Effects of co-administration of fluoxetine andrisperidone on properties of peritoneal and pleuralmacrophages in rats subjected to the forcedswimming test

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Effects of co-administration of fluoxetine and risperidone on properties of peritoneal and pleural macrophages in rats subjected to the forced

swimming test

Adam Roman¹, Justyna Kuœmierczyk¹, Ewa Klimek¹, Zofia Rogó¿², Irena Nalepa¹

1Department of Brain Biochemistry,²Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

Correspondence: Adam Roman, e-mail: roman@if-pan.krakow.pl

Abstract:

Background: Literature data show that administration of atypical antipsychotic drug, risperidone (RIS), enhances antidepressive action of fluoxetine (FLU). As antidepressive treatments also regulate immune functions, we examined whether combined administration of FLU and RIS to rats subsequently subjected to a forced swimming test (FST) modifies parameters of macrophage activity that are directly related to their immunomodulatory functions, i.e., arginase (ARG) activity and nitric oxide (NO) synthesis.

Methods: Antidepressive action of the drugs was assessed with FST. Peritoneal and pleural cells were eluted and selected parame- ters of immunoreactivity were assessed colorimetrically.

Results: We found that the concomitant administration of FLU (10 mg/kg) and RIS (0.1 mg/kg) produced antidepressive-like effects in the FST, whereas the drugs were ineffective if administered separately. Stress related to the FST affected immune cell redistribution and changed some of the metabolic and immunomodulatory properties of macrophages. FLU administered to rats at a suboptimal dose for antidepressive action potently influenced macrophage immunomodulatory properties and redirected their activity toward anti- inflammatory M2 functional phenotype, as manifested by changes in the ARG/NO ratio. These effects resulted from a direct cellular in- fluence of the drug, as well as its action via neuroendocrine pathways, as evidenced in peritoneal and pleural cells. Addition of RIS did not augment immunomodulatory action of FLU, though the combination showed antidepressant-like activity in the FST.

Conclusions: Our results suggest that when the drugs were administered together, FLU was potent enough to redirect macrophages toward M2 activity. It is also postulated that drug-induced changes in the immune system are not so closely related to antidepressant-like effects or might be secondary to those produced in the neuroendocrine system.

Key words:

fluoxetine, risperidone, peritoneal and pleural macrophages, nitric oxide, arginase activity, forced swimming test, stress

Abbreviations: ARG – arginase, CV – crystal violet, FLU – fluoxetine, FST – forced swimming test, iNOS – inducible nitric oxide synthase, LPS – lipopolysaccharide, NBT – nitrotetrazolium blue chloride, NO – nitric oxide, PMA – phorbol 12-myristate 13-acetate, RIS – risperidone, TNF – tumor necrosis factor, WBCs – white blood cells

Introduction

Depression is a severe, multifactorial medical condition that includes abnormalities of affect and mood,

Pharmacological Reports 2012, 64, 13681380 ISSN 1734-1140

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cognition, psychomotor activity, and neurovegetative and immune functions. Serotonergic and noradrenergic neurotransmission deficiencies play an important role in depression pathology, and almost all antidepressants act on monoamine systems. However, enhanced noradrenergic or serotoninergic neurotransmission does not seem to be solely responsible for the therapeutic action of these drugs. Several non-monoamine neurotransmitter and neuromodulatory systems, as well as immuno-endocrine mechanisms, are involved in the pathomechanism of depression, and they may be regarded as potential targets of antidepressive treatment [34, 47].

Currently used antidepressant drugs are only par- tially effective, and significant attention is given to the development of more efficient pharmacotherapies.

One option is combined treatment with first-line antidepressants and other drugs with various mode of action, e.g., N-methyl-D-aspartate (NMDA) receptor antagonists [40], cyclooxygenase inhibitors [23] and atypical antipsychotics [35]. Risperidone (RIS) is an atypical antipsychotic and its use in treating depressive disorders has been reported [14, 51]. The drug influences a wide spectrum of noradrenergic, serotoninergic and dopaminergic systems, which may explain its clinical efficacy [35].

Macrophages play an important role in the immune system. They are innate immune cells with well- established roles in the primary response to patho- gens. They also coordinate the adaptive immune response, inflammation, resolution, and tissue repair [21, 52]. Regarding their role in the immune system, macrophages can be divided at least in two functional subpopulations, which are named M1 and M2 for their associations with immune responses, Th1 and Th2, respectively [20]. Their functional dichotomy is reflected by different surface marker patterns, different cytokines released and also by the balance of two main arginine metabolism enzymes: arginase (ARG) and inducible nitric oxide synthase (iNOS) [20, 22].

