Pharmacological Reports Copyright © 2010 2010, 62, 11481058 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences Modulating effects of nonselective and selective phosphodiesterase inhibitors on lymphocyte subsets and humoral immune response in mice Marianna Szczypka, Bo¿ena Obmiñska-Mrukowicz Department of Biochemistry, Pharmacology and Toxicology, 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 regulate the activity of immune cells by increasing intracellular levels of cyclic nucleotides. The aim of this study was to determine the effects of milrinone, a selective PDE3 inhibitor, sildenafil, a selective PDE5 inhibitor, and aminophylline, a nonselective PDE inhibitor, on lymphocyte subsets and humoral immune response in mice when administered in vivo. Aminophylline (20 mg/kg, im), milrinone (1 mg/kg, im) or sildenafil (1 mg/kg, po) were administered to mice either once or five times at 24 h intervals. Some mice were immunized with a sheep red blood cell (SRBC) suspension administered ip either 2 h after the single dose or 2 h after the second of the five doses. In non-immunized mice treated five times with PDE inhibitors, the subsets of T lymphocytes in the thymus and T and B lymphocytes in the spleen and mesenteric lymph nodes were determined 12, 24 or 72 h after the last dose. The humoral immune response was determined on days 4, 7 and 14 after SRBC injection in SRBC-immunized mice treated with PDE inhibitors. A modulating effect of the drugs on lymphocyte subpopulations was observed. The greatest impact was observed in splenocyte subpopulations, and resulted in decreased percentages of B cells (CD19+) and increased percentages of T cells (CD3+, CD4+, CD8+). No effect or slight influence of the drugs on anti-SRBC hemagglutinins was observed, but the number of plaque-forming splenocytes was increased. The drugs under investigation did not show a significant immunosuppressive effect. Key words: aminophylline, milrinone, sildenafil, PDE inhibitors, lymphocyte subsets, humoral immune response Abbreviations: cAMP – cyclic adenosine monophosphate, cell activity, including the activity of inflammatory CD – cluster of differentiation, cGMP – cyclic guanosine mono- and immune cells. Phosphodiesterase (PDE) catalyzes phosphate, IL – interleukin, im – intramuscular injection, the hydrolysis of cyclic nucleotides and thereby ip – intraperitoneal injection, PBS – phosphate buffered saline downregulates the intracellular levels of these second solution, PDE – phosphodiesterase, PFC – plaque forming sple- messengers. The PDE isozymes are classified into at nocytes, po – per os, by mouth, 2-ME – 2-mercaptoethanol, SRBC – sheep red blood cells least 11 families that vary in molecular structure, sen- sitivity to endogenous and exogenous regulators, in- tracellular location, affinity to cAMP and/or cGMP and sensitivity to selective inhibitors [4, 6]. For in- Introduction stance, PDE4 is highly selective for cAMP, PDE3 fa- vors cAMP but inactivates both cyclic nucleotides, and PDE5 preferentially inactivates cGMP [41]. The levels of intracellular cyclic nucleotides (cAMP In human T lymphocytes, membrane-bound PDE3 and cGMP) play an important role in the regulation of and cytosolic PDE4 are equally active [18, 40]. The 1148 Pharmacological Reports, 2010, 62, 11481058 PDE inhibitors and lymphocyte subsets and humoral response Marianna Szczypka and Bo¿ena Obmiñska-Mrukowicz soluble fraction also contains PDE7 isozyme [18]. animal care (NIH publication No 86–23, revised Marginal activities of PDE1, PDE2 and PDE5 have 1985), and national laws regarding the protection of been detected in human CD4+ and CD8+ lymphocyte animals were observed. The study protocol was ap- homogenates [40]. However, no differences in PDE proved by the Local Ethics Committee in Wroc³aw, isozyme patterns between CD4+ and CD8+ lympho- Poland (No. 16/04). cytes have been observed [40]. Cytosolic PDE4 is a major cyclic-nucleotide-meta- Drugs and treatment bolizing enzyme in human B lymphocytes, though PDE7 activity has also been found in the soluble frac- The PDE inhibitors aminophylline (20 mg/kg, im, tion. Low activity of PDE3 isozyme have been de- Aminophyllinum, Pliva Kraków, Poland), milrinone tected in the particulate fraction. However, activities (1 mg/kg, im, Corotrope, Sanofi-Synthelabo, Paris, of PDE1, PDE2 and PDE5 in B lymphocytes have not France), and sildenafil (1 mg/kg, po, Pfizer, Sand- been detected [17]. wich, UK) were administered either once or five The influence of selective PDE4 inhibitors and times at 24 h intervals. Mice in the control group re- nonselective PDE inhibitors on the activity of im- ceived phosphate buffered saline (PBS) solution (In- mune cells, including lymphocytes, has been studied stitute of Immunology and Experimental Therapy, extensively [8, 13, 21, 24, 26, 28, 35, 38, 42]. As Wroc³aw, Poland) instead of PDE inhibitors. The vol- a major PDE isozyme in immune cells, PDE4 is ume of each dose was 0.1 ml per animal. Each experi- a principal molecular target for drugs that modulate mental group consisted of eight mice. immunological and inflammatory processes by in- Some mice were immunized with 0.2 ml of 10% creasing intracellular cyclic nucleotide levels [6, 9, sheep red blood cell (SRBC) suspension (4 × 108 12, 41]. Many authors have demonstrated beneficial cells/mouse) given ip 2 h after the single dose or 2 h effects of selective PDE4 inhibitors in the treatment after the second of five doses. The sheep blood was of airway diseases and some autoimmune diseases [2, collected, using sterile techniques, into Alsever’s so- 5, 6, 9, 10, 12]. Fewer reports can be found on the im- lution (prepared in our laboratory) containing glucose munotropic effects of selective inhibitors of other iso- (2.05%), sodium citrate (0.8%), sodium chloride zymes, most of which were conducted in vitro. (0.42%) and citric acid (0.055%) (Archem, Wroc³aw, In our previous study, the effects of a single ad- Poland) and was kept at 4°C for at least 3 days. The ministration of aminophylline, a nonselective PDE in- SRBC suspension in PBS was prepared fresh just hibitor (at a dose of 20 mg/kg, im), milrinone, a selec- prior to use. tive PDE3 inhibitor (at a dose of 1 mg/kg, im), or silde- nafil, a selective PDE5 inhibitor (at a dose of 1 mg/kg, Measurements po), on lymphocyte subsets were determined [39]. The aim of the present investigation was to study The following measurements were taken: (i) the total the effects of five administrations of aminophylline, number of lymphocytes in the thymus, spleen and milrinone or sildenafil on lymphocyte subsets and to mesenteric lymph nodes, (ii) the weight ratio of or- assess the effects of one or five doses of these drugs gans calculated according to the following formula: on humoral immune response in mice. weight of organ (g)/body weight of mouse (g) × 100, (iii) the lymphocyte subpopulations in lymphatic or- gans, (iv) the number of plaque forming cells (PFC) in spleen, and (v) anti-SRBC hemagglutinin titers in Materials and Methods the serum. Animals Assay of subpopulations of thymocytes, splenocytes and lymphocytes of lymph nodes The studies were conducted on 8-week-old female Balb/c mice, each weighing 18–22 g. Experimental Mice were anesthetized with halothane (Narcotan, animals were obtained from the Breeding Center of Zentiva, Prague, Czech Republic) 12, 24 or 72 h after Laboratory Animals of the Institute of Occupational the last drug administration. Thymuses, spleens and Medicine, £ódŸ, Poland. The principles of laboratory mesenteric lymph nodes were removed and placed in Pharmacological Reports, 2010, 62, 11481058 1149 Petri dishes containing sterile, ice-cold PBS. The sus- Jerne et al. [22] with some modifications [32]. The pended cells were released from the lymphatic organs number of PFC in SRBC-immunized mice treated by passage through a nylon mesh and then centrifuged once or five times with PDE inhibitors was deter- (3000 × g, 15 min., 4°C) on a layer of Ficoll 400 (Sigma)/ mined on days 4 and 7 after injection of SRBC. Uropolinum 75% (sodium amidotrizoate, Polpharma S.A., Starogard Gdañski, Poland) in a 1:3 ratio at Determination of anti-SRBC antibodies a density of 1.076. After centrifugation, the cells were in the serum collected from the interphase and washed twice with 4°C PBS supplemented with 1% bovine serum albu- Blood samples were taken by retro-orbital bleeding min (BSA, Sigma). After the second wash, the cells 7 from halothane-anesthetized mice. The sera were were resuspended in PBS with 1% BSA at 1 × 10 obtained by centrifugation of the blood (3500 × g, cells/ml. The viability of each cell suspension, as de- 15 min) and then inactivated at 56°C for 30 min. The termined by trypan blue dye exclusion, was 90–95%. total (IgM + IgG) and 2-mercaptoethanol resistant (IgG) Cells in suspension were stained with monoclonal rat serum hemagglutinin titers were defined by the active anti-mouse CD4:FITC/CD8:RPE dual color reagent hemagglutination test, carried out on microplates, as (Serotec, Kidlington, UK) or monoclonal rat anti-mouse described by Adler [1]. Anti-SRBC hemagglutinin CD19:FITC/CD3:RPE dual color reagent (Serotec, titers in SRBC-immunized mice treated once or five Kidlington, UK) according to the manufacturer’s in- times with PDE inhibitors were determined on days 4, structions. Cells were incubated at 4°C for 30 min and 7 and 14 after SRBC injection. then washed three times with ice-cold PBS. Fluores- cence was analyzed using a flow cytometer (FACS Calibur, Becton-Dickinson Biosciences). Lymphocyte Statistical analysis marker distribution was analyzed using CellQuest 3.1f software. The data obtained in the study were analyzed statisti- t CD subsets (CD4-CD8-, CD4+CD8+, CD4+ and CD8+ cally using Student’s -test.