Journal of Neuroimmunology 269 (2014) 94–97

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Reversal of stroke induced lymphocytopenia by levodopa/ benserazide treatment Enida Kuric ⁎, Karsten Ruscher ⁎ Laboratory for Experimental Brain Research, Division of Neurosurgery, Department of Clinical Sciences, Lund University, BMC A13, S-22184 Lund, Sweden

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Article history: Received 15 October 2013 Received in revised form 7 February 2014 Accepted 19 February 2014 Keywords: Dopamine Immunodepression Levodopa Stroke recovery T-cells

a b s t r a c t Evidences exist that dopamine is involved in T-cell homeostasis. Here, we investigated if daily treatment with levodopa (20 mg/kg)/benserazide (15 mg/kg) regulates the number of T-cells in the blood of Sprague Dawley rats subjected to transient occlusion of the middle cerebral artery (tMCAO). T-cells and associated inflammatory cytokines were analyzed in the blood one week after tMCAO. Treatment with levodopa/benserazide completely recovered T-helper cell depletion in animals subjected to tMCAO whereas T-cell associated cytokines remained unaffected after stroke, however, cytokines were downregulated in levodopa treated sham operated animals. Together, we demonstrate that dopamine has beneficial effects on poststroke immunodepression. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Poststroke immunodepression is detrimental, reflecting the high incidence of life threatening infections and mortality in stroke patients (Klehmet et al., 2009; Dirnagl et al., 2007). The phenomenon has also been found in experimental models where selective lymphocytopenia has been confirmed and atrophy of lymphoid organs was observed (Prass et al., 2003; Ruscher et al., 2013). Moreover, changes of the immune system have been attributed to a hyperactive state of the sympathetic nervous system via activation of the hypothalamic pituitary adrenal (HPA) axis and activation of beta-adrenergic receptors on effector cells (Prass et al., 2003; Offner et al., 2006; Dirnagl et al., 2007). Thus, the communication between the central nervous system and the immune system evidently is mediated by the release of catecholamines (Basu and Dasgupta, 2000). Conditions, such as schizophrenia and Parkinson's disease, show reduced levels of CD4+ T-cells (Steiner et al., 2010) together with alterations of dopamine 3 receptor expression on these cells (Ilani et al., 2001; Nagai et al., 1996). These findings suggest the involvement of dopamine signaling in T-cell response and homeostasis. We recently have bed to bench translated results from a clinical trial (Scheidtmann et al., 2001) and shown that levodopa/benserazide significantly improves functional recovery in ⁎ Corresponding authors at: Laboratory for Experimental Brain Research, Wallenberg Neuroscience Center, Lund University, BMC A13, S-22184 Lund, Sweden. Tel.: +46 46 2220609; fax: +46 46 2220615. E-mail addresses: [email protected] (E. Kuric), [email protected] (K. Ruscher).

http://dx.doi.org/10.1016/j.jneuroim.2014.02.009 0165-5728/© 2014 Elsevier B.V. All rights reserved.

rats after experimental stroke (Ruscher et al., 2012). The present study was conducted to test if enhanced functional recovery in rats treated with levodopa/benserazide is of relevance for poststroke immunodepression and in particular if the treatment results in changes of Tcells and inflammatory molecules in the blood of rats subjected to tMCAO. 2. Materials and methods 2.1. Transient middle cerebral artery occlusion (tMCAO) All animal experiments were approved by Malmö–Lund ethical committee and carried out in agreement with the ARRIVE guidelines. Male Sprague Dawley rats (325–350 g, Harlan Scandinavia, Denmark) were subjected to tMCAO for 105 min as described previously (Ruscher et al., 2013). In total 68 rats were operated whereof 18 died during the first 48 h after tMCAO. 2.2. Drug treatment protocol All experiments have been performed in a blinded fashion to the investigator. Two days post-tMCAO, rats were randomized based on sensorimotor deficits that were assessed by the rotating pole test (Ruscher et al., 2009) and Garcia test (Encarnacion et al., 2011). Only animals with sufficient sensorimotor deficits were assigned into the treatment groups and treated for 5 days with daily intraperitoneal injections of 20 mg/kg levodopa combined with 15 mg/kg benserazide

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(Sigma Aldrich, Deisenhofen, Germany) or saline, starting at day 2 posttMCAO. Number of animals included in the study: sham vehicle (n = 8), sham levodopa (n = 7), tMCAO vehicle (n = 10) and tMCAO levodopa/ benserazide (n = 12).

were acquired and forward side scatter (FSC) threshold was set to 20,000. Data analysis was performed by using the FlowJo (7.6.5 version, Tree Star, Ashland, OR, USA) software. T-cells were identified by CD3 antigen and further analyzed for CD4, CD8 and CD25 (Fig. 1).

2.3. Serum preparation and cytokine analysis

2.6. Statistics

Blood from animals was sampled into heparinized syringes by intracardial puncture. Following incubation at room temperature for 30 min, the blood was centrifuged at 1000 ×g and supernatant serum was used for cytokine analyses. We analyzed the following cytokines: interleukin-1beta (IL-1β), chemokine (C-X-C motif) ligand 1 (CXCL1), IL-4, IL-5, interferon-gamma (IFN-γ) and IL-13 according to manufacturer's protocol (Mesoscale, Gaithersburg, USA).