M1 macrophages possess pro-inflammatory activity.

They release a panel of Th1-type cytokines, including interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF), and produce huge amounts of nitric oxide (NO) downstream of iNOS induction in response to stimulation with the bacterial cell wall component lipopolysaccharide (LPS). Excessive activity of M1 macrophages and subsequent overproduction of pro- inflammatory cytokines are implicated in the induction of depression [33]. Conversely, M2 macrophages

are regulatory and tissue repairing cells. They release anti-inflammatory and regulatory cytokines (IL-4, IL-10, IL-13) and possess high ARG activity. Macro- phages are antigen-presenting cells, and they exert a profound impact on immune response direction and intensity [22]. Thus, the ARG to NO ratio mirrors of macrophage inflammatory status and an indirect re- flection of the inflammatory tendency of the immune system. Macrophages are very flexible cells that are able to continuously change their functional phenotype in response to microenvironmental stimuli [29].

It is now generally accepted that immune abnormalities play an important role in the pathogenesis of depressive disorders [18, 33], and normalization of immune functions seems to be an important component of the therapeutic actions of antidepressant drugs [12]. Antidepressant drugs may modulate immune system function, and many of them, as well as non- pharmacological therapies, exert immunosuppressive and/or anti-inflammatory action [16, 42, 43]. Fluoxet- ine (FLU), a selective serotonin reuptake inhibitor, is a first-line antidepressant drug. Its anti-inflammatory and immunosuppressive actions are well established.

FLU has been shown to increase synthesis of suppressive IL-10 by splenocytes [16], lower the number of macrophages at an inflammation site, decrease TNF levels in vivo and its synthesis in vitro [45], and attenu- ate symptoms of experimentally induced arthritis [46].

Conversely, the effect of RIS on immune function is poorly understood. Nevertheless, Sugino et al. [49] reported that RIS decreased the serum level of pro- inflammatory cytokines, TNF and IL-6, and increased the level of anti-inflammatory IL-10 in LPS-treated mice. Additionally, it has been shown that RIS decreases synthesis of NO and pro-inflammatory cytoki- nes IL-1b, IL-6 and TNF in cultured microglia [13].

The positive clinical effects observed when RIS is given with antidepressive medication [14, 51] and the immunomodulatory properties of FLU and RIS prompted us to investigate if combined treatment of FLU, a first-line antidepressant, and RIS, an adjunc- tive agent, modulates macrophage activity, especially their regulatory functions. Our previous study showed that combined treatment with FLU and amantadine, an NMDA receptor antagonist, increased the ARG/NO ratio in peritoneal macrophages and shortened immobility time in a forced swimming test (FST), which in- dicated that the drug combination had antidepressive action [38, 44]. However, as these results were obtained in peritoneal cells, which were exposed to the

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high drug concentration, in the present study, the activity of pleural macrophages was also assessed.

These cells, being beyond reach of the direct drug action, are mainly influenced by neuroendocrine regulation.

Materials and Methods

Animals

The experiments were conducted on male Wistar rats (250–300 g) obtained from Charles River Laborato- ries (Sulzfeld, Germany) and kept under standard animal housing conditions (room temperature of 23°C, 12/12 h light/dark cycle, lights on at 7:00) with food and water ad libitum. The animals were randomly di- vided into 5 groups (7 rats in each) and acclimatized for at least 1 week. Experiments were performed in accordance with the European 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 Pharmacology, Polish Academy of Sciences in Kraków.

Drug administration and FST

FLU (fluoxetine hydrochloride; Pliva, Kraków, Po- land) was dissolved in sterile water. RIS (Tocris, Bris- tol, UK) was suspended in a 1% aqueous solution of Tween 80. The drugs were administered intraperito- neally (ip) at 10 mg/kg (FLU) or 0.1 mg/kg (RIS) three times (24, 5 and 1 h before the FST). Such a schedule of drugs administration was established by Porsolt et al. [30] as the most effective for detection of antidepressive action. The doses were chosen based on a preliminary experiment and did not produce antidepressant-like effects in the FST when administered alone. The control group was injected with dis- tilled water (vehicle). The combined treatment group received FLU and RIS at the same doses and times as stated above, and RIS was injected 30 min after the administration of FLU. An additional control group was treated with vehicle and was not subjected to the FST. The animals were subjected to the FST accord- ing to procedure originally described by Porsolt et al.

