison to AMA

alone treatment (Fig. 1).

Synthesis of NO

Rate of NO synthesis was analyzed in basal condi-

tions and after stimulation of the cells with LPS.

There were no differences between FST-subjected

control and non-stressed control rats in both spontane-

ous and LPS-induced synthesis of NO (Fig. 2). FLU

administered alone or in combination with AMA in-

duced statistically significant changes in spontaneous

and LPS-induced NO synthesis (main effect of FLU:

F(1,25) = 12.59, p < 0.01 and F(1,25) = 7.89, p < 0.01,

respectively). Analysis of individual differences be-

tween groups showed that treatment with FLU alone

caused a statistically significant decrease in basal NO

release in comparison to FST-treated control group

(p < 0.05). Opposite changes were induced by AMA.

The drug, given individually, increased the LPS-

induced synthesis of NO as compared with FST-

treated control group (p < 0.05). In rats co-injected

with AMA + FLU, the basal and LPS-induced synthe-

sis of NO were statistically lower (both p < 0.01) in

comparison to the AMA alone treated group (Fig. 2).

ARG activity

Neither FST nor drug treatment changed the basal

ARG activity (Fig. 3). Also, no difference in LPS-

induced ARG activity between FST-subjected con-

trols and non-stressed control rats was observed. In

macrophages derived from rats treated with AMA

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alone or in combination with FLU, a statistically sig-

nificant change in LPS-induced ARG activity was

noted (main effect of AMA: F(1,25) = 29.84; p <

0.001). In the mentioned groups this parameter was

significantly higher as compared with FST-treated con-

trols (p < 0.001). Though FLU alone slightly increased

the LPS-induced ARG activity, the effect was negligi-

ble and the ARG activity in this group was signifi-

cantly lower (p < 0.01) than in rats treated concomi-

tantly with AMA and FLU (Fig. 3).

ARG activity / NO synthesis ratio

The activities of ARG and inductible nitric oxide syn-

thase (iNOS) reflect the functional polarization of

macrophages. Therefore, we investigated whether

AMA and FLU administered separately or jointly in-

fluenced the relationship between those enzymes un-

der basal conditions and after stimulation of cells with

LPS. The ratio of ARG activity to NO synthesis

(ARG/NO) was calculated as shown in Table 1. In

non-stimulated cell cultures, this parameter did not

differ among groups and its values ranged from 0.90 ±

0.226 in non-stressed control rats to 2.58 ± 0.929 in

the group co-injected with AMA + FLU. After stimu-

lation with LPS, the ratio was increased, especially in

the group co-injected with AMA + FLU and the main

effect of FLU reached statistical significance at

F(1,25) = 4.34; p < 0.05. In rats treated with the com-

bination of AMA and FLU, an elevation of the

ARG/NO ratio was significantly higher as compared

with animals injected with vehicle and subjected to

FST (p < 0.01), as well as with groups receiving AMA

or FLU separately (both p < 0.05) (Tab. 1).

Discussion

The main finding of the present study is that the co-

administration of AMA and FLU at doses ineffective

for antidepressant-like activity in FST when adminis-

tered alone, caused an increase of ARG/NO ratio in

macrophages stimulated with LPS. This phenomenon

indicates a redirection of immune response toward

anti-inflammatory processes, as one can anticipate in

the case of effective antidepressive treatment. Other

results of the present study correlate and also support

the notion about anti-inflammatory response. Com-

bined treatment with AMA and FLU decreased cellu-

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Group n Non-stimulated cells Cells stimulated with LPS

ARG activity[OD540]

Nitrite concentration[OD540]

ARG activity/NOsynthesis ratio

ARG activity[OD540]

Nitrite concentration[OD540]

ARG activity/NOsynthesis ratio

Control 6 0.331 ± 0.091 0.394 ± 0.056 0.90 ± 0.226 0.996 ± 0.229 0.480 ± 0.048 2.00 ± 0.334

