Pharmacological Reports Copyright © 2012 2012, 64, 179184 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences
Modulation of Th1/Th2 cytokine production by selective and nonselective phosphodiesterase inhibitors administered to mice
Marianna Szczypka1, Sebastian Ploch2, Bo¿ena Obmiñska-Mrukowicz1
1 2 Department of Biochemistry, Pharmacology and Toxicology, IT Lab, Faculty of Veterinary Medicine, Wroc³aw University of Environmental and Life Sciences, Norwida 31, PL 50-375 Wroc³aw, Poland
Correspondence: Marianna Szczypka, e-mail: [email protected]
Abstract: Phosphodiesterase (PDE) inhibitors can modulate the functions of immune cells, including T lymphocytes, due to increased intracel- lular levels of cyclic nucleotides. The drugs (aminophylline, milrinone and sildenafil) were administered once or five times at 24 h intervals at the following doses: 20 mg/kg, im, 1 mg/kg, im and 1 mg/kg, po, respectively. Th1 and Th2 cytokine levels (IL-2, IFN-g, IL-4, IL-5, TNF) were determined 12, 24 or 72 h after the last administration of the drugs. A commercial BDTM Cytometric Bead Array Mouse Th1/Th2 Cytokine Kit (CBA) was used to determine the levels of Th1/Th2 cy- tokines in the serum. Neither of the PDE inhibitors under investigation administered once changed IFN-g, TNF and IL-4 production. A single dose of aminophyl- line decreased the production of IL-2 (after 12 h). A single dose of milrinone did not affect Th1/Th2 cytokine secretion. Sildenafil administered once decreased the production of IL-2 (after 72 h). Atemporary enhancement in the level of IL-5 was observed 12 h after a sin- gle dose of sildenafil. No changes in Th1 and Th2 cytokine production were observed after five doses of PDE inhibitors under investigation. These results indicate that nonstimulated lymphocytes Th1 and Th2 exhibited a slight sensitivity to aminophylline and sildenafil. The drugs under investigation were ineffective inhibitors of Th1/Th2 cytokine production.
Key words: aminophylline, milrinone, sildenafil, Th1/Th2 cytokines, T lymphocytes
Abbreviations: cAMP – cyclic adenosine monophosphate, terase (PDE) isozymes downregulate the activity of cGMP – cyclic guanosine monophosphate, IFN – interferon, cAMP and cGMP due to catalyzed hydrolysis of these IL – interleukin, im – intramuscular injection, PBS – phosphate second messengers. buffered saline solution, PDE – phosphodiesterase, po – per os, Similar activity of PDE4 (in the soluble fraction) by mouth, TNF – tumor necrosis factor and PDE3 (in particulate fraction) were observed in human T lymphocytes [12, 25]. The activity of PDE7 [12] as well as low activity of PDE1, PDE2 and PDE5 Introduction were also detected [25]. As has been reported, PDE isozyme pattern in CD4+ and CD8+ lymphocytes is similar [25]. PDE3 hydrolyzes both cyclic nucleo- The changes in intracellular cyclic nucleotide levels tides, but favors cAMP as substrate; cGMP is pre- are an important regulatory factor of immune cell ferred as substrate for PDE5, while PDE4 selectively functions, including lymphocytes T. Phosphodies- inactivates cAMP [26].