Data are expressed as mean values ± SEM. Differences were tested with analysis of variance (ANOVA) and followed by Bonferroni correction or Fisher's LSD test was indicated. In all experiments p b 0.05 was considered significant.

2.4. Immune cell isolation from the blood Two hundred microliters of heparinized blood was incubated with Red Blood Cell lysis buffer (eBioscience, San Diego, CA, USA). Following the addition of phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS, Life Technologies, Stockholm, Sweden) cells were centrifuged (300 ×g, 4 °C), the supernatant was discarded and cells were resuspended in PBS containing 2% FBS. 2.5. Analysis of T-cells by fluorescence activated cell sorter (FACS) One hundred microliters of immune cells from the blood was stained with lymphocytic markers; mouse anti-rat cluster of differentiation (CD) 3 fluorescein isothiocyanate conjugated, mouse anti-rat CD4 allophycocyanin conjugated, anti-rat CD8 phycoerythrin conjugated and anti-rat CD25 peridinin chlorophyll protein cyanine 5.5 conjugated antibody (all diluted to 1:200; BD Biosciences, USA). Cells were fixed with (Cytofix/Cytoperm™, BD Biosciences) and analyzed 48 h later. Fluorescence minus one (FMO) and unstained cells were used as controls. Analysis and data collection were performed by using FACS AriaIII and FACS Diva software (BD Biosciences). Up to 200,000 events

3. Results Recent studies demonstrate that dopamine has a significant impact on the immune system; in particular the T-cell mediated response (Basu and Dasgupta, 2000; Sarkar et al., 2010). Together with previous reports (Ilani et al., 2001; Nagai et al., 1996; Steiner et al., 2010) we hypothesized that levodopa/benserazide may affect peripheral immunodepression induced by tMCAO. Confirming previous results (Ruscher et al., 2013) stroke induced lymphocytopenia was observed 7 days after tMCAO (Fig. 2A). Compared to sham operated rats (sham vh 51.75% ± 8.22%, sham LD 52.17% ± 10.56%) a significant decrease of total CD3+ T-cells (MCAO vh 25.72% ± 5.56%) was detected in rats subjected to tMCAO (Fig. 2A) expressed as ratio to the total number of cells in the blood. In contrast, levodopa/benserazide treatment normalized CD3+ T-cell counts (MCAO LD 42.6% ± 2.14%) to levels found in the blood of sham operated rats (Fig. 2A). A representative histogram of the treatment effect is shown in Fig. 2B. Importantly, levodopa/benserazide treatment per se had no effect on CD3+ T-cells in sham operated animals. Quantification by FACS revealed that in particular the CD3+CD4+ Tcells (T-helper cells) were reduced after tMCAO (Fig. 2C, Table 1) and this population of cells was restored by levodopa/benserazide treatment. All other subpopulations of T-cells were not affected by tMCAO or treatment with levodopa/benserazide, respectively (Fig. 2D,

Fig. 1. Gating strategy. Representative scatter plots and gating strategies for T-cell analysis in blood from rats subjected to tMCAO. Cells were first gated for lymphocytes (SSC-A vs. FSC-A) and singlets (FSC-W vs. FSC-A). Thereafter, surface expression of CD3 or CD4 was determined from this gated population. Abbreviations; forward side scatter (FSC), Side scatter (SSC), fluorescence minus one (FMO).

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Fig. 2. Number of T-cells in the blood after tMCAO. (A) Total T-cells in the blood defined by CD3+ expression. Data are expressed as means ± SEM and statistical analysis has been performed with two-way ANOVA followed by Bonferroni post hoc correction. # indicates significant difference between sham operated animals and MCAO VH. * indicates a difference between MCAO VH and MCAO LD. (B) Representative histogram illustrating differences in CD3+ cell counts between vehicle and levodopa/benserazide treated rats after tMCAO. (C) T-helper cells defined by CD3+CD4+ expression shown as percentage ± SEM of the total CD3+ population. Differences were tested with two-way ANOVA and Fisher's Least Significant Difference test. (D) Distribution of T-cell phenotypes shown as percentages of the total CD3+ population. (E) Number of events counted in respective groups tested with two-way ANOVA, n.s. — not significant. Data are shown as medians with Q1 and Q3 and whiskers. VH — vehicle, LD — levodopa/benserazide treatment. p b 0.05 was considered significant.