FST 6 0.613 ± 0.315 0.488 ± 0.071 1.40 ± 0.624 1.324 ± 0.262 0.561 ± 0.087 3.37 ± 1.518

AMA 6 0.633 ± 0.114 0.560 ± 0.030 1.11 ± 0.166 2.758 ± 0.307 0.748 ± 0.081 3.75 ± 0.415 #

FLU 6 0.470 ± 0.129 0.340 ± 0.046 1.47 ± 0.526 1.907 ± 0.127 0.498 ± 0.025 3.90 ± 0.381 #

AMA + FLU 6 0.866 ± 0.272 0.352 ± 0.037 2.58 ± 0.929 2.988 ± 0.182 0.474 ± 0.031 6.40 ± 0.414 **

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lar adherence, a property of pro-inflammatory macro-

phages. Concurrently, it did not alter the basal meta-

bolic activity of the cells (reduction of resazurin) or

the synthesis of superoxide anion, another parameter

of cellular activity, not engaged directly in immuno-

modulatory functions of macrophages.

Involvement of macrophages in the pathomecha-

nisms of depressive disorders was raised for the first

time almost two decades ago [35]. Since then, the

overactivity of pro-inflammatory M1 type macro-

phages has been considered to be an important

mechanism of induction of depressive states [21, 28].

Macrophages are an extremely heterogeneous popula-

tion among the cells of the immune system [20]. Re-

garding functional properties, two main subsets of

macrophages can be distinguished. Mirroring Th1/Th2

nomenclature, these subpopulations are frequently re-

ferred to as M1 (pro-inflammatory) and M2 (anti-

inflammatory), respectively [19]. This polarization is

reflected, among others, by the balance between two

main enzymes of arginine metabolism, iNOS and

ARG [22, 24]. In response to some stimuli, e.g., LPS,

macrophages undergo activation and disclose diverse

functional properties. M1 macrophages express iNOS

and produce NO and pro-inflammatory cytokines of

the Th1 type; IL-1, IL-12 and TNF [19]. M2 macro-

phages express mainly ARG and metabolize arginine

to ornithine and urea. Furthemore, they release

cytokines of the Th2 type, including IL-4, IL-10, and the

transforming growth factor �, displaying anti-inflam-

matory and/or immunosuppressive action. In addition,

functional properties of macrophages are not static and can

undergo changes in response to environmental stimuli [19,

36]. Macrophages are also able to direct the immune re-

sponse toward M1/Th1 or M2/Th2 type [24].

Regarding participation of macrophages in the

pathomechanisms of depressive disorders and action

of antidepressants, it can be assumed that efficient an-

tidepressive treatment may influence the inflamma-

tory status of macrophages. Indeed, numerous papers

describe an anti-inflammatory and/or immunosup-

pressive action of both antidepressants and non-

pharmacological antidepressive treatments, e.g., elec-

troconvulsive shock [17, 18, 32, 33]. Results of the

current study support and considerably broaden such

an assumption. We found that although AMA and

FLU administered separately can influence some

properties of macrophages, only combined treatment

with these drugs was able to modify an important im-

munoregulatory feature of macrophages, i.e., the bal-

ance between two main pathways of arginine metabo-

lism as reflected by ARG/NO ratio.

Interestingly, joint administration of AMA and

FLU at the doses used in the current study revealed

antidepressive-like effects in the FST, whereas treat-

ments with these drugs separately were ineffective

[29]. In addition, a similar effect of combined treat-

ment with AMA and a tricyclic antidepressant drug,

imipramine (at doses ineffective when administered

separately), was reported by Kubera et al. [16]. Thus,

increase of ARG/NO ratio in macrophages in rats ad-

ministered jointly with AMA and FLU observed in

the present study seems to be closely related to the an-

tidepressive effect observed in the FST.

Although AMA is an NMDA receptor antagonist, it

also interferes with monoaminergic systems affecting

dopaminergic, noradrenergic and serotonergic compo-

nents [15]. This fact may explain some contradiction

existing in data concerning AMA effects on the im-

mune system, as some data suggest the M1/Th1 type

of immune response induced by the drug [37] while

others indicate the immune response of the M2/Th2

type [16]. Wandinger et al. [37] reported a stimulatory

effect of AMA on ex vivo production of IL-2 con-

comitantly with an increase of interferon gamma in

patients with idiopathic Parkinson’s disease, suggest-

ing support of the M1/Th1 type of immune response.

