Modulating effects of nonselective and selective phosphodiesterase inhibitors on lymphocyte subsets and humoral immune response in mice

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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: marianna.szczypka@up.wroc.pl

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 administeredin 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 administeredip 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, CD – cluster of differentiation, cGMP – cyclic guanosine monophosphate, IL – interleukin, im – intramuscular injection, ip – intraperitoneal injection, PBS – phosphate buffered saline solution, PDE – phosphodiesterase, PFC – plaque forming splenocytes,po – per os, by mouth, 2-ME – 2-mercaptoethanol, SRBC – sheep red blood cells

Introduction

The levels of intracellular cyclic nucleotides (cAMP and cGMP) play an important role in the regulation of

cell activity, including the activity of inflammatory and immune cells. Phosphodiesterase (PDE) catalyzes the hydrolysis of cyclic nucleotides and thereby downregulates the intracellular levels of these second messengers. The PDE isozymes are classified into at least 11 families that vary in molecular structure, sensitivity to endogenous and exogenous regulators, intracellular location, affinity to cAMP and/or cGMP and sensitivity to selective inhibitors [4, 6]. For in- stance, PDE4 is highly selective for cAMP, PDE3 fa- vors cAMP but inactivates both cyclic nucleotides, and PDE5 preferentially inactivates cGMP [41].

In human T lymphocytes, membrane-bound PDE3 and cytosolic PDE4 are equally active [18, 40]. The

Pharmacological Reports 2010, 62, 11481058 ISSN 1734-1140

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soluble fraction also contains PDE7 isozyme [18].

Marginal activities of PDE1, PDE2 and PDE5 have been detected in human CD4⁺and CD8⁺lymphocyte homogenates [40]. However, no differences in PDE isozyme patterns between CD4⁺ and CD8⁺ lymphocytes have been observed [40].

Cytosolic PDE4 is a major cyclic-nucleotide-meta- bolizing enzyme in human B lymphocytes, though PDE7 activity has also been found in the soluble fraction. Low activity of PDE3 isozyme have been detected in the particulate fraction. However, activities of PDE1, PDE2 and PDE5 in B lymphocytes have not been detected [17].

The influence of selective PDE4 inhibitors and nonselective PDE inhibitors on the activity of immune cells, including lymphocytes, has been studied extensively [8, 13, 21, 24, 26, 28, 35, 38, 42]. As a major PDE isozyme in immune cells, PDE4 is a principal molecular target for drugs that modulate immunological and inflammatory processes by increasing intracellular cyclic nucleotide levels [6, 9, 12, 41]. Many authors have demonstrated beneficial effects of selective PDE4 inhibitors in the treatment of airway diseases and some autoimmune diseases [2, 5, 6, 9, 10, 12]. Fewer reports can be found on the im- munotropic effects of selective inhibitors of other isozymes, most of which were conductedin vitro.

In our previous study, the effects of a single administration of aminophylline, a nonselective PDE inhibitor (at a dose of 20 mg/kg,im), milrinone, a selec- 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, po), on lymphocyte subsets were determined [39].

The aim of the present investigation was to study the effects of five administrations of aminophylline, milrinone or sildenafil on lymphocyte subsets and to assess the effects of one or five doses of these drugs on humoral immune response in mice.

Materials and Methods

Animals

The studies were conducted on 8-week-old female Balb/c mice, each weighing 18–22 g. Experimental animals were obtained from the Breeding Center of Laboratory Animals of the Institute of Occupational Medicine, £ódŸ, Poland. The principles of laboratory

animal care (NIH publication No 86–23, revised 1985), and national laws regarding the protection of animals were observed. The study protocol was ap- proved by the Local Ethics Committee in Wroc³aw, Poland (No. 16/04).

Drugs and treatment

The PDE inhibitors aminophylline (20 mg/kg, im, Aminophyllinum, Pliva Kraków, Poland), milrinone (1 mg/kg, im, Corotrope, Sanofi-Synthelabo, Paris, France), and sildenafil (1 mg/kg, po, Pfizer, Sand- wich, UK) were administered either once or five times at 24 h intervals. Mice in the control group received phosphate buffered saline (PBS) solution (In- stitute of Immunology and Experimental Therapy, Wroc³aw, Poland) instead of PDE inhibitors. The vol- ume of each dose was 0.1 ml per animal. Each experimental group consisted of eight mice.

