International Immunopharmacology 12 (2012) 342–349
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Differential effects of acyclic nucleoside phosphonates on nitric oxide and cytokines in rat hepatocytes and macrophages Petra Kostecká a, b,⁎, Antonín Holý c, Hassan Farghali b, Zdeněk Zídek a, Eva Kmoníčková a, d a
Institute of Experimental Medicine, Academy of Sciences, Vídeňská 1083, 14220 Prague 4, Czech Republic Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Albertov 4, 12800 Prague 2, Czech Republic Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo nám. 2, 16610 Prague 6, Czech Republic d Institute of Pharmacology and Toxicology, Faculty of Medicine in Pilsen, Charles University, Karlovarská 48, 30166 Pilsen, Czech Republic b c
a r t i c l e
i n f o
Article history: Received 17 April 2011 Received in revised form 29 November 2011 Accepted 5 December 2011 Available online 21 December 2011 Keywords: Acyclic nucleoside phosphonates Cytokines Nitric oxide Rat hepatocytes
a b s t r a c t Acyclic nucleoside phosphonates (ANP) are virostatics effective against viruses like hepatitis B virus and human immunodeﬁciency virus. Our previous reports indicated immunomodulatory activities of ANP in mouse and human innate immune cells. Recently, evidence has increased that hepatocytes may play an active role in immune regulation of the liver homeostasis or injury. In this study we investigated possible immunomodulatory effects of ANP on rat hepatocytes and macrophages. Nitric oxide (NO) production and secretion of cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, IL-18, IFN-γ, TNF-α and GM-CSF) were analyzed under in vitro conditions. Test compounds included: 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; adefovir); 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP); (R)- and (S)-enantiomers of 9-[2-(phosphonomethoxy)propyl] adenine [(R)-PMPA; tenofovir] and [(S)-PMPA]; 9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine [(R)PMPDAP] and [(S)-PMPDAP]. The group of test compounds also included their N6-substituted derivatives. Some of ANP which are able to induce NO production and cytokine secretion in cultured macrophages possess the same immunobiological activity in isolated hepatocytes. The extent of responses is in range of LPS/IFN-γ stimulation in both types of cells. The effects of active ANP on NO expression and cytokine secretion are doseand time-dependent. Interestingly, the spectrum of detected cytokines induced by ANP is broader in hepatocytes. The results also conﬁrm immunomodulatory effects of some ANP on rodent macrophages. Moreover, we demonstrate for the ﬁrst time immunobiological reactivity of primary rat hepatocytes induced by exogenous ANP compounds. The potential of hepatocytes to synthesize cytokines can contribute to better understanding of liver immune function and can serve for pharmacological intervention in liver diseases. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Acyclic nucleotide analogues were originally developed as antivirals effective against replication of both DNA and retroviruses . Among of them, tenofovir (9-[2-(phosphonomethoxy)propyl]adenine; (R)PMPA) is broadly prescribed for HIV patients and later was approved for treatment of hepatitis B. We have shown recently  that a number of ANP derivatives are endowed with the potential to activate secretion of cytokines including the anti–HIV effective chemokines in murine macrophages. This activity is associated with up-regulation of NO biosynthesis mediated by iNOS. Immunostimulatory effect of the same derivatives of ANP was manifested in hPBMC (human peripheral
Abbreviations: ANP, Acyclic nucleoside phosphonates; NO, Nitric oxide; LPS, Lipopolysaccharide; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase. ⁎ Corresponding author at: Institute of Experimental Medicine, Academy of Sciences, Vídeňská 1083, 14220 Prague 4, Czech Republic. Tel.: + 420 241 062 716; fax: + 420 241 062 720. E-mail address: [email protected]
(P. Kostecká). 1567-5769/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2011.12.005
blood mononuclear cells) by secretion of cytokines (TNF-α, IL-10) and chemokines (RANTES, MIP-1α) . Analogous results were obtained in rat peritoneal cell model. Nevertheless we revealed some differences in reactivity of this type of animal cells. Interestingly, in our screening system for immunomodulatory potential of drugs , we have noticed, unlike mice, that rat macrophages considerably enhanced NO after the compounds without any other immune stimulus such as IFN -γ. Further, in vitro secretion of IL10 was not observed in rat peritoneal cells treated by ANP. It has been shown that liver contributes to the immune status of whole organism. NO and cytokines has been recognized as modulators of the immune response. In the liver, inducible NO and cytokines are important signal molecules involved in regulation of organ damage or repair. These processes are orchestrated by non-parenchymal and parenchymal liver cells. In hepatocytes, it is well known that administration of LPS (+ TNF-α, IFN-γ, IL-1β), which mimics bacterial infection and inﬂammation, govern the expression of the iNOS gene with massive production of NO. A balance between the quantity, duration and timing of NO expression may be beneﬁcial or detrimental in liver function . Although there are plethora data describing the inﬂuence of various
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cytokines on hepatocyte functions, less is known about the possibility of hepatocytes to express cytokines. Up to now, induction of cytokines and chemokines (IL-8, IP-10, MIG, MIP-1, MIP-2, MIP3, KC)  has been observed in hepatocytes during liver inﬂammation. In contrast to other types of immune cells in liver, there is still poor information about synthesis, secretion and roles of various classes of cytokines in parenchymal cells (hepatocytes). Based on the data on NO and cytokine regulation of liver functions and our data on immunostimulatory (NO production and cytokine secretion) action of ANP in rat macrophages, we decided to perform the present study on isolated hepatocytes with following goals: 1) whether compounds with proven immunomodulatory potential on immune cells are able to induce NO production in other type of cells possessing molecular components for NO production based on iNOS and 2) whether ANP will be able to induce cytokine secretion in primary rat hepatocytes. Immunomodulatory potential of ANP should be considered for experimentally and clinically oriented studies of liver diseases and during drug development.