[30]. In brief, on a first day, the rats were placed in

a cylinder containing water (25 ± 0.5°C, level 25 cm above the bottom of the tube) for 15 min. On the second day, the procedure was repeated, but the rats were kept in water for 5 min. Each of rats was tested in a fresh water. The total duration of immobility was measured during the second trial. 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 and pleural macrophages

Peritoneal macrophages were prepared as described previously [44]. The pleural cavity was washed out with 4 ml of ice-cold phosphate buffered saline (PBS;

Biomed, Lublin, Poland). Both peritoneal and pleural cell suspensions were centrifuged at 300 × g for 3 min and resuspended in 1 ml of culture medium. The cells were then counted with a hematology analyzer (Aba- cus Junior Vet; Diatron Messtechnik GmbH, Vienna, Austria), and cell suspension concentrations were ad- justed with culture medium to 1 × 10⁶ total white blood cells (WBC) per ml. The culture medium consisted of RPMI 1640 (Biomed, Lublin, Poland) supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories GmbH, Pasching, Austria), 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, 8 mM HEPES and 50 µM 2-mercapto- ethanol (all from Sigma-Aldrich, St. Louis, USA).

The cells were placed in 96-well, flat-bottomed culture plates (TPP, Trasadingen, Switzerland) in a vol- ume of 100 µl per well and cultured under standard conditions (37°C, 5% CO₂, 95% humidity) for 1 h.

Cell viability exceeded 90%, as assessed by propid- ium iodide (PI) staining and flow cytometry [6]. Cell suspensions with lower viability were excluded from further analysis. The whole peritoneal and pleural cell populations were used for assays.

Differential cell count and cell number

Peritoneal and pleural cell composition was analyzed with a 3-part WBC differential in a hematology analyzer (Abacus Junior Vet) set to “rat”. The analyzer works using the impedance method and differentiates WBCs into three categories: lymphocytes, granulocytes and other cells. Macrophages are counted as granulocytes. They predominate among peritoneal and pleural cell populations [11] and will be referred to as macrophages. The WBC number reflects the to-

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tal number of cells (mln) obtained from individual cavities. The hematology analyzer data were proc- essed with DiatronLab 1.72 software (Diatron Mes- stechnik GmbH, Vienna, Austria) run on a PC-compatible machine.

Assessment of metabolic activity and adherence

Cellular metabolic activity was assessed in basal conditions with the resazurin reduction test as described previously [44], but absorbance was measured at 570 nm with reference wavelength at 600 nm, using a Multiskan Spectrum microplate reader under the control of Multiskan Spectrum Software v. 1.1 (both Thermo Labsystems, Helsinki, Finland) run on a PC- compatible machine.

Cellular adherence was assessed with crystal violet (CV) staining as described in detail elsewhere [44]

and measured colorimetrically at 570 nm.

Nitrotetrazolium blue chloride (NBT) reduction test

The capacity for superoxide anion (O₂^-) synthesis was assessed with a previously described NBT reduction test [44]. This assay was performed in basal conditions and after stimulation with 2 µg/ml (final concentration) phorbol 12-myristate 13-acetate (PMA; Sigma- Aldrich, St. Louis, USA). Absorbance was measured at 630 nm.

NO assay

NO synthesis by macrophages was assessed as accu- mulation of nitrites in culture medium during a 24-h incubation period using Griess’s reaction, as described previously [44]. The measurement was made in basal conditions and in cultures stimulated with 1 µg/ml (final concentration) of LPS from E. coli, se- rotype 0111:B4 (Sigma-Aldrich, St. Louis, USA) and colorimetrically assessed at 540 nm.

ARG activity assay

ARG activity was evaluated after a 24-h incubation of macrophages, which were non-stimulated or stimu- lated with LPS (E. coli, serotype 0111:B4) at a final concentration of 1 µg/ml, as described in detail elsewhere [44]. ARG catalyzes the hydrolysis of arginine

to ornithine and urea. Urea levels were quantified with 1-phenyl-1,2-propanedione-2-oxime (Sigma- Aldrich, St. Louis, USA) and colorimetrically assessed at 540 nm.

Statistical analysis

Statistica 9.0 software (Statsoft, Tulsa, USA) run on a PC-compatible computer was used to analyze the data.