Some mice were immunized with 0.2 ml of 10%

sheep red blood cell (SRBC) suspension (4 × 10⁸ cells/mouse) givenip 2 h after the single dose or 2 h after the second of five doses. The sheep blood was collected, using sterile techniques, into Alsever’s solution (prepared in our laboratory) containing glucose (2.05%), sodium citrate (0.8%), sodium chloride (0.42%) and citric acid (0.055%) (Archem, Wroc³aw, Poland) and was kept at 4°C for at least 3 days. The SRBC suspension in PBS was prepared fresh just prior to use.

Measurements

The following measurements were taken: (i) the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes, (ii) the weight ratio of organs calculated according to the following formula:

weight of organ (g)/body weight of mouse (g) × 100, (iii) the lymphocyte subpopulations in lymphatic organs, (iv) the number of plaque forming cells (PFC) in spleen, and (v) anti-SRBC hemagglutinin titers in the serum.

Assay of subpopulations of thymocytes, splenocytes and lymphocytes of lymph nodes

Mice were anesthetized with halothane (Narcotan, Zentiva, Prague, Czech Republic) 12, 24 or 72 h after the last drug administration. Thymuses, spleens and mesenteric lymph nodes were removed and placed in

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Petri dishes containing sterile, ice-cold PBS. The sus- pended cells were released from the lymphatic organs by passage through a nylon mesh and then centrifuged (3000 × g, 15 min., 4°C) on a layer of Ficoll 400 (Sigma)/

Uropolinum 75% (sodium amidotrizoate, Polpharma S.A., Starogard Gdañski, Poland) in a 1:3 ratio at a density of 1.076. After centrifugation, the cells were collected from the interphase and washed twice with 4°C PBS supplemented with 1% bovine serum albu- min (BSA, Sigma). After the second wash, the cells were resuspended in PBS with 1% BSA at 1 × 10⁷ cells/ml. The viability of each cell suspension, as determined by trypan blue dye exclusion, was 90–95%.

Cells in suspension were stained with monoclonal rat anti-mouse CD4:FITC/CD8:RPE dual color reagent (Serotec, Kidlington, UK) or monoclonal rat anti-mouse CD19:FITC/CD3:RPE dual color reagent (Serotec, Kidlington, UK) according to the manufacturer’s in- structions. Cells were incubated at 4°C for 30 min and then washed three times with ice-cold PBS. Fluores- cence was analyzed using a flow cytometer (FACS Calibur, Becton-Dickinson Biosciences). Lymphocyte marker distribution was analyzed using CellQuest 3.1f software.

CD subsets (CD4^-CD8^-, CD4⁺CD8⁺, CD4⁺and CD8⁺ in thymocytes; CD19⁺, CD3⁺, CD4⁺ and CD8⁺ in splenocytes and mesenteric lymph node cells), CD4⁺/ CD8⁺ratio, total cell number and organ weight ratio were determined at 12, 24 or 72 h after the last dose in non-immunized mice treated with five doses of PDE inhibitors.

Determination of PFC

The mice were anesthetized with halothane and killed by cervical dislocation. The spleens were removed and placed into Hank’s saline (Institute of Immunol- ogy and Experimental Therapy, Wroc³aw, Poland).

The lymphocytes from the spleens were isolated as described above. After centrifugation at 4°C, the splenocytes were collected from the interface and washed twice in Hank’s saline. Erythrocytes in the suspen- sions of splenocytes were lysed using 0.84% ammo- nium chloride at 37°C for 5 min. After the second wash, the splenocytes were resuspended in Hank’s saline to a concentration of 1 × 10⁶cells/ml. Viability of the isolated splenocytes, as determined by trypan blue dye exclusion, was > 95%. The splenocytes produc- ing hemolytic anti-SRBC antibodies (PFC) were iden- tified by local hemolysis in agar gel, as described by

Jerne et al. [22] with some modifications [32]. The number of PFC in SRBC-immunized mice treated once or five times with PDE inhibitors was determined on days 4 and 7 after injection of SRBC.

Determination of anti-SRBC antibodies in the serum

Blood samples were taken by retro-orbital bleeding from halothane-anesthetized mice. The sera were obtained by centrifugation of the blood (3500 × g, 15 min) and then inactivated at 56°C for 30 min. The total (IgM + IgG) and 2-mercaptoethanol resistant (IgG) serum hemagglutinin titers were defined by the active hemagglutination test, carried out on microplates, as described by Adler [1]. Anti-SRBC hemagglutinin titers in SRBC-immunized mice treated once or five times with PDE inhibitors were determined on days 4, 7 and 14 after SRBC injection.

Statistical analysis

The data obtained in the study were analyzed statisti- cally using Student’st-test. The differences were con- sidered significant at p < 0.05.

Results

The effects of PDE inhibitors on thymocyte subsets

The administration of aminophylline five times augmented the percentage of single-positive CD8⁺thymic cells 12 h after the last dose of drug. An increase in the percentage of immature CD4⁺CD8⁺thymic cells (double-positive cells) was observed 72 h after the last aminophylline administration, and it corresponded with reduced percentages of single-positive CD4⁺and CD8⁺cells. Treatment with milrinone did not change the surface marker expression of thymocytes. Admin- istration of sildenafil temporarily decreased the percentage of CD4⁺CD8⁺thymocytes, which corresponded with an increased percentage of single-positive CD8⁺ cells and consequently resulted in a decreased CD4⁺/CD8⁺ratio, as compared to the control group.

This effect was only observed 24 h after the last dose of sildenafil (Tab. 1).

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The effects of PDE inhibitors on splenocyte and mesenteric lymph node cell subsets

Aminophylline modulated splenocyte subsets as manifested by a decreased percentage of B cells (CD19⁺) and increased percentage of T cells (CD3⁺, CD4⁺and/or CD8⁺). A similar effect of sildenafil on the splenocyte subpopulations was observed. These

changes were maintained for 72 h. A decrease in the weight ratio of the spleen was noted 72 h after the last dose of sildenafil. Exposure to milrinone also decreased the percentage of CD19⁺ splenocytes (but only 24 h after the last dose) and increased the percentage of CD4⁺spleen cells (after 24 and 72 h).

Aminophylline also diminished the percentage of CD19⁺cells in the mesenteric lymph nodes, but this

Tab. 1.The percentage of thymocyte subpopulations in mice treated five times with PDEinhibitors. The mean values (n = 8) and standard deviations are presented

Group Index 12 h 24 h 72 h

Control group total number of cells (mln) 49.8 ± 11.0 55.0 ± 14.4 56.7 ± 23.7

weight ratio 0.14 ± 0.03 0.25 ± 0.11 0.23 ± 0.13

% CD4^-CD8^- 4.66 ± 2.10 3.15 ± 0.69 2.86 ± 1.00

% CD4⁺CD8⁺ 79.07 ± 3.18 80.89 ± 2.61 80.89 ± 1.51

% CD4⁺ 12.96 ± 3.64 12.51 ± 1.98 13.06 ± 1.39

% CD8⁺ 3.32 ± 0.58 3.46 ± 0.52 3.12 ± 0.27

CD4⁺/CD8⁺ 4.12 ± 1.67 3.65 ± 0.47 4.21 ± 0.60

Aminophylline total number of cells (mln) 61.1 ± 22.3 61.9 ± 19.4 44.5 ± 14.7

weight ratio 0.15 ± 0.05 0.28 ± 0.15 0.18 ± 0.06

% CD4^-CD8^- 3.92 ± 0.72 2.33 ± 0.57* 2.58 ± 0.65

% CD4⁺CD8⁺ 77.12 ± 2.70 80.42 ± 2.03 83.44 ± 1.29**

% CD4⁺ 14.29 ± 2.09 13.80 ± 1.81 11.27 ± 1.14*

% CD8⁺ 4.67 ± 0.36** 3.45 ± 0.33 2.72 ± 0.26**

CD4⁺/CD8⁺ 3.07 ± 0.47 4.04 ± 0.65 4.17 ± 0.43

Milrinone total number of cells (mln) 48.1 ± 17.6 62.7 ± 12.8 53.5 ± 14.2

weight ratio 0.15 ± 0.04 0.26 ± 0.07 0.23 ± 0.06

% CD4^-CD8^- 4.09 ± 1.17 2.71 ± 0.71 2.88 ± 0.97

% CD4⁺CD8⁺ 79.37 ± 4.32 81.30 ± 1.31 82.30 ± 3.11

% CD4⁺ 12.38 ± 2.29 12.36 ± 0.97 12.03 ± 1.64

% CD8⁺ 4.16 ± 1.39 3.64 ± 0.68 2.87 ± 0.79

CD4⁺/CD8⁺ 3.13 ± 0.64 3.51 ± 0.79 4.33 ± 0.64

Sildenafil total number of cells (mln) 38.2 ± 12.1 52.7 ± 8.8 38.9 ± 8.8

weight ratio 0.13 ± 0.06 0.22 ± 0.04 0.19 ± 0.05

% CD4^-CD8^- 3.35 ± 1.09 3.99 ± 0.89 3.47 ± 0.48

% CD4⁺CD8⁺ 78.91 ± 3.98 77.66 ± 2.43* 80.37 ± 2.15

% CD4⁺ 13.36 ± 0.68 13.43 ± 1.48 12.69 ± 1.84

% CD8⁺ 4.37 ± 1.22 4.92 ± 0.61** 3.48 ± 0.45

CD4⁺/CD8⁺ 3.18 ± 0.68 2.74 ± 0.16** 3.68 ± 0.58

* p < 0.05 as compared to the control group, ** p < 0.01 as compared to the control group

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Tab. 2.The percentage of splenocyte subpopulations in mice treated five times with PDEinhibitors. The mean values (n = 8) and standard deviations are presented

weight ratio 0.52 ± 0.07 0.64 ± 0.10 0.67 ± 0.12

% CD19⁺ 78.06 ± 2.73 78.46 ± 6.10 73.16 ± 1.24

% CD3⁺ 16.16 ± 2.19 14.6 ± 5.23 18.25 ± 1.57

% CD4⁺ 10.65 ± 1.46 10.66 ± 3.00 12.76 ± 0.54

% CD8⁺ 3.45 ± 0.68 3.62 ± 2.11 3.88 ± 0.52

CD4⁺/CD8⁺ 3.15 ± 0.45 3.39 ± 1.01 3.33 ± 0.37

Aminophylline total number of cells (mln) 95.8 ± 19.5 80.5 ± 25.0 66.3 ± 18.0

weight ratio 0.53 ± 0.05 0.62 ± 0.23 0.61 ± 0.06

% CD19⁺ 70.73 ± 4.72** 64.66 ± 5.10** 70.98 ± 2.88

% CD3⁺ 21.53 ± 3.30** 26.23 ± 3.73** 20.02 ± 2.39

% CD4⁺ 12.52 ± 2.00 18.31 ± 2.44** 15.61 ± 1.39**

% CD8⁺ 6.52 ± 1.74** 7.15 ± 1.79** 4.84 ± 0.68

CD4⁺/CD8⁺ 2.00 ± 0.47** 2.66 ± 0.56 3.25 ± 0.30

Milrinone total number of cells (mln) 109.1 ± 10.2* 108.6 ± 18.7* 69.4 ± 25,5

weight ratio 0.50 ± 0.04 0.66 ± 0.06 0.61 ± 0.06

% CD19⁺ 75.24 ± 4.07 72.42 ± 4.32* 71.64 ± 1.95

% CD3⁺ 16.85 ± 1.62 18.17 ± 2.91 18.84 ± 1.61

% CD4⁺ 11.64 ± 1.57 14.24 ± 1.72* 14.87 ± 1.36*

% CD8⁺ 3.63 ± 0.68 4.69 ± 1.02 3.86 ± 0.39

CD4⁺/CD8⁺ 3.26 ± 0.57 3.10 ± 0.41 3.87 ± 0.39**

Sildenafil total number of cells (mln) 120.3 ± 9.1** 124.0 ± 14.2** 48.7 ± 14.0**

weight ratio 0.46 ± 0.03 0.59 ± 0.06 0.55 ± 0.08*

% CD19⁺ 70.85 ± 3.71** 68.92 ± 3.31** 69.46 ± 3.24*

% CD3⁺ 21.17 ± 3.33** 23.46 ± 3.03** 20.44 ± 1.98*

% CD4⁺ 15.99 ± 1.99** 18.01 ± 2.42** 14.82 ± 1.89

% CD8⁺ 5.85 ± 1.54** 5.91 ± 1.06* 5.83 ± 1.01**

CD4⁺/CD8⁺ 2.83 ± 0.49 3.08 ± 0.31 2.57 ± 0.31**

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effect was found only 12 h after the last dose of aminophylline. Administration of aminophylline also augmented the percentage of T mesenteric lymph node cells (CD3⁺, CD4⁺and CD8⁺). A decrease in the weight ratio of mesenteric lymph nodes was observed 72 h after the last dose of aminophylline. In sildenafil-treated mice, a temporary increase in the percentage of CD4⁺cells and the CD4⁺/CD8⁺ratio was

found after 24 h. A decrease in the percentage of CD19⁺cells and a rise in the percentage of CD3⁺cells in the mesenteric lymph nodes was also noted after 72 h. Milrinone had a different effect on the subsets of cells within the mesenteric lymph nodes. This drug reduced the percentage of CD8⁺cells, which corresponded with an increased CD4⁺/CD8⁺ratio (Tab. 2 and 3).

Tab. 3.The percentage of mesenteric lymph node cell subpopulations in mice treated five times with PDEinhibitors. The mean values (n = 8) and standard deviations are presented

weight ratio 0.51 ± 0.12 0.71 ± 0.15 0.73 ± 0.14

% CD19⁺ 63.01 ± 5.02 62.12 ± 12.38 65.49 ± 2.04

% CD3⁺ 33.41 ± 5.66 32.77 ± 12.45 30.28 ± 1.93

% CD4⁺ 24.39 ± 2.27 21.45 ± 5.69 25.57 ± 1.06

% CD8⁺ 13.05 ± 3.76 14.88 ± 7.39 7.04 ± 1.00

CD4⁺/CD8⁺ 1.99 ± 0.51 1.71 ± 0.65 3.69 ± 0.52

Aminophylline total number of cells (mln) 96.0 ± 41.9 62.0 ± 14.7* 52.1 ± 42.7

weight ratio 0.58 ± 0.10 0.71 ± 0.32 0.53 ± 0.19 *

% CD19⁺ 55.4 ± 4.87* 56.34 ± 3.70 62.47 ± 3.93

% CD3⁺ 40.75 ± 4.96* 40.31 ± 3.67 33.44 ± 3.59

% CD4⁺ 26.21 ± 1.78 27.32 ± 2.17* 27.04 ± 2.30

% CD8⁺ 16.84 ± 2.57* 13.31 ± 2.64 8.61 ± 1.59*

CD4⁺/CD8⁺ 1.58 ± 0.20 2.11 ± 0.38 3.20 ± 0.42

Milrinone total number of cells (mln) 97.1 ± 23.1 68.1 ± 26.6 98.55 ± 45.43*

weight ratio 0.53 ± 0.14 0.83 ± 0.21 0.74 ± 0.09

% CD19⁺ 65.80 ± 2.75 62.83 ± 2.25 68.06 ± 3.20

% CD3⁺ 30.54 ± 2.50 33.18 ± 4.06 28.02 ± 3.28

% CD4⁺ 22.54 ± 2.21 24.85 ± 2.85 24.04 ± 3.31

% CD8⁺ 9.24 ± 2.49* 10.21 ± 0.66 4.87 ± 1.05**

CD4⁺/CD8⁺ 2.55 ± 0.53* 2.47 ± 0.32* 5.07 ± 0.89**

Sildenafil total number of cells (mln) 67.7 ± 15.2 86.8 ± 28,5** 56.7 ± 30.6

weight ratio 0.44 ± 0.10 1.03 ± 0,39 0.67 ± 0.08

% CD19⁺ 58.86 ± 6.06 59.36 ± 4.32 58.18 ± 7.39*

% CD3⁺ 37.67 ± 5.72 37.05 ± 4.46 35.51 ± 6.10*

% CD4⁺ 26.46 ± 3.25 26.93 ± 1.44* 26.84 ± 1.97

% CD8⁺ 12.74 ± 4.01 11.85 ± 2.64 10.34 ± 4.62

CD4⁺/CD8⁺ 2.22 ± 0.49 2.35 ± 0.41* 2.96 ± 1.01

* p< 0.05 as compared to the control group, ** p< 0.01 as compared to the control group

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The effects of one administration of PDE inhibitors on the humoral immune response

One administration of all the PDE inhibitors under investigation did not change the number of splenocytes that produced anti-SRBC antibodies (PFC) on day 4 but did increase the number of PFC on day 7 after immunization. No changes in the hemagglutinin titers after a single administration of aminophylline were observed. A single injection of milrinone increased the total anti-SRBC hemagglutinin titers only on day 14 after administration. One administration of sildenafil augmented the 2-ME-resistant anti-SRBC hemagglutinin titers on day 4 but decreased total

anti-SRBC antibodies on day 7 after immunization (Tab. 4).

The effects of five administrations of PDE inhibitors on humoral immune response

Administration of aminophylline or milrinone five times increased the number of PFC on day 4 after immunization, whereas sildenafil administered five times did not change the number of PFC. Treatment five times with aminophylline induced a decrease in the total anti-SRBC hemagglutinin on day 14. Admin- istration of milrinone five times did not influence anti-SRBC antibodies. Sildenafil administered five

Tab. 4.The humoral immune response in SRBC-immunized mice after a single administration of PDEinhibitors. The mean values (n = 8) and standard deviations are presented

Group

PFC/10⁶cells

Anti-SRBC hemagglutinin (log₂titer)

Total 2-ME resistant

4 day 7 day 4 day 7 day 14 day 4 day 7 day 14 day

Control group 2055.0 ± 431.4 333.3 ± 161.6 9.00 ± 0.93 9.57 ± 0.53 10.00 ± 2.07 3.13 ± 1.13 5.25 ± 1.49 7.63 ± 1.30 Aminophylline

1´ 20 mg/kg 2144.3 ± 625.7 670.0* ± 379.5 9.14 ± 1.07 9.75 ± 1.49 11.50 ± 0.93 3.50 ± 1.41 4.25 ± 1.75 7.63 ± 1.06 Milrinone

1´ 1 mg/kg 2145.7 ± 392.7 834.3* ± 442.7 9.38 ± 1.85 9.38 ± 1.06 12.25 * ± 0.89 3.00 ± 1.07 4.25 ± 0.46 8.25 ± 1.16 Sildenafil

1´ 1 mg/kg 2292.5 ± 695.8 728.7* ± 388.2 8.13 ± 1.46 8.25** ± 1.04 11.67 ± 0.82 5.50 ** ± 1.07 4.50 ± 1.77 8.00 ± 1.10

Tab. 5.The humoral immune response in SRBC-immunized mice treated five times with PDEinhibitors. The mean values (n = 8) and standard deviation are presented

Group

PFC/10⁶cells

Anti-SRBC hemagglutinin (log₂titer)

Total 2-ME resistant

4 day 7 day 4 day 7 day 14 day 4 day 7 day 14 day

Control group 2052.5 ± 666.1 221.2 ± 115.9 8.38 ± 1.51 8.13 ± 0.99 10.5 ± 1.69 5.25 ± 1.28 5.86 ± 1.07 8.50 ± 1.60 Aminophylline

5´ 20 mg/kg 5165.0* ± 2616.8 223.7 ± 78.4 8.40 ± 1.34 8.00 ± 1.51 8.50* ± 1.69 5.25 ± 1.28 6.88 ± 0.83 7.88 ± 1.36 Milrinone

5´ 1 mg/kg 4743.7* ± 2691.9 312.5 ± 177.7 8.86 ± 2.04 7.75 ± 0.71 10.5 ± 2.07 4.88 ± 2.17 5.63 ± 2.13 10.13 ± 1.64 Sildenafil

5´ 1 mg/kg 3026.2 ± 1346.9 360.0 ± 173.5 6.63* ± 1.51 8.50 ± 2.00 9.63 ± 1.69 5.25 ± 1.16 5.75 ± 1.98 7.00 ± 1.51

* p < 0.05 as compared to the control group

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times suppressed total anti-SRBC hemagglutinin on day 4 after immunization (Tab. 5).

Discussion

The results obtained in our previous study showed that a single administration of aminophylline, milrinone or sildenafil increased the percentage of double- positive (CD4⁺CD8⁺) thymocytes, with correspond- ing decreases in the percentages of single-positive CD4⁺and CD8⁺thymic cells 12 h after drug administration [39]. In the present study, similar changes were only observed 72 h after the last dose of aminophylline. Interestingly, the administration of sildenafil five times temporarily exerted an opposite effect:

a decreased percentage of CD4⁺CD8⁺cells and an increased percentage of CD8⁺cells 24 h after the last dose. Generally, the influence of the drugs on thymocyte subsets was more pronounced after a single dose than after five doses.

The changes in thymocyte subsets demonstrate that aminophylline, milrinone and sildenafil influence T cell maturation in the thymus. Cyclic AMP is one of the factors that induces programmed cell death in thymocytes [19, 25, 31]. Therefore, PDE inhibitors can stimulate thymocyte apoptosis by inhibiting degrada- tion of cAMP. It was also found that TNF-a enhanced cAMP-induced thymocyte apoptosis [3, 20, 23]. Both nonselective, i.e., pentoxifylline [35, 44], and selective PDE drugs, i.e., PDE4 and PDE3 inhibitors [33, 34, 42], reduced TNF-a production, but the nonselec- tive drugs and selective PDE3 inhibitors were less potent [33, 42, 43]. Therefore, it seems quite likely that PDE inhibitors can modulate thymocyte maturation also by influencing TNF-a synthesis. Interestingly, zaprinast, a selective PDE5 inhibitor, exerts a slightly stimulating effect on TNF-a release [33]. This oppo- site influence of a selective PDE5 inhibitor on TNF-a production can partially explain the differences observed between the effects of sildenafil and the other two drugs under investigation on thymocyte subsets.

The results obtained in our previous studies showed that a single administration of aminophylline, milrinone or sildenafil increased the percentage of B lymphocytes (CD19⁺) and decreased the percentages of T lymphocytes (CD3⁺, CD4⁺, CD8⁺) in the mesenteric lymph nodes [39]. In the present study, an oppo-

site effect on the lymphocyte subsets in mesenteric lymph nodes was observed 12 h after the last dose of aminophylline and 72 h after the last dose of sildenafil. However, milrinone decreased the percentage of CD8⁺cells, as in our previous study. Additionally, we had previously found that a single administration of PDE inhibitors had only a slight effect on splenocyte subsets. A decrease in the percentage of T cells 72 h after a single injection of aminophylline was observed [39]. In the present experiment, aminophylline had the opposite effect. A single administration of sildenafil did not change the splenocyte subsets [39], but when it was administered five times, it markedly decreased the percentage of B cells and increased the percentage of T cells. This significant effect of sildenafil was observed even though only marginal activity of the PDE5 isozyme was detected in T cells [40]. In- terestingly, PDE5 inhibitors can modulate cell functions by increasing not only levels of cGMP but also cAMP, as elevated cGMP inhibits inactivation of cAMP [30]. Generally, a single administration of PDE inhibitors had a more significant impact on the lymphocyte subsets of the mesenteric lymph nodes than five doses. However, a more potent influence of the splenocyte subsets was observed when the drugs were administered five times. The differences between the effects observed after a single dose vs. five doses of the drugs suggest that their influence on the lymphocyte subsets was changing during the course of the treatment.

Not much information is available about the influence of selective PDE inhibitors on B lymphocyte function. Coqueret et al. [7] reported that selective PDE4 inhibitors (rolipram and Ro 20-1724 at a concentration of 10 µM) reduced IL-4-induced IgE production by peripheral blood mononuclear cellsin vi- tro. According to them, this effect resulted from the failure of co-stimulatory signals from monocytes be- cause there was no inhibition of IgE secretion in purified B lymphocytes. In the same study, neither the selective PDE3 inhibitors (milrinone and SK&F 94- 836) nor a selective PDE5 inhibitor (SK&F 96-231) changed IgE synthesis. Strannegard and Strannegard [37] also suggested an indirect influence of cAMP- elevating substances on IgE productionvia T lymphocytes. They found that the nonselective PDE inhibitor, theophylline, suppressed spontaneous IgE secretion by peripheral blood mononuclear cells, but only at high concentrations (10^–3– 10^–4M). Interestingly, an increase in IgE production was observed at low con-

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centrations of the drug (10^–8– 10^–12 M). The results from the present experiment show negligible or no inhibitory influence of the drugs on anti-SRBC hemagglutinins. The temporary stimulating effect on the number of plaque forming splenocytes vs. a decreas- ing percentage of B cells and an increasing percentage of T cells in the spleen suggest that the influence of aminophylline, milrinone and sildenafil on B lymphocytes is rather indirect. For this reason, even if B cells do not have PDE5 isozyme, PDE5 inhibitors can modulate the activity of these cells indirectly, i.e.,via T cells.

It seems quite likely that the modulating effect of PDE inhibitors on lymphocyte subpopulations and humoral immune response is related to their influence on synthesis and release of cytokines. Schmidt et al.

[36] conducted in vitro studies and reported that the nonselective PDE inhibitors theophylline, 3-isobutyl- 1-methylxanthine (IBMX) and enprofylline and a selective PDE4 inhibitor, rolipram, enhanced anti- CD3-induced IL-5 production by murine Th2 cells (with only a marginal effect on IL-4 production). The PDE3 inhibitors motapizone and milrinone affected IL-5 production to a lesser degree, whereas a selective PDE5 inhibitor, zaprinast, had no effect on IL-5 synthesis [36]. Some authors [5, 43] have reported that Th1 lymphocytes are more sensitive to the action of PDE4 or PDE3 inhibitors than Th2 lymphocytes. Be- cause PDE4 or PDE3 inhibitors decreased IL-2 and IFN-g secretion with little or no inhibitory effect on IL-4 and IL-5 production. This influence can partially explain the absence of an inhibitory effect of aminophylline, milrinone and sildenafil on humoral immune response. In contrast, Crocker et al. [8] conducted in vitro studies and found that theophylline, a nonse- lective PDE inhibitor, and Ro20-1724, a selective PDE4 inhibitor, suppressed the synthesis and release of IL-4 and IL-5. Lin et al. [27] conducted in vivo studies and found that administration of aminophylline (5 or 25 mg/kg,ip) to ovoalbumin-sensitized rats decreased Th2 cytokine mRNA expression but increased Th1 cytokine mRNA expression.

The results obtained by other authors show that PDE inhibitors modulated the proliferative response of lymphocytes. Gantner et al. [17] investigated the influence of a selective PDE3 inhibitor motapizone, and the selective PDE4 inhibitors rolipram and piclamilast on IL-4/LPS-induced proliferation of B lymphocytes. They found that motapizone did not

change the proliferative response of B cells. Interest- ingly, rolipram and piclamilast at concentrations greater than 1 µM increased the proliferation of B lymphocytes. In contrast, selective PDE4 inhibitors and nonselective PDE drugs suppressed proliferative activity of T lymphocytes [15, 16, 18, 29]. Selective PDE3 or PDE5 inhibitors had little or no inhibitory influence [14–16, 18, 29]. When a combination of PDE3 and PDE4 selective drugs was used, additional suppressive effects on T lymphocyte proliferation were observed [5, 11, 15, 18, 29].

In summary, aminophylline, milrinone and sildenafil had a modulating effect on lymphocyte subsets, as they increased and/or decreased the number of specific CD antigens on the surface of thymocytes, splenocytes and lymph node cells. The most significant effect observed in the present study was a decrease in the percentage of B cells and an increase in the percentage of T cells in splenocyte subsets. Only a slight modulating influence on humoral immune response in SRBC-immunized mice was observed. These effects depended on the type of the drug and the number of doses administered. The drugs under investigation did not show an immunosuppressive effect similar to the immunosupressive action of other PDE inhibitors, such as selective PDE4 inhibitors.

Acknowledgment:

This study was supported by the Polish State Committee for Scientific Research; Ministry of Science and Higher Education, (Grant No. 2 P06K 014 28).

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Received:

December 14, 2009; in revised form: June 29, 2010.