exclude possible bacterial contamination using chromogenic Limulus Amoebocyte Lysate assay (Kinetic-QCL; BioWhittaker, Walkersville, MD, USA). The highest 100 μM concentrations of ANP in culture wells contained less than 10 pg/ml of LPS, an amount that is ineffective to activate NO production and cytokine secretion . LPS (Escherichia coli O111:B4) and rat recombinant IFN-γ used in experiments were purchased from Sigma (Prague, Czech Republic). Other chemicals were obtained from standard sources or as described in methods.
2. Materials and methods
2.3. Isolation and culture of rat hepatocytes
2.1. Acyclic nucleoside phosphonates and other chemicals
Primary hepatocytes were isolated from untreated animals using the standard two phase collagenase perfusion method . Separated cells were washed twice through sedimentation then counted and cell viability was examined by trypan blue exclusion method. For further studies hepatocytes with cell viability greater than 90% were used. Freshly isolated hepatocytes were seeded (0.66 × 106 cells/ml) into collagen-coated cell culture plates (Costar, Cambridge, MA, USA) in William's medium E, supplemented with gentamicin (50 μg/ml), Lglutamine (2 mM) and 10% heat-inactivated fetal bovine serum, and maintained at 37 °C, 5% CO2 in humidiﬁed Heraeus incubator for 24 hours in presence or absence of test compounds. At earlier time we used one of the most comprehensive approaches for hepatocyte puriﬁcation by centrifugation in Percoll that depends on the use of low centrifugal speed to sediment more than 99% pure hepatocytes. For the purpose of the present kind of experiments; we found that hepatocyte culture prepared with or without centrifugation in Percoll resulted in comparative nitrite or cytokine production.
All used acyclic nucleoside phosphonates (ANP) were synthesized at the Institute of Organic Chemistry and Biochemistry (Czech Academy of Sciences, Prague, Czech Republic). The synthesis procedure was described previously . The group of test ANP comprised of acyclic phosphonates of adenine (A) and 2,6-diaminopurine (DAP) containing [2-hydroxy-3-(phosphonomethoxy)ethyl] (PME) or [2-hydroxy-3(phosphonomethoxy)propyl] (PMP) in the N9 position of the heterocyclic base. Besides the nonsubstituted prototype compounds (PMPA, PMPDAP, PMEA, and PMEDAP), also their N6-substitued derivates were used. The ANP are summarized in Table 1 according their chemical structure. Stock solutions (5 mM) were prepared in incomplete phenol redfree RPMI-1640 medium (Sigma-Aldrich, Prague, Czech Republic) and sterile ﬁltered through nonpyrogenic 0.22-μm ﬁlters (Costar, Cambridge, MA, USA). They were kept at −20 °C and before use diluted to the required concentration in complete culture medium (described below). All used ANP were checked for lipopolysaccharide (LPS) to
2.2. Animals Outbreed male rats of Wistar strain, 200–250 g of body weight, were purchased from Velaz, (Lysolaje, Czech Republic). Rats were allowed to tap water and standard granulated diet ad libitum. They were kept in transparent plastic cages and maintained in an Independent Environmental Air Flow Animal Cabinet (ESI Flufrance, Wissous, France). Lighting was set on 6–18 h, temperature at 22 °C. All protocols were approved by the Institution Animal Ethics Committee.