Our finding of increased NO synthesis in rats treated

with AMA alone is in line with Wandinger’s observa-

tion. On the other hand, the present study showed that

administration of AMA alone also induced an in-

crease of ARG activity, thus suggesting a supportive

role for the M2/Th2 immune response. Since ARG ac-

tivity is regulated (among others) by adrenergic recep-

tors [9], one can speculate that the aforementioned effect

of AMA is linked to the noradrenergic component of its

pharmacological action.

In the present study we found that administration

of FLU alone resulted in a significant decrease in NO

synthesis by macrophages together with a substantial,

but statistically insignificant, rise in ARG activity,

and also potent suppression of cellular adherence.

These changes are indicative of a decrease of inflam-

matory properties of macrophages and a shift toward

M2 type of immune response. Moreover, they

corroborate the observations of other authors that re-

ported modulatory effects of FLU on various immune

factors and its anti-inflammatory properties [8, 17, 38].

In light of our data and the literature cited above,

we propose that AMA alone stimulates both M1/Th1

�� 1075


and M2/Th2 type of immune response as reflected by

increased NO synthesis and ARG activity in macro-

phages. Administration of FLU alone mainly sup-

presses M1/Th1 type of immunoreactivity, as indi-

cated by diminished synthesis of NO by macro-

phages, decreased cellular adherence and only a slight

increase in the ARG activity. None of these treat-

ments, however, was sufficient to increase ARG/NO

ratio. Only a concomitant administration of the drugs,

exerting a synergistic effect, was efficient enough to

increase the ARG/NO ratio and to direct the immune

response toward M2/Th2 type.

It can be expected that stress related to FST or in-

jection with vehicle may alter the metabolic activity

of cells as well as their immune function, as such phe-

nomenon have been reported [11, 13, 16]. In addition,

in the current study the macrophages were collected

two hours after the last treatment and conceivable

harmful direct effects of the drugs on the cells cannot

be excluded. Thus, we assessed some parameters of

cellular activity, not directly related to the above men-

tioned immunomodulatory action, i.e., the ability to

reduce resazurin or NBT. Resazurin is a compound

widely used for the assessment of various parameters

of the metabolic activity of cells, including cell viabil-

ity [6], cytotoxicity [25], proliferation [4] and mito-

chondrial function [2, 3]. Resazurin is reduced by the

mitochondrial respiratory chain and its redox poten-

tial allows the respiratory chain to function in a condi-

tion close to completion. Thus the reduction of resa-

zurin may reflect overall metabolic activity of the

cells related to respiratory processes. However, we

found that none of the treatments applied in the pres-

ent study altered the overall metabolic activity of

macrophages, as revealed by unaltered reduction of

resazurin. Also, the FST-related stress did not affect

immune function of macrophages. This discrepancy

may be related to the type of stress applied and the

cell population studied [11, 13, 16].

Another parameter assessed in the present study

was the ability of macrophages to reduce NBT. This

tetrazolium salt is widely used for detection and quan-

tification of superoxide anion synthesized in the pro-

cess called “oxidative burst” [7, 10]. In response to

some stimuli, e.g., LPS or PMA, formation of super-

oxide anion substantially increases and participates in

cellular cytotoxicity. This process in one of the fea-

tures of M1 type macrophages [19]. It has been shown

that AMA stimulates oxidative burst in guinea pig

neutrophils in vitro [5]. Again, we did not find any ef-

fect of applied treatments on the basal and PMA-

induced synthesis of superoxide anion. The discrep-

ancy between our data and results reported by other

authors may be related to a different cell population

and to distinct experimental conditions used [14, 26].

Thus, none of the treatments applied in the present

study affected basal metabolic properties of the cells

and intracellular host defense as reflected by the non-

altered reduction of resazurin and NBT.

In conclusion, our results show that co-administration

of AMA and FLU at doses ineffective as antidepres-

sants (10 mg/kg) when used separately, resulted in an

increase of ARG/NO ratio in macrophages, and thus

suggest a redirection of immune response toward the

anti-inflammatory process. This capability may be

partially responsible for the antidepressive properties

of the combined treatment of AMA and FLU.

Acknowledgments:

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