Normality of variable distribution and homogeneity of variances were checked by Shapiro-Wilk and Leve- ne’s tests, respectively. Behavioral data were evaluated by a one-way analysis of variance (ANOVA) fol- lowed by Dunnett’s test. Remaining data were analyzed in two steps. The rats treated with vehicle and subjected to the FST (VEH group) were compared to additional controls that were treated with vehicle and were not subjected to FST (control group) with Stu- dent’s t-test (or Mann-Whitney’s test when assump- tions of parametric analysis were not fulfilled).

Groups treated with the drugs or vehicle and subjected to the FST were evaluated with two-way ANOVA (FLU × RIS) with unequal n HSD (Honestly Significant Difference) Tukey’s post-hoc test, when appropriate. When ANOVA’s assumptions were not fulfilled, the Kruskal-Wallis ANOVA (by ranks) for multiple comparisons was used. All of the colorimet- ric assays were conducted either in quadruplets or sextuplets and the data are expressed in absorbance units as optical density (OD) at the respective wavelength. The data are given as the means of 5–7 rats

± standard error of the mean (SEM) or standard devia- tion (SD) when mentioned in the text. p-values lower than 0.05 were regarded as statistically significant.

Results

Antidepressant effect in the FST

Neither FLU (10 mg/kg) nor RIS (0.1 mg/kg) modi- fied immobility time in the FST when given separately. However, combined treatment with FLU and RIS significantly shortened (p < 0.05) rat immobility time in the FST when compared to the vehicle-treated group, indicating that the combination had an antidepressant-like effect (Fig. 1).

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Number of WBCs and peritoneal and pleural cell population compositions

The total number of WBCs and the peritoneal cell population composition, including macrophages and lymphocytes, are shown in Table 1. The percentage of the other cell category in peritoneal and pleural cell populations did not differ significantly across groups (data not shown).

Submitting the vehicle-treated rats to the FST increased (p < 0.01) the total number of WBCs in their peritoneal cavities when compared to the control group (i.e., injected with vehicle and not subjected to FST). The drugs administered separately had no effect, but when administered jointly, they decreased this parameter (p < 0.01 vs. VEH group) to the value of the control rats.

The FST did not change the peritoneal cell popula- tion composition (VEH vs. control group) (Tab. 1).

Administration of FLU alone increased the percent- age of macrophages (p < 0.05) and decreased that of lymphocytes (p < 0.01). Treatment with RIS did not induce any changes, but combined treatment normal- ized the percentages of macrophages and lymphocytes to the level of the VEH group.

The number of WBCs in the pleural cell population was 0.98 ± 0.39 (the mean ± SD), and there was no difference between groups (data not shown). The mean percentage of macrophages and lymphocytes were 72.39 ± 9.03 and 20.32 ± 8.85, respectively, and the values did not differ significantly among groups (data not shown).

Metabolic activity and adherence

There was no statistically significant difference between the control and VEH groups in the metabolic

VEH FLU RIS FLU + RIS

0 60 120 180 240 300

Groups

Immobilitytime[s]±SEM

# Fig. 1. Immobility time of the rats in the FST. The rats were administered with vehicle (VEH), fluoxetine (FLU) and/or risperidone (RIS), and subjected to FST; # p < 0.05 vs. vehicle-treated group (VEH)

Tab. 1. Total number of WBC and composition of peritoneal cell population in rats administered with vehicle (VEH), fluoxetine (FLU) and/or risperidone (RIS) and/or subjected to FST

Group n WBC (mln/rat) ± SEM Macrophages (%) ± SEM Lymphocytes (%) ± SEM

Control 6 2.59 ± 0.33 72.10 ± 3.70 21.01 ± 4.68

VEH 7 9.34 ± 1.79 ** 64.31 ± 3.97 31.55 ± 4.35

FLU 6 4.09 ± 0.44 81.65 ± 2.69^## 10.20 ± 2.14^#

RIS 7 5.80 ± 1.14 72.64 ± 2.63 21.08 ± 1.93

FLU + RIS 5 2.49 ± 0.69^## 70.63 ± 4.50 23.14 ± 5.67

** p < 0.01 vs. control group (injected with vehicle and not subjected to FST); ## p < 0.01 and # p < 0.05 vs. vehicle-treated and FST-subjected group (VEH)

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activity of peritoneal cells (Fig. 2). In groups treated with FLU (alone or in combination with RIS), a decrease of resazurin reduction ability was observed (main effect of FLU: F(1,21) = 17.34, p < 0.001). Analysis of individual differences between groups showed that basal metabolic activity was further decreased by combined treatment with FLU and RIS, but the result was only statistically significant (p < 0.01) in comparison to the RIS-treated group (significance not marked in Fig. 2).