2.4. Preparation of rat peritoneal macrophages and their culture Table 1 List of the compounds used in the study. Compound
PMEA ([2-hydroxy-3-(phosphonomethoxy)ethyl]adenine) N6-cyclopropyl-PMEA N6-cyclopentyl-PMEA PMEDAP ([2-hydroxy-3-(phosphonomethoxy)ethyl]2,6-diaminopurine) N6-cyclopropyl-PMEDAP N6-cyclohexylmethyl-PMEDAP N6-cycloheptyl-PMEDAP N6-cyclooctyl-PMEDAP N6-dimethylaminoethyl-PMEDAP N6-isobutyl-PMEDAP N6-pyrrolidino-PMEDAP (R)-PMPA ((R)-[2-hydroxy-3-(phosphonomethoxy)propyl]adenine) (R)-PMPDAP ((R)-[2-hydroxy-3-(phosphonomethoxy)propyl]2,6diaminopurine) N6-cyclopropyl-(R)-PMPDAP 6 N -cyclopentyl-(R)-PMPDAP N6-isobutyl-(R)-PMPDAP N6-pyrrolidino-(R)-PMPDAP (S)-PMPA ((S)-[2-hydroxy-3-(phosphonomethoxy)propyl]adenine) N6-cyclopropyl-(S)-PMPA N6-pyrrolidino- (S)-PMPA (S)-PMPDAP ((S)-[2-hydroxy-3-(phosphonomethoxy)propyl]2,6diaminopurine) N6-cyclopropyl-(S)-PMPDAP N6-pyrrolidino-(S)-PMPDAP
A1 A2 A3 B1 B2 B3 B4 B5 B6 B7 B8 C1 D1 D2 D3 D4 D5 E1 E2 E3 F1 F2 F3
Untreated animals were killed by cervical dislocation and i.p. injected with 16 ml of sterile saline. Collected cells were washed and resuspended in phenol red RPMI-1640 medium containing gentamicin, glutamine, 10% fetal bovine serum and 5 × 10 − 5 M 2-mercaptoethanol (Sigma, Prague, Czech Republic). Then cells were plated into 96-well round-bottom microplates (Costar, Cambridge, MA, USA) in 100 μl volumes, 2 × 106 cells/ml. Adherent cells (macrophages) were isolated by incubating the cells for 2 h at 37 °C, 5% CO2, and subsequently shaking the plate and washing the wells three times to remove non-adherent cells. Cultures were kept at 37 °C, 5% CO2 in humidiﬁed Heraeus incubator for 24 h in presence or absence of test compounds. 2.5. Detection of NO production Cell cultures were incubated 24 h in presence of test compounds or LPS and IFN-γ as positive controls. The concentration of nitrites in supernatants was taken as a measure of NO production . It was detected in 96-well plate as individual cell-free supernatants (50 μl) incubated 5 min at room temperature with 50 μl of Griess reagent containing 1% sulphanilamide, 0.1% naphtylendiamine and 2.5% H3PO4. The absorbance of samples was measured at 540 nm by using a microplate spectrophotometer (Tecan, Austria). The nitrite levels were extrapolated from nitrite calibration curve.
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2.6. Cytokine assay Cells were cultured in presence of ANP for 24 h. The cell-free supernatants were analyzed using Luminex's xMAP™ technology which combines the principle of a sandwich immunoassay with ﬂuorescent-bead-based technology allowing multiplex analysis of up to 100 different analytes in a single microplate well . Cytokines were measured using commercial kits in 96-well microplate format according to the protocol provided by manufacturer (R&D Systems, Minneapolis, MN, USA). Limited set of cytokines for rat was available, which offered IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, IL-18, IFN-γ, TNF-α and GM-CSF. Results were gained using the Luminex® 200™ System (Luminex Corporation, Austin, TX, USA). Analysis of data was done using ﬁve-parametric-curve ﬁtting in STarStation software (Applied Cytometry, Dinnington, UK). In some cases, secretion of cytokines was determined by enzyme– linked immunoabsorbent assay (ELISA) kits, following the manufacturer instructions (R&D Systems, Minneapolis, MN, USA). 2.7. Determination of cell viability Cell viability was determined using colorimetric assay based on degradation of tetrazolium salt WST-1 (Roche Diagnostics, Mannheim, Germany) which reﬂect the activity of mitochondrial dehydrogenases in viable cells. Hepatocyte and macrophage cultures were cultured 24 hours with test compounds as described above. After that, the solution of WST-1 was added and cells were cultured further for additional 3 hours. The optical density at 450–690 nm was recorded using microplate spectrophotometer (Tecan, Austria). Data of treated cells were compared with untreated cells and dead cells, killed by the presence of 5% Triton. 2.8. Statistical analysis Data were analyzed for statistical signiﬁcance of difference between test and control samples. Analysis of variance (ANOVA) with subsequent Dunnett's multiple comparison test and graphical presentation of data were done using the Prism program (GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Production of NO NO production in rat hepatocytes and macrophages was measured after 24 h cultivation with test compounds with or without any other immune stimulus. In untreated rat hepatocytes and macrophages, slight differences of NO levels were found (Fig. 1). Spontaneous production of NO in rat macrophages is marginal while the levels of NO accumulated in supernatants of isolated hepatocytes reached the value 15.5 ± 2.3 μM (average over all experiments ± S.E.M.). Twenty three derivates of ANP were examined in macrophages and hepatocytes (Fig. 1). The test compounds in graphs are arranged in an increasing order of NO production in macrophages (Fig. 1A). More than one half of test derivatives are able to enhance NO production in resident peritoneal cells, eight of them signiﬁcantly. The most effective compounds (B5, B7, D2, D3) increased NO up to 65 μM, i.e. about 1300% (Fig. 1A). These active ANP signiﬁcantly augmented nitrite levels in hepatocytes (Fig. 1B). The maximal accumulation of NO in supernatant reached to 400% of control values of untreated hepatocytes. Compounds which are unable to induce NO production in macrophages are also unable to stimulate NO in hepatocytes. Fