Gender-specific immunological effects of the phosphodiesterase 5 inhibitor sildenafil in healthy mice

Contents lists available at Molecular Immunology Gender-specific immunological effects of the phosphodiesterase 5 inhibitor sildenafil in healthy mice Svetlana Karakhanova , Yuhui Yang , Julia Link , Sabine Soltek , Katharina von Ahn , Viktor Umansky , Jens Werner , Alexandr V. Bazhin a Department of General Surgery, University of Heidelberg, Heidelberg, Germany b Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China c Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany d Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany Phosphodiesterase 5 (PDE5) is a pharmacological target in erectile dysfunction, pulmonary hypertension Received 29 April 2013 and in other indications. In tumor-bearing mice an inhibition of PDE5 with sildenafil prolongs survival Received in revised form 17 June 2013 of the animals through the augmentation of antitumor immunity, indicating the immunomodulatory Accepted 27 June 2013 properties of this drug. Effects of sildenafil on the immune system in healthy organisms are poorly inves- tigated. In this work we showed that chronic application of sildenafil in healthy mice leads to opposite gender-dependent effects on NK cells, subpopulations of CD4+ and CD8+ T cells, activated conventional T cells, and to a decrease in Gr-1+CD11b+ immature myeloid cells. Besides, sildenafil treatment decreases Gender-specific effects the serum concentration of interleukin-6. Ex vivo cultivation of isolated splenocytes with sildenafil results in an increase in CD4+ T cells and a concomitant decrease in B cells and central memory CD8+ T cells. Ex Phosphodiesterase 5 vivo modulatory properties of sildenafil are not gender-specific, indicating the importance of sildenafil's pharmacokinetics for it immunomodulatory activity in vivo. While the PDE5 expression is equal in the splenocytes from both genders, splenocytes from female mice possess higher basal level of cGMP com- pared to the male ones. Moreover, cultivation of splenocytes obtained from female but not male mice with sildenafil leads to an increase in cGMP concentration, making sildenafil's pharmacodynamics also responsible for gender-specific effects of the drug. Thus, this work secures conclusive evidence that the PDE5 inhibitor sildenafil possesses immunomodulatory properties and these effects are gender-specific.
Immunological clinical trials are needed to prove the potential immunomodulatory effects of sildenafil 2013 Elsevier Ltd. All rights reserved.
cyclic nucleotides and can be classified according to their tissue expression, biochemical properties, regulation, and the sensitiv- Cyclic guanosine monophosphate (cGMP) was one of the first ity to various pharmacological agents (PDE-5 molecules described as the cellular second messenger (together with PDE6 and PDE9) belongs to the group of cGMP- Its intracellular concentration was shown to specific PDEs, with two ubiquitous isoforms, PDE5A1 and PDE5A2, be regulated through the cGMP synthesis by guanylyl cyclases while PDE-5A3 is specific for smooth muscle cells ( (GC, EC 4.6.1.2) and the degradation via dual-specific or cGMP- specific phosphodiesterases (PDE, EC 3.1.4.17). PDE represent a PDE5 is a pharmacological target in erectile dysfunction and pul- family of eleven enzymes (PDE1-PDE11) that specifically hydrolyze monary hypertension ( There is a large body of evidence demonstrating that inhibition of PDE5 can also be important for other indications: cognition and stroke (cardio- cGMP, cyclic guanosine monophosphate; FACS, fluorescence activated cell sorting; GC, guanylyl cyclases; IL, interleukin; mAbs, monoclonal anti- vascular diseases (inhibition of tumor cell growth bodies; NK cells, nature killer cells; NO, nitric oxide; PDE5, phosphodiesterase 5; TCR, (and other disorders, in which the benefit of T cell receptor.
PDE5 inhibition is not easy to explain. Therefore, clinical usage of ∗ Corresponding author at: University Hospital Heidelberg, Im Neuenheimer Feld PDE5 suppression makes the development of PDE5 inhibitors very 430, 69120 Heidelberg, Germany. Tel.: +49 6221 5636932; fax: +49 6221 568240.
attractive for clinic and pharmacologic industry. At present, sil- E-mail address: (A.V. Bazhin).
denafil is the first-line therapeutic for erectile dysfunction Equal contribution.
0161-5890/$ – see front matter 2013 Elsevier Ltd. All rights reserved.
S. Karakhanova et al. / Molecular Immunology 56 (2013) 649–659 and is newly approved for the treatment of pulmonary days later and their spleens were removed to prepare a splenocyte single cell suspension as described elsewhere ( Recently, we and another group have showed that chronic appli- cation of sildenafil to tumor-bearing mice prolonged survival of 2.4. FACS analysis of murine splenocytes the animals through the augmentation of anti-tumor immunity This unexpected effect Freshly isolated spleen cell suspension was resuspended in the was due to an inhibition of myeloid-derived suppressor cell (MDSC) stain buffer (PBS and 1 mM EDTA). The cells were counted and the function and, as a consequence, to a partial restoration of the T cell density was adjusted to 4 × 107/mL. The unspecific binding caused receptor (TCR) ␨-chain expression in T cells and of their prolifera- by the Fc receptors was blocked by incubating the cells with anti- tion (It was concluded that mouse CD16/CD32 antibody (1 ␮L for 2 × 106 cells) at 4 ◦C in the the PDE5 inhibitor sildenafil possesses certain immunomodulatory dark for 10 min. Then 50 ␮L cell suspension (2 × 106 cells) was incu- properties in tumor-bearing mice.
bated with 50 ␮L of the stain buffer containing various mAbs at Taking in account the potential immunoregulatory property of 4 ◦C in the dark for 15 min. The amount of each antibody had been sildenafil in tumor-bearing mice, it is important to know whether titrated before the experiment. After two-time washing with the this drug can induce immunological changes in healthy organ- stain buffer, the cells were resuspended in 100 ␮L of the stain buffer isms as well, since sildenafil is a very common medicament, and transferred to a 5 ml polystyrene tube containing 300 ␮L of the which sales peaked at about 2 million US dollars in 2008. So stain buffer for FACS analysis. Foxp3 staining buffer set was used for far, there are only two investigations dealing with effects of PDE intracellular staining, according to the manufacturer's instruction.
inhibitors in healthy mice: The following panels with the different combinations of anti- that sildenafil increased the production of inter- bodies were used in our experiments: (1) panel for CD4+ T and leukin (IL)-1␤ and nitric oxide (NO) by peritoneal macrophages ex CD8+ T cells. Different subsets of CD4+ T cells were characterized as vivo, increased the percentage of phagocytosing granulocytes and naïve T cells (CD62L+CD45RBhi), effector T cells (CD62L−CD45RBhi), decreased the percentage of phagocytosing monocytes. In the sec- central memory T cells (Tcm, CD62L+CD45RBlo) and effector mem- ory T cells (Tem, CD62L−CD45RBlo); the corresponding subsets of demonstrated that sildenafil decreased percentage of B cells while CD8+ T cells were naïve T cells (CD62L+CD44−), effector T cells increased percentage of T cells (CD3+, CD4+ and CD8+ cells) ex vivo.
(CD62L−CD44−), central memory T cells (Tcm, CD62L+CD44+), effec- Nevertheless, sildenanfil's effects on specific lymphocyte subpop- tor memory T cells (Tem, CD62L−CD44+); (2) panel for Treg/NK/NKT ulations and other leukocyte subsets were not assessed, especially cells. Treg cells were characterized as Foxp3+CD25+ within the CD3+CD4+ T cell population. NK cells (CD3−NK1.1+) and NKT cells Therefore, the aim of this work was to investigate immuno- (CD3+NK1.1+) were identified, and their activation state was mea- logical effects of sildenafil both in vivo and ex vivo. Results of sured by the expression of CD69; (3) panel for dendritic cells.
our work present conclusive evidence that this PDE5 inhibitor Mature conventional dendritic cells (cDC, CD11chiCD11b+) and indeed possesses immunomodulatory properties and these effects mature plasmacytoid dendritic cells (pDC, CD11cintCD45R+) were further characterized by the high expression of MHC-II molecules (I-Ab). The activation state of both DC subsets were measured 2. Materials and methods
by the expression of CD80 (B7-1) and CD86 (B7-2); (4) Panel for granulocyte/macrophage/monocyte. Here CD11b+Gr-1+ cells were analyzed. All the gates were set according to the corresponding fluorescence minus one (FMO) control.
The following anti-mouse directly conjugated monoclonal anti- bodies (mAbs) were used for the fluorescence activated cell sorting 2.5. In vitro treatment of murine splenocytes with sildenafil (FACS) staining: rat or hamster anti-mouse antibodies CD4-Pacific Blue, CD3e-V500, CD45RB-PE, CD8a-PerCP-Cy5.5, CD62L-APC, CD45R-Pacific Blue, CD80-FITC, I-A[b]-PE, CD11b-PerCP-Cy5.5, After the preparation of splenocyte single-cell suspension, cells CD11c-APC, CD86-PE-Cy7, Gr-1-APC-Cy7, NK1.1-PE, CD69-PerCP- were seeded in the 6-well plate at the density of 106 cells/mL. Then Cy5.5, and CD25-APC (BD Bioscience, Germany). Fc receptor the cells were treated with different concentrations of sildenafil block (anti-mouse CD16/CD32), and Foxp3-FITC were purchased citrate (75 nM, 750 nM and 7.5 ␮M) or vehicle control in 5% CO2 at from eBioscience (Germany). CD19-PE was from Immunotools 37 ◦C for 24 h. Afterwards all the cells, including the adherent cells, (Germany). Sildenafil citrate was obtained from Sigma–Aldrich were recovered for FACS analysis, or for RNA isolation, or cGMP (Germany). Revatio® 20 mg tablets (contained sildenafil) were pur- chase by Pfizer (Germany).
2.6. RNA isolation and real-time RT-PCR analysis Total RNA from splenocytes was isolated using RNeasy mini kit C57BL/6 mice (6–8 weeks) were purchased from Charles River (Qiagen, Germany) according to the manufacturer's instructions.
(Germany) and kept under specific pathogen-free condition in the RNA concentrations were determined using a NanoPhotometerTM animal facility of University Heidelberg (IBF, Heidelberg). Animal Pearl (SERVA Electrophoresis, Germany). Real-time RT-PCR anal- experiments were carried out after approval by the authorities ysis was done as described elsewhere (using the SYBR-Green system and primers for PDE5a, ˇ-actin and GAPDH provided by Qiagen (Germany), and measured using a Light-Cycler 2.3. Sildenafil treatment of mice (Roche, Germany). For each experiment, a melting curve analy- sis and a gel electrophoresis of PCR products were performed to Drinking water for C57BL/6 mice was supplemented with sil- exclude primer dimers. Data were analyzed using the compara- denafil (compounded from pulverized Revatio® tablets) to yield a tive Ct method (Measurements were dose of 20 mg/kg body weight per day. The mice were sacrificed 21 performed in triplicate.
S. Karakhanova et al. / Molecular Immunology 56 (2013) 649–659 Fig. 1. CD4+ T lymphocytes and their subpopulations in spleen from male and female mice after sildenafil treatment. Data from four independent experiments are presented
as whiskers plots (n = 10), *p < 0.05; ***p < 0.001, control group vs. treatment group.
S. Karakhanova et al. / Molecular Immunology 56 (2013) 649–659 Fig. 2. CD8+ lymphocytes and their subpopulations in spleen from male and female mice after sildenafil treatment. Data from four independent experiments are presented
as box-and-whiskers plots (n = 10), *p < 0.05; control group vs. treatment group.
S. Karakhanova et al. / Molecular Immunology 56 (2013) 649–659 Fig. 3. Treg, Tcon, activated Tcon, NK and NKT cells in spleen from male and female mice treated with sildenafil. Data from four independent experiments are presented as
whiskers plots (n = 10), *p < 0.05; control group vs. treatment group.
S. Karakhanova et al. / Molecular Immunology 56 (2013) 649–659 A 6ytescoku
of le
%+b 2
Fig. 4. Gr-1+CD11b+ leukocytes in spleen from all (A), and male and female (B) mice treated with sildenafil. Data from four independent experiments are presented as
box-and-whiskers plots (n = 20 for (A), and n = 10 for (B)), *p < 0.05; control group vs. treatment group.
2.7. cGMP measurement by means, SE, median, 25% and 75% percentiles, and were presented as box-and-whiskers plots or as column bar graphs. D'Agostino and cGMP was measured with the DetectX® Cyclic GMP Colorimet- Pearson omnibus normality tests were conducted to estimate the ric EIA Kit from ArborAssays (USA) in a regular format as described distribution of data. The null hypothesis (mean values were equal) by the manufacturer. Briefly, harvested cells (about 107 cells) were versus the alternative hypothesis (mean values were not equal) lysed with the provided diluent. 50 ␮L of the lysates or standards was tested for more than two groups by one-way ANOVA with the (provided with the kit) were added into the IgG pre-coated wells.
Dannett's post hoc test, and for two groups by unpaired two-tailed After incubation with different kit reagents at room temperature, t-test for normal distributed variants or by Mann–Whitney test for the plates were read on a plate reader. Standard concentrations of nonparametric distributed data.
cGMP (from 0.5 pmol/mL to 32 pmol/mL) were simultaneously pro- ceeded to estimate cGMP concentration in the probes of interest.
Each sample was measured in duplicate, and the final concentration 3. Results
of cGMP normalized on cell numbers was determined.
3.1. Different in vivo effects of sildenafil on immune cell 2.8. Analysis of cytokines with a luminex assay populations in healthy male and female mice Analysis of IL-2, IL-1␤, IL-6, IL-10 and VEGF was performed as To investigate the immunological effects of sildenafil, we per- described elsewhere (using MILLIPLEX® formed an immunophenotyping of splenocytes from healthy mice MAP Kit (Millipore GmbH, Schwalbach/TS, Germany). Briefly, 25 ␮L by FACS analysis. In these experiments, healthy wild-type mice serum were incubated with color-coded beads coated with the cap- were treated with 20 mg/kg of sildenafil per day for 3 weeks.
ture antibodies for the respective cytokines overnight at 4 ◦C. After Upon the treatment, mice were visibly neither physiologically washing, the beads were incubated with biotinylated secondary nor behaviourally affected. In addition, mice did not show any detection antibodies for each cytokine for 1 h at room tempera- splenomegaly or microsplenia. The percentage of T cells, DC, Gr- ture, followed by the incubation with streptavidin-phycoerythrin 1+CD11b+ leukocytes, NK and NKT cells were analyzed. The gating for 30 min at room temperature. Finally, measurement using strategy for leukocytes is shown in Fig. S1.
Luminex® 100/200 System was performed. According to the First, the percentage of T cells and their memory/naïve state standard curves, the concentration of the respective cytokines was were accessed. In the whole population of our experimental mice calculated and presented in pg/mL.
including equal proportion of male and female animals, we did not find any difference in the percentage of T cells or in their 2.9. Statistical analysis memory/naïve state between treated and control mice (Fig. S2).
Surprisingly, analyzing our data based on the mouse gender, we All statistical analyses were performed using GraphPad Prism observed some different effects of sildenafil in male and female Version 5.01. Distributions of continuous variables were described mice (Whereat, sildenafil treatment has a tendency S. Karakhanova et al. / Molecular Immunology 56 (2013) 649–659 Fig. 5. Serum cytokine (IL2, IL6, IL10, IL1␤ and VEGF) level of mice treated with sildenafil. Data from four independent experiments are presented as box-and-whiskers plots
(n = 16–18 for all animals, and n = 8–9 for different genders), *p < 0.05; control group vs. treatment group.
S. Karakhanova et al. / Molecular Immunology 56 (2013) 649–659 Fig. 6. MNC and leukocyte subpopulations after in vitro cultivation with sildenafil of splenocytes obtained from all (A), male and female (B) mice, and (C) CD8+ Tcm-cells in
all animals and in both genders. Data from two independent experiments are presented as column bar graphs (n = 8 for (A, C), and n = 4 for (B, C)), *p < 0.05, **p < 0.01 and ***p < 0.001; control group vs. treatment group.
to decrease the percentage of CD4+ T cells and to increase the per- the whole population of treated mice but not in male mice sepa- centage of CD8+ T cells in male but not in female mice rately analyzed (Furthermore, we also found a tendency of Concerning the memory/naïve state of T cells, we found opposite an increase in IL-2 in serum of female but not of male mice after sil- effects of sildenafil on CD4+ T cell subpopulations in males and denafil treatment No influence of sildenafil on serum levels females, i.e. a decrease in percentage of Tem and Tcm in males while of IL-10, IL-1␤ or VEGF was observed an increase in percentage of these cells in females (Besides, Thus, sildenafil has some immunomodulatory properties in we observed an increase in percentage of naïve CD4+ T cells after healthy mice, which are gender-specific. This sex-related effects the treatment in male mice. In the subpopulations of CD8+ T cells of sildenafil could be manifested due to the gender-dependent dif- merely a decrease in percentage of the CD8+ Tcm cells was found in ferences in pharmacokinetics (resorption, distribution, metabolism and excretion of sildenafil) or in pharmacodynamics (biochemical With regard to Treg, conventional T cells (Tcon), activated Tcon, effects of sildenafil).
NK and NKT cells, sildenafil treatment showed no effect on these cell populations in the whole gender-mixed population (Fig. S3).
Gender-specific analysis of the data revealed different effects of 3.2. Direct effects of sildenafil on splenocytes ex vivo sildenafil on activated Tcon and NK cells: there was a decrease in the male population while an increase in female mice However, Next, we aimed to understand whether pharmacokinetics, phar- there was still no effect of sildenafil either on Tcon, Treg or NKT cells macodynamics or both are responsible for such gender-dependent effects of sildenafil. For this purpose, we isolated splenocytes from Treatment with sildenafil did not affect the percentage of DC healthy mice (male and female), cultivated them ex vivo for 24 h in the whole mouse population or in gender-specific one (Fig. S4).
with different concentrations of sildenafil (75 nM, 750 nM and Moreover, the treatment did not change also the maturation and 7.5 ␮M) and finally performed a FACS analysis. We found that activation state of these cells irrespective of the mouse gender (Figs.
sildenafil induced no cytotoxicity of mononuclear cells (MNC) This drug did not affect CD8+ T cells, but at the highest Since sildenafil can abolish immunosuppressive effects of Gr- concentration (7.5 ␮M), increased the percentage of CD4+ T cells 1+CD11b+ immature myeloid cells in tumor-bearing mice and diminished the amount of B cells (These effects were wanted to investigate this subpopulation in healthy not gender-specific, since the splenocytes from both genders - male mice after sildenafil treatment. In the whole population of ani- and female manifested the same features after sildenafil treatment mals sildenafil did not affect the percentage of these cells However, such treatment led to a decrease in percentage of Gr- In respect to the naïve/memory phenotype of T lymphocytes, we 1+CD11b+ cells in the spleen of female but not of male mice found that sildenafil at 7.5 ␮M concentration reduced the amount Next we performed an analysis of different factors (IL-2, IL-6, IL- both in the whole population, and separately in 10, IL-1␤ and VEGF) in serum of the experimental animals male or female mice (Sildenafil did not affect other sub- Sildenafil diminished the IL-6 level both in the female group and in populations of CD8+ T-cells and subpopulations of CD4+ T-cells (Fig.
S. Karakhanova et al. / Molecular Immunology 56 (2013) 649–659 Fig. 7. Relative expression of PDE5a (A) and basal cGMP level (B) in splenocytes obtained from male and female mice, and effects of sildenafil on basal cGMP expression in
different genders (C). Data from two independent experiments are presented as box-and-whiskers plots or as column bar graphs (n = 5 for A and n = 8 for B and C), *p < 0.05 and ***p < 0.001; control group vs. treatment group.
S7). We also found no ex vivo effects of sildenafil on Tcon, activated sildenafil might have different effects in different genders because Tcon, Treg, NK and NKT cells (Fig. S7).
of the various basal level of cGMP. To prove this assumption, we iso- Since the ex vivo effects of sildenafil are not gender-dependent lated splenocytes from male and female mice, cultivated them with we assumed that the in vivo differences of sildenafil could be due different concentrations of sildenafil (75 nM, 750 nM and 7.5 ␮M) to different pharmacokinetics properties of sildenafil in male and and finally measured the cellular cGMP level. Interestingly, we female animals.
found an increase in cGMP level (because of PDE5 inhibition) only in splenocytes isolated from female mice as compared to untreated 3.3. Gender-specific effects of sildenafil on the basal cGMP level in splenocytes of healthy mice Thus, pharmacodynamics of sildenafil could also be responsible for different effect of the drug in different genders.
In the last part of the present work we dealt with pharmaco- dynamics effects of sildenafil, i.e. with the biochemical features of this drug. Since sildenafil is a specific inhibitor of PDE5, which hydrolyzes cGMP, this should lead to a reduced level of this cellular Taking in the account the immunomodulatory properties of One possibility could be that murine immune cells have differ- sildenafil in tumor-bearing mice and the high frequency of silden- ent PDE5 expression levels in male and female animals. To prove afil prescription by medicines, the main aim of this work was to this hypothesis, we performed a real-time PCR analysis of the PDE5a investigate immunological effects of sildenafil in healthy mice. Our gene expression in isolated splenocytes from male and female mice.
date demonstrated that sildenafil has immunomodulatory effects The level of PDE5a expression in splenocytes was equal in both in vivo. These effects are (i) different in male and female mice, and genders Thus, we could exclude the difference in the (ii) the gender-dependent differences are due to the pharmacoki- PDE5a expression level in the murine splenocytes as a reason for the netics and pharmacodynamics of this drug.
observed gender-specific effects. Another possibility could be that We showed in vivo that sildenafil possessed a modest immuno- different genders have various basal level of cGMP that would affect suppressive effect in male mice reflected by a decrease in biochemical effects of sildenafil. We measured the basal cGMP level frequencies of activated Tcon and NK cells. On the contrary, in in murine splenocytes and found that male mice have lower level female mice we observed an immunostimulatory effect of sil- of cGMP as compared to female animals (suggesting that denafil represented by an increase in frequencies of activated S. Karakhanova et al. / Molecular Immunology 56 (2013) 649–659 Tcon, NK cells and an elevated IL-2 levels in serum, while the Sure, the man is not a mouse, and we cannot directly transfer our frequency of Gr-1+CD11b+ immature myeloid cells (as a counter- data in murine system to the human one. Therefore, clinical studies part of myeloid-derived suppressor cells in tumor-bearing hosts in men as well as in women should be performed to investigate the and the IL-6 concentration in potential imunomodulatory properties of sildenafil in humans.
serum were decreased. Moreover, the balance between naïve and memory subpopulations of CD4+ T cells was differently affected by sildenafil in male and female mice. While we observed a decrease in percentage of CD4+ Tem and CD4+ Tcm cells in males, these sil- We thank Mr. Markus Herbst, Ms. Tina Maxelon and Ms. Inna denafil effects were opposite in females. Since we did not observe Schwarting for their excellent technical assistance. This work was such effects of sildenafil in ex vivo experiments, we could exclude supported by a grant from B. Braun Stiftung and Heidelberger its direct effect on the expression of subpopulation markers used Stiftung Chirurgie to AVB.
in our panels (except CD62L and CD44 as markers for CD8+ Tcm).
Thus, we conclude that sildenafil indeed shifts the balance between immune stimulatory and suppressive cell, and influences the dis- Appendix A. Supplementary data
tribution of subpopulations in the memory T cell compartment in healthy mice.
Supplementary data associated with this article can be found, In general, differences in immunological parameters between in the online version, at the genders are already known (and could be explained by endocrine and genetic differences between males and females (for review see (The endocrine influence on immune system can be illustrated by the fact that testosterone reduces the NK cell activity (production of proinflam- Bazhin, A.V., Schadendorf, D., Owen, R.W., Zernii, E.Y., Philippov, P.P., Eichmüller, matory cytokines increasing the synthesis of anti-inflammatory cytokines (On the other Boswell-Smith, V., Spina, D., Page, C.P., 2006. hand, estrogens regulate differentiation of DC and other antigen- presenting cells Corbin, J.D., Francis, S.H., Webb, D.J., 2002. In addition, it has also been proposed that estrogens could D'Agostino, P., Milano, S., Barbera, C., Di Bella, G., La Rosa, M., Ferlazzo, V., Farruggio, affect the immunosuppressive function of Treg under partial con- R., Miceli, D.M., Miele, M., Castagnetta, L., Cillari, E., 1999. trol of the immune regulatory molecule B7-H1 From the genetic point of view, some genes on the X chromo- Escribese, M.M., Kraus, T., Rhee, E., Fernandez-Sesma, A., Lopez, C.B., Moran, T.M., some play a role in functions of the immune system (for review see Therefore, we as well can say that "immune Fish, E.N., 2008. cells have a sex" that could be manifested in differ- ent gender-mediated effects of some drugs on immune cells. It Francis, S.H., Busch, J.L., Corbin, J.D., Sibley, D., 2010. has been shown that melatonin possesses sex-dependent effects on the development of systemic lupus erythematosus ( Gabrilovich, D.I., Nagaraj, S., 2009. Caliani Thus, our gender-specific effects of sildenafil are in line with the above-mentioned publications.
Hou, J., Zheng, W.F., 1988. Based on our data, we concluded that the mechanisms underling Hunt, C.M., Westerkam, W.R., Stave, G.M., 1992. the different effects of sildenafil are represented by the pharma- cokinetics and pharmacodynamics of the drug. From the clinical Hyland, R., Roe, E.G., Jones, B.C., Smith, D.A., 2001. pharmacology it is known that anti-hypertensive drugs have sex- related effects due to their gender specific pharmacokinetics and Jimenez-Caliani, A.J., Jimenez-Jorge, S., Molinero, P., Fernandez-Santos, J.M., pharmacodynamics (In general, pharmacoki- Martin-Lacave, I., Rubio, A., Guerrero, J.M., Osuna, C., 2006. netics depends on the gender-differences in expression of gene coding for hepatic drug metabolizing enzymes (i.e. cytochrome P450, glutathione transferase etc.) ( Klein, S.L., 2012. Sildenafil is cleared predominantly in hepatic Libert, C., Dejager, L., Pinheiro, I., 2010. microsomes by CYP3A4 At the same time, human CYP3A4 displayed higher expression at the mRNA and pro- Lin, P.Y., Sun, L., Thibodeaux, S.R., Ludwig, S.M., Vadlamudi, R.K., Hurez, V.J., Bahar, tein levels in women than in men Therefore, R., Kious, M.J., Livi, C.B., Wall, S.R., Chen, L., Zhang, B., Shin, T., Curiel, T.J., 2010.
men and women should have also different bioavailability of the drug, and consequently, they can have a different reaction to sil- Meyer, C., Sevko, A., Ramacher, M., Bazhin, A.V., Falk, C.S., Osen, W., Borrello, I., Kato, denafil. In line with this suggestion, ex vivo experiments showed M., Schadendorf, D., Baniyash, M., Umansky, V., 2011. no differences in effects of sildenafil in male and female spleno- cytes, underlying the importance of the pharmacokinetics in the sex-dependent variability of these effects in healthy mice.
Omori, K., Kotera, J., 2007. However, we could not exclude that sildenafil may interfere Paharkova-Vatchkova, V., Maldonado, R., Kovats, S., 2004. with all steps of cGMP metabolism, manifesting in the difference of cGMP levels from male and female splenocytes. Indeed, we showed that splenocytes from female mice possess higher basal cGMP level Raja, S.G., 2006. than the male ones. It is tempting to speculate that not only PDE5, Rettew, J.A., Huet-Hudson, Y.M., Marriott, I., 2008. inhibited by sildenafil, is important for cGMP level in splenocytes, but also the anabolic pathway of cGMP metabolism, namely GC Sanchez Luna, M., Franco, M.L., Bernardo, B., 2012. could also have an influence on the cGMP level what in turn would affect the reaction to sildenafil.
S. Karakhanova et al. / Molecular Immunology 56 (2013) 649–659 Sarfati, M., Mateo, V., Baudet, S., Rubio, M., Fernandez, C., Davi, F., Binet, J.L., Sutherland, E.W., Rall, T.W., 1958. Delic, J., Merle-Beral, H., 2003. Szczypka, M., Obminska-Mrukowicz, B., 2010a. Scandlyn, M.J., Stuart, E.C., Rosengren, R.J., 2008. Szczypka, M., Obminska-Mrukowicz, B., 2010b. Schmidt, C.J., 2010. Schmittgen, T.D., Livak, K.J., 2008. Ueno, K., Sato, H., 2012. Serafini, P., Meckel, K., Kelso, M., Noonan, K., Califano, J., Koch, W., Dolcetti, L., Bronte, Waxman, D.J., Holloway, M.G., 2009. V., Borrello, I., 2006. Yang, Y., Karakhanova, S., Soltek, S., Werner, J., Philippov, P.P., Bazhin, A.V., Shevchenko, I., Karakhanova, S., Soltek, S., Link, J., Bayry, J., Werner, J., Umansky, V., Bazhin, A.V., 2012. Zhang, L., Zhang, R.L., Wang, Y., Zhang, C., Zhang, Z.G., Meng, H., Chopp, M., 2005.

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