The basal metabolic activity of pleural cells was 0.073

± 0.054 (the mean ± SD), and there were no statistically significant differences between groups (data not shown).

The ability of macrophages to adhere to plastic was assessed by CV staining. Administration of vehicle and exposure to the FST decreased peritoneal cell adherence (p < 0.01) in comparison to control rats injected with vehicle and not subjected to the FST (Fig. 2). The groups treated with RIS showed increased adherence (p < 0.01) in comparison to the VEH group and decreased adherence in the group coadministered with FLU and RIS, but the result reached statistical significance (p < 0.001) only in comparison to the RIS-treated group (significance not marked in Fig. 2), so it did not differ significantly from that observed in the VEH group.

Pleural cell adherence was 1.175 ± 0.467, and no statistically significant differences between groups were observed (data not shown).

O₂^–synthesis

Synthesis of O₂was assessed using the NBT reduction test both in basal conditions and after stimulation

with PMA. The mean absorbances ± SD of basal and PMA-induced reduction of NBT by peritoneal cells were 0.177 ± 0.049 and 0.282 ± 0.101, respectively.

In pleural cells these values were: 0.159 ± 0.061 and 0.258 ± 0.130, respectively. No significant differences between groups were observed (data not shown).

NO release

NO synthesis was assessed both in basal conditions and in cultures stimulated with LPS, and the results are shown in Figure 3. LPS-induced peritoneal cells had higher NO synthesis (p < 0.05) in VEH group than that in controls (Fig. 3A). There was no difference between these groups in basal NO synthesis. In rats receiving FLU with or without RIS, both basal and LPS-induced NO release were significantly decreased [main effect of FLU: H(1, n = 25) = 12.89, p < 0.001 and F (1, 21) = 36.10, p < 0.001, respectively]. RIS administration did not alter basal or LPS-induced NO release by peritoneal cells.

In pleural cells, both basal and LPS-induced NO synthesis were increased in the VEH group (p < 0.05) in comparison to control rats (Fig. 3B). There were no statistically significant differences between groups treated with vehicle and/or the drugs and subjected to the FST.

ARG activity

ARG activity was measured in basal conditions and in LPS-stimulated cells. Basal ARG activity in peritoneal cells obtained from VEH group rats did not differ from that observed in controls (Fig. 4A). An increase

Control VEH FLU RIS FLU + RIS

0.00 0.04 0.08 0.12 0.16 0.20

0.0 0.3 0.6 0.9 1.2 1.5 Metabolic activity Adherence

Group

Metabolicactivity[OD570-600]±SEM Adherence[OD570]±SEM

##

***

Fig. 2. Metabolic activity and adher- ence of peritoneal cells as assessed by reduction of resazurin and CV staining, respectively. The control group was injected with vehicle and was not subjected to the FST. Remaining groups were treated with vehicle and/or drugs and subjected to the FST.

*** p < 0.001 vs. control group; ## p <

0.01 vs. VEH. For metabolic activity, the main effect of FLU was significant at F (1, 21) = 17.34, p < 0.001, as re- vealed by two-way ANOVA

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SynthesisofNO[OD540]±SEM

Control VEH FLU RIS FLU + RIS

0 0.1 0.2 0.3 0.4

Basal LPS-induced A

*

0 0.1 0.2 0.3 0.4

Group

B

Fig. 3. Spontaneous (open bars) and LPS- induced (grey bars) NO release by peritoneal (A) and pleural (B) cells. Description of the groups is as in Figure 2, * p < 0.05 vs. control group. For basal and LPS-induced NO release by peritoneal cells (A), the main effect of FLU was significant at H(1, n = 25) = 12.89, p <

0.001 and F (1, 21) = 36.10, p < 0.001, respectively

ARGactivity[OD540]±SEM

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Group

B

*

0.0 0.5 1.0 1.5 2.0 2.5

Basal LPS-induced A

** Fig. 4. Spontaneous (open bars) and LPS-

induced (grey bars) ARG activity in peritoneal (A) and pleural (B) cells. Description of the groups is as in Figure 2, * p < 0.05, ** p < 0.01 vs.control group. For basal ARG activity in peri- toneal cells (A) and LPS-induced ARG activity in the pleural ones (B) the main effects of FLU were significant at F (1, 21) = 14.80, p < 0.001 and H(1, n = 21) = 11.16, p < 0.001, respectively

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in this parameter was observed in groups treated with FLU alone or in combination with RIS [main effect of FLU: F (1, 21) = 14.80, p < 0.001]. In cultures stimulated with LPS, a statistically significant (p < 0.01) difference was observed only between VEH and control groups. In the former group, this parameter was higher than in controls (Fig. 4A). There were no differences between groups treated with vehicle and/or drugs and subjected to the FST.

In pleural cells, ARG activity was highly variable within groups, especially in LPS-stimulated cultures (Fig. 4B). Basal ARG activity did not change regard- less of treatment. In cultures stimulated with LPS, an increase in ARG activity (p < 0.05) was observed in the VEH group compared with the control. This parameter was also significantly higher in groups treated with FLU alone or in combination with RIS [main effect of FLU: H(1, n = 21) = 11.16, p < 0.001].

ARG/NO ratio

We investigated whether FLU and RIS administered separately or concomitantly influenced the main path-

ways of arginine metabolism, which mark the functional polarization of macrophages both in basal conditions and after stimulation with LPS. The ARG/NO ratio was calculated by the formula: ARG activity (OD450)/NO release (OD540), and the results are shown in Figure 5. The basal condition ratio in peritoneal cells obtained from VEH group rats was comparable to that of the control group. In cultures stimulated with LPS, the ratio was higher in the VEH group (p < 0.05) than in controls (Fig. 5A). Treatment with FLU alone or in combination with RIS increased the ratio both in basal conditions and in LPS- stimulated peritoneal cells [main effect of FLU: H(1, n = 25) = 18.00; p < 0.001 and F (1, 21) = 8.04, p <

0.01, respectively]. Treatment with RIS alone had no effect.

In pleural cells, the ARG/NO ratio was highly variable. The ratio did not differ significantly across groups in basal conditions (Fig. 5B). In the cells stimulated with LPS, an increased ratio was noted in groups treated with FLU alone or with RIS [main effect of FLU: F (1, 17) = 10.23, p < 0.01]. The effect of RIS was statistically insignificant.

ARG/NOratio±SEM

0 3 6 9 12

Basal LPS-induced A

0 3 6 9 12 15

Group

B

Fig. 5. ARG/NO ratio in basal conditions (open bars) and in LPS-stimulated (grey bars) perito- neal (A) and pleural (B) cells. The ARG/NO ra- tio was calculated the formula: ARG activity (OD450)/NO release (OD540). Description of the groups is as in Figure 2, * p < 0.05 vs. con- trol group. For basal and LPS-induced ARG/NO ratio in peritoneal cells (A) the main effect of FLU was significant at H(1, n = 25) = 18.00, p <

0.001 and F (1, 21) = 8.04, p < 0.01, respectively. For the ratio in LPS-stimulated pleural cells (B) the main effect of FLU was significant at F (1, 17) = 10.23, p < 0.01

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Discussion

In the current study, we aimed to assess properties of macrophages derived from rats treated with FLU and RIS separately or concomitantly in the context of antidepressive-like drug action, which was assessed in the FST. We examined peritoneal and pleural cells separately to differentiate potential effects of direct drug action (in the case of peritoneal cells) from indi- rect effects, either via neuroendocrine pathways or their presence in blood circulation (effects in pleural cells). Although peritoneal and pleural cavities are independent anatomical and functional compartments [25], macrophages obtained from these sites have similar functional and phenotypic properties [11]. We examined macrophages from animals treated with vehicle and/or drugs and subjected to the FST, which is a stressful procedure that increases serum corticosterone [38, 41]. Therefore, an additional control group, treated with vehicle and not subjected to the FST, was included to assess effects of FST-related stress on macrophage properties.

We found that FLU and RIS administered separately did not alter immobility time in the FST but ad- ministering both drugs together shortened it, thereby revealing antidepressant-like activity. These results suggest that an addition of RIS to the treatment poten- tiates the antidepressive-like action of FLU. A similar efficiency of combination therapy was recently reported in mice [37]. Moreover, a synergistic effect of FLU and other drugs was observed in other animal studies [38, 39], and combined treatment with various selective serotonin reuptake inhibitors and RIS have been successfully tested in clinics [14, 51].

The participation of immunological factors in the pathophysiology of depressive disorders is well established [18, 33]. Special attention has been focused on macrophages because of their excessive activity and ability to release pro-inflammatory mediators (cytokines, prostaglandins) that are involved in the induction of depressive states. The results of the present study showed that the antidepressant FLU exerted potent modulatory effects on macrophages when administered at a suboptimal dose for antidepressive action.

Specifically, it enhanced the ARG/NO ratio both in basal and LPS-stimulated conditions. These effects cannot be attributed simply to direct drug action on the cells because increased ARG activity and ARG/NO ratio in LPS-stimulated cells were also observed in

pleural macrophages, which were collected from an anatomical compartment distinct from the peritoneal cavity where the drugs were delivered. At the same time, we did not observe any effect on the ability of macrophages to reduce NBT. This compound is com- monly used for detection and quantification of superoxide anion synthesized by phagocytes in the “oxida- tive burst” process [2], which is one of main mechanisms of bactericidal activity. Thus, the reduction of NBT reflects, to some extent, the activity of macrophages in innate immunity [3]. Taken together, the observed phenomena suggest that administration of FLU can direct macrophages toward anti-inflammatory activity but preserves their capability as effector cells of innate immunity.

FLU exerts widespread and complicated effects on the immune system. It may act centrally [26] or directly on immunocytes [45, 46], by elevating extracellular serotonin levels [27], in addition to serotonin- independent pathways [46]. FLU has a potent inhibitory influence on inflammatory processes. If adminis- tered in vivo, it increases synthesis of suppressive IL-10 by splenocytes [16] and decreases synthesis of pro-inflammatory cytokines IL-6, TNF and IFN-g [45, 46]. If added to culture media, FLU decreases the release of NO and prostaglandin by human synovio- cytes [50] and NO synthesis and iNOS mRNA expres- sion in microglial cells [17].

In the present study, we showed that FLU administered in a dose ineffective for antidepressive action in rats suppressed NO synthesis and increased ARG activity, which resulted in a higher ARG/NO ratio in peritoneal macrophages. These effects, at least in part, may be the result of direct drug action on the cells, especially shortly after injection into the peritoneal cavity. Moreover, our observed increase of ARG activity and the ARG/NO ratio in LPS-stimulated pleural cells may be related to the central, indirect action of FLU, as these cells were not directly exposed to high drug concentrations.

In the present study, we did not observe any significant effect of RIS administration on macrophage immunomodulatory status, as assessed by NO synthesis and ARG activity. It should be noted that the immunomodulatory action of RIS is poorly recognized. The results have been inconsistent and are complex due to the multidirectional action of the drug on noradrenergic, serotoninergic and dopaminergic systems, which also have receptors on immune cells (reviewed in [10]). In animal studies, Sugino et al. [49] reported

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decreased serum levels of TNF and IL-6 and increased IL-10 after a single administration of RIS in an LPS-induced inflammatory state. Regarding the direct effect of RIS on immunocytes, it has been shown that this compound decreased synthesis of NO and pro-inflammatory cytokines IL-1b , IL-6 and TNF by IFN-g-activated microglial cells [13]. On the other hand, Quincozes-Santos et al. [32] found elevated NO synthesis by astroglial cells cultured in the presence of RIS without other stimulatory agents. Taken together, these limited data suggest that RIS is immunosuppressive.

In our experiments, although the concomitant injection of RIS and FLU had an antidepressant-like effect in the FST, the combination did not augment the immunomodulatory action of FLU. Thus, the results suggest that in these circumstances, the immunomodulatory action of FLU is potent enough to redirect macrophages toward M2 activity, while an eventual anti-inflammatory effect of RIS was either too weak to be detected with the methods used or was masked by the effects of FLU. However, it cannot be excluded that reinforcement of macrophage anti-inflammatory activity is not closely related to the antidepressant- like effects. Alternatively, changes induced in the immune system might be secondary to those produced in the neuroendocrine system.

We also assessed cellular properties that are not directly related to macrophage immunomodulatory activity, i.e., overall metabolic activity and the ability to adhere to plastic surfaces. These parameters may in- terfere with those involved in immunomodulation, such as the effect of stress due to the FST and injection, as well as possible noxious direct drug action on the cells, which cannot be excluded. These phenomena may have an effect because the macrophages were collected two hours after drug administration and one hour after FST.