Table 1). As shown in Fig. 2E, the number of cell counts did not differ between the experimental groups verifying that the evaluation was not influenced by the number of analyzed cells. In addition, we measured levels of inflammatory cytokines in the serum of respective animals. In sham operated animals a significant down-regulation of not only Th1 cytokines, IFN-γ and IL-1β but also IL-4, IL-5, and Th2 cytokines were detected by levodopa/benserazide treatment (Fig. 3). After tMCAO, no differences were observed between the two treatment conditions,

however, all cytokines were lower than in the sham vh group (Fig. 3) except the levels for CXCL1. We found a significant difference between sham vehicle treated animals and animals subjected to tMCAO and treated with levodopa/benserazide. Interleukin 13 was not detected in the serum (data not shown). Together, we show that levodopa/ benserazide treatment normalizes the number of T-helper cells in the blood after tMCAO but does not affect their further response, measured as the secretion of corresponding cytokines after tMCAO.

Table 1 T-cell populations in the blood. CD3+CD4+

CD3+CD8+

CD3+CD25+

CD3+CD4+CD25+

CD3+

MCAO blood MCAO vh MCAO LD

15602 ± 4127⁎⁎ 31132 ± 7899⁎

7882 ± 1928 12865 ± 3330

0 5±2

188 ± 91 420 ± 151

1449 ± 593 1159 ± 267

Sham blood Sham vh Sham LD

24586 ± 4271 24629 ± 3863

12340 ± 2168 10679 ± 2777

2 ± 14 2 ± 12

315 ± 96 225 ± 108

1017 ± 748 6100 ± 4436

Number of T-cell populations in the blood from rats subjected to tMCAO for 105 min and treated with either saline (MCAO vh) or levodopa/benserazide (MCAO LD) and respective sham operated animals (sham vh, sham LD). Results are shown as absolute numbers ± SEM. ⁎ Denotes p b 0.05 versus MCAO vh. ⁎⁎ Denotes p b 0.05 versus sham LD. Two-way ANOVA followed by LSD.

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Acknowledgments We thank Professor Tadeusz Wieloch for valuable discussions and suggestions. We also thank Gunilla Gidö, Carin Sjölund and Anna Hammarberg for excellent technical assistance. This study was supported by the Swedish Research Council (grant 2011-2684 and grant 20112652), the EU 7th workprogram through the European Stroke Network (grant No 201024), the Hans-Christian and Alice Wachtmeister Foundation, the Swedish Brain Fund, the Thorsten och Elsa Segerfalk stiftelse and the Crafoord Foundation. References

Fig. 3. Analysis of cytokines in the serum 7 days after tMCAO. Serum from rats subjected to tMCAO or sham operation was treated with either vehicle (saline) or levodopa (20 mg/kg)/benserazide (15 mg/kg), was obtained as described in the Materials and methods section and analyzed by a multiplex immunoassay using a SECTOR Imager 6000 reader. Data are expressed as means, picograms per 0.1 mL serum ± SEM and differences were tested with two-way ANOVA followed by Bonferroni post hoc correction or Fisher's Least Significant Difference test (italic). Letters denote p values: a) p = 0.002; b) p = 0.015; c) p = 0.022; d) p = 0.017; e) p = 0,008; f) p = 0.001; g) p = 0.015; h) p = 0.018; i) p = 0.001; j) p = 0.018; k) p = 0.038; and l) p = 0.031. p b 0.05 was considered significant. VH — vehicle, LD — levodopa/benserazide.

4. Discussion The present study was conducted to investigate if treatment with levodopa/benserazide has an effect on poststroke immunodepression. We observed recovery of stroke induced reduction of T-helper cells by levodopa/benserazide. Normalized levels of T-cells might be due to direct activation by dopamine since those cells express functional dopamine receptors (Bergquist et al., 1994) and an increase of dopamine may lead to mobilization of T-cells from lymphoid tissues into the circulation. Such a mechanism might be due to direct actions of levodopa converted to dopamine in the periphery. Despite the co-administration of benserazide to prevent decarboxylation of levodopa, increased dopamine levels can be detected in the plasma (Boomsma et al., 1989). Previous in vitro studies also support this idea showing that dopamine inhibits activated T-cells, negatively regulates T-cell proliferation and it inhibits the release of IFN-γ and IL-4 by stimulated T-cells (Ghosh et al., 2003). In contrast, dopamine can activate resting T-cells which leads to increased migration and homing together with an increased release of chemokines as well as the up-regulation of integrins to promote cellular adhesion (Levite et al., 2001; Watanabe et al., 2006; Strell et al., 2009). Thus, dopamine seems to have a dual role in the activation and direction of T-cells, dependent on their activity status. Although we have observed a reduction of inflammatory cytokines in the serum of rats subjected to tMCAO we did not detect differences in the levels between vehicle and levodopa/benserazide treated rats. Hence, our data confirmed previous findings demonstrating reduced levels of the Th1 cytokines IFN-γ and IL-1β in the blood after stroke (Prass et al., 2003). Downregulation of Th1 and Th2 cytokines in sham operated rats treated with levodopa/ benserazide indicates that despite the fact that there were no differences in the absolute numbers of T-cells between vehicle and levodopa/ benserazide treated rats dopamine significantly reduced the activation of these cells since T-cells account for a significant amount of cytokines in the periphery in blood. Indeed, the activity and functional status of Tcells after stroke differ from control conditions (Vogelgesang et al., 2010). Together, our data suggest that dopamine attenuates poststroke immunodepression.

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