Pharmacological Reports, 2012, 64, 179184 179 Currently, isozyme PDE4, highly selective for Drugs and treatment cAMP, is a major molecular target for the drugs modulating the activity of immune and inflammatory The drugs were administered once or five times at 24 h cells through PDE inhibition. The effects of selective intervals, aminophylline (Aminophyllinum, Pliva, PDE4 and nonselective PDE inhibitors on the activity Kraków, Poland) at a dose of 20 mg/kg, im, milrinone of lymphocytes, including cytokine synthesis, have (Corotrope, Sanofi-Synthelabo, Paris, France) at a dose been studied extensively [5, 7, 14, 17, 20, 24, 26]. of 1 mg/kg, im and sildenafil (Viagra, Pfizer, Sand- Th1/Th2 cytokines are regulatory factors in the im- wich, UK) at a dose of 1 mg/kg, po. Parallely, mice in mune system, therefore, modulation of cytokine pro- the control group were treated with phosphate buff- duction by PDE inhibitors can play a role in immuno- ered saline solution – PBS (Institute of Immunology and Experimental Therapy, Wroc³aw, Poland) instead logical processes. It has been found that PDE inhibi- of the drugs. The volume of each dose was 0.1 ml per tors change Th1/Th2 cytokine production [2, 5, 6, 9, animal. Each experimental group consisted of 8 mice. 11, 22, 27], most of these studies were conducted in vitro. Some authors demonstrate beneficial effects of nonselective PDE and selective PDE4 inhibitors in Measurements the treatment of some autoimmune diseases, due to decreased synthesis of Th1 cytokines [1, 6, 21]. Se- The mice were anesthetized with halothane (Narcotan, lective PDE4 inhibitors also have anti-inflammatory Zentiva, Prague, Czech Republic). The blood samples were taken from retro-ocular artery. The sera were ob- properties (e.g., inhibition of cytokine release from tained by blood centrifugation (3,500 × g, 15 min). inflammatory cells), resulting in clinical implications A commercial BDTM Cytometric Bead Array Mouse of these drugs [3, 10, 13]. Th1/Th2 Cytokine Kit (CBA) (lot: 68585, BD Biosciences Little information is still available on the immuno- Pharmingen, San Diego, USA) was used to determine the tropic influence of selective inhibitors of other isozymes, levels (pg/ml) of interleukin-2 (IL-2), interferon-g (IFN-g), such as selective PDE3 or PDE5 inhibitors, and most of interleukin-4 (IL-4), interleukin-5 (IL-5) and tumor necro- these studies were conducted in vitro [2, 8, 9, 22]. sis factor (TNF) in serum samples, according to the manu- The purpose of the present investigation was to de- facturer’s instructions. Fluorescence was analyzed using termine the effects of milrinone, selective PDE3 in- a flow cytometer (FACS Calibur, Becton-Dickinson Bio- hibitor, sildenafil, selective PDE5 inhibitor and sciences, Heidelberg, Germany) and cytokine level was de- aminophylline, nonselective PDE inhibitor, adminis- termined using a BD CBA Software. tered in vivo, on cytokine production by T helper 1 The parameters were determined 12, 24 and 72 h (Th1) and T helper 2 (Th2) lymphocytes in non- after the last administration of PDE inhibitors. stimulated mice.
Statistical analysis
The data obtained in the study do not meet the Materials and Methods assumptions of parametric tests. Statistical analysis was performed using a Kruskal-Wallis test (non- parametric analogue of one-way ANOVA). The dif- Animals ferences were considered significant at p > 0.05. The data were analyzed with Statistica 9.0 software. The studies were conducted on female Balb/c mice, each weighing 18–22 g (8 weeks of age). The mice were obtained from a Breeding Center of Laboratory Animals at the Institute of Occupational Medicine, Results £ódŸ, Poland. The principles of laboratory animal care (NIH publication No. 86-23, revised 1985) as The effects of single doses of PDE inhibitors well as the specific national laws on the protection of on Th1 and Th2 cytokine production animals were followed. The study protocol was ap- proved by the Local Ethics Committee in Wroc³aw, Neither of the PDE inhibitors under investigation ad- Poland (No. 16/04). ministered once was able to change IFN-g, TNF and
180 Pharmacological Reports, 2012, 64, 179184 The effects of PDE inhibitors on Th1/Th2 cytokine production Marianna Szczypka et al.
*o^ * *
Fig. 1. The Th1/Th2 cytokine levels after a single administration of PDE inhibitors. The mean value (n = 8) and standard deviation. * p < 0.05 as compared to the control group; º p < 0.05 as compared to the aminophylline group; ^ p < 0.05 as compared to the milrinone group
IL-4 production. A single dose of aminophylline de- creased the production of IL-2 (after 12 h). A single Discussion dose of milrinone did not affect Th1/Th2 cytokine se- cretion. Sildenafil administered once decreased the Th1/Th2 cytokines are regulatory factors in several production of IL-2 (after 72 h). A temporary enhance- immunological processes. Generally, Th1 lympho- ment in IL-5 was observed 12 h after a single dose of cytes produce mainly IL-2 and IFN-g, being stimula- sildenafil (Fig. 1). tors of cell-mediated response, whereas cytokines from Th2 lymphocytes (e.g., IL-4 and IL-5) promote humoral immune response [4, 15]. TNF is secreted The effects of PDE inhibitors administered five both by Th1 and Th2 cells [4]. The balance of times on Th1 and Th2 cytokine production Th1/Th2 cytokine production is a major factor affect- ing the immune response. The activity of Th1 lym- No changes in Th1 and Th2 cytokine production were phocytes predominates in autoimmune diseases, while observed after five doses of PDE inhibitors under in- an increase in Th2 activity is observed in allergic dis- vestigation (Fig. 2). orders [18, 19, 23].
Pharmacological Reports, 2012, 64, 179184 181 Fig. 2. The Th1/Th2 cytokine levels after five-time administration of PDE inhibitors. The mean value (n = 8) and standard deviation
The results obtained in earlier studies showed did not change the cytokine production by nonstimu- a modulating effect of PDE inhibitors on the cytokine lated T lymphocytes. The results obtained by Gantner production by lymphocytes Th1 and Th2. Essayan et et al. [11] in the study conducted in vivo show that al. [9] conducted in vitro studies and reported that both rolipram and motapizone, selective PDE3 inhibi- rolipram (selective PDE4 inhibitor) downregulated tor (0.1–10 mg/kg) suppressed a release of IFN-g and gene expression for IL-4, IL-5, IFN-g and also re- TNF as initiated by injection of T cell mitogens (con- duced IL-4 and IFN-g secretion. They also found canavalin A or staphylococcal enterotoxin B). These a suppressive effect of rolipram on proliferative re- differences suggest that sensitivity of the lymphocytes sponse of Th1 and Th2 lymphocytes (Th2 cells were to PDE inhibitors depends on the lymphocyte status. more sensitive to this action). In the same study, Some authors in the study conducted in vitro show siguazodan (selective PDE3 inhibitor) had no impact that Th1 lymphocytes are more sensitive to the inhibi- on these parameters. It was also found that rolipram tory action of PDE inhibitors than Th2 lymphocytes. inhibited phytohemagglutinin- and anti-CD3-induced It has been found that nonselective PDE inhibitors, se- proliferation of lymphocytes T and IL-2 secretion, lective PDE4 or selective PDE3 inhibitors decrease whereas SK&F 95654 (selective PDE3 inhibitor) was IL-2 and IFN-g release from phytohemagglutinin- inactive [12]. Similarly, in the present study con- stimulated human peripheral blood mononuclear cells, ducted in vivo, a selective PDE3 inhibitor, milrinone, whereas secretion of IL-4 and IL-5 is suppressed only
182 Pharmacological Reports, 2012, 64, 179184 The effects of PDE inhibitors on Th1/Th2 cytokine production Marianna Szczypka et al.
by high concentrations of a PDE4 inhibitor [27]. observed only after a single administration of these A similar effect, i.e., inhibition of Th1 cytokine secre- drugs. No changes were observed after five doses of tion by mesopram (selective PDE4 inhibitor), with no PDE inhibitors. These results show that the influence influence on Th2 cytokine production was observed of PDE inhibitors on Th1/Th2 cytokine production by Dinter et al. [6]. Bielekova et al. [2] also found that was balanced during the course of the treatment. In rolipram (at concentrations of 0.1, 1.0 and 10.0 µM) general, the drugs under investigation were ineffec- or a combination of rolipram and cilostamide, selec- tive inhibitors of Th1/Th2 cytokine production. tive for PDE3 (but not cilostamide used alone) de- creased IFN-g production. However, neither rolipram nor a combination of rolipram and cilostamide exhib- Declaration of interest: ited a significant inhibitory effect on IL-4 secretion. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Interestingly, cilostamide used alone at a high concen- tration (10 µM) had a stimulatory impact on IL-4 pro- Acknowledgment: This study was supported by the Polish State Committee for duction. Similarly, in the present study, neither of the Scientific Research; Ministry of Science and Higher Education, PDE inhibitors under investigation was able to sup- (Grant No. 2 P06K 014 28). press Th2 cytokine release (IL-4, IL-5), but a single dose of aminophylline or sildenafil temporarily de- creased the production of Th1 cytokine – IL-2. An opposite effect on cytokine gene expression References: was reported by Lin et al. [16] who conducted the studies in vivo. They found that aminophylline admin- 1. 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