Metabolic activity was measured with the resazurin reduction assay. This compound is reduced by the mitochondrial respiratory chain and is widely used for assessing various parameters of cellular metabolic activity [28]. We found that FLU treatment decreased resazurin metabolism in peritoneal cells, and this effect seemed to be due to direct drug influence because no changes were noticed in pleural cell parameters.

Though some authors have reported that FLU and RIS do not have a direct cytotoxic effect on cultured cells [9, 13], negative effects of FLU and RIS on respiratory processes have been observed [1, 19].

In the present study, only RIS administered alone increased peritoneal cell adherence, no effect was observed in pleural macrophages. Again, this suggests direct drug action on the cells. We have previously shown [44] that FLU administration decreases cellular adherence of peritoneal cells, but this was not observed in the present study for unknown reasons.

However, a weak inhibitory effect of FLU was observed; FLU administered concomitantly with RIS re- versed the stimulatory effect of RIS on this parameter.

Additionally, Quincozes-Santos et al. [31] reported decreased adherence of a cultured astroglial cell line only at high RIS concentrations. Overall, these reports suggest that the drugs affect cellular adherence, and this parameter may affect the data presented here.

We found that stress related to the FST had a profound effect on macrophages and resulted in general macrophage activation, especially in the peritoneal compartment, where milky spots, a primary source of peritoneal cells, are more reactive than in pleural cavity [25]. Several authors have reported a stimulatory effect of acute stress on various aspects of immunity (reviewed in [7]). Thus, acute stress decreases macrophage adherence [48], enhances innate immunity and improves recovery from inflammation, largely due to increased NO release [4]. Our results indicating that stress related to the FST activates macrophages cor- roborate with previously published data.

The FST substantially increased the number of the cells in peritoneal cavities, and co-administration of FLU and RIS decreased it. Neither effect was found for pleural cells. These phenomena may result from the stress related to the FST procedure and the inter- play between FLU and stress hormones, as well the abundant source of immune cells in the peritoneal cavity, i.e., the omental and mesenteric milky spots [5]. In the pleura, milky spots are confined to some ar- eas of the parietal leaflet and seem to be less reactive to systemic immunological stimuli than omental spots [25]. Acute stress can mobilize immune system cells from stores or sites of origin and drive their redistribution throughout different compartments of the body, mainly by increased release of the adrenal gland hormones corticosterone and adrenaline (reviewed in [8]). Interestingly, increased corticosterone in the circulation of FST-subjected rats has been reported, and FLU administration seemed to alleviate this effect [38, 41]. Moreover, it was shown that chronic administration of RIS at ultra-low doses (0.1 mg/kg) re- versed the stress-induced plasma corticosterone in-

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crease [15]. These reports suggest that the influence of FLU and RIS on the stress-evoked redistribution of immune cells may be more complicated and cannot be simplified to drug action on corticosterone levels.

In the current study, FLU administration increased the percentage of peritoneal macrophages. FLU blocks a serotonin transporter, the molecule which is present on neurons and immune cells as well. Sero- tonin, a classical neurotransmitter, is involved in im- mune regulation via receptors expressed on broad range of immune cells [36]. Serotonin is a chemo- attractant for immature dendritic cells, which are myeloid lineage cells that are closely related to macrophages [24]. Direct effects of FLU on the immune system may partly explain increased levels of extracellular serotonin. Thus, the changes in peritoneal cell composition observed in the present study may be the result of the chemo-attractant action of serotonin in the peritoneal environment.

In summary, the present study showed that an antidepressant drug, FLU, administered to rats at a suboptimal dose for antidepressive action in the FST, potently influences macrophage immunomodulatory properties and is able to redirect them toward the anti-inflammatory M2 functional phenotype, as reflected by changes in the ARG/NO ratio. These effects were due to the direct action of the drug on the cells, as well as to modulation via neuroendocrine pathways. Because combined treatment with the atypical antipsychotic RIS did not augment the immunomodulatory action of FLU, it seems that in this par- ticular drug combination, the immunomodulatory action of FLU is strong enough to redirect macrophages toward M2 activity and masks action of RIS. Our results also suggest that drug-induced changes in the immune system are not so closely related to the antidepressant-like effects or might be secondary to those produced in the neuroendocrine system.

Acknowledgments:

This work was supported by statutory funds from the Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland and in part by grant no. POIG.01.01.02-12-004/09 co-financed by the European Regional Development Fund.

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Received: April 27, 2012; in the revised form: July 5, 2012;

accepted: July 27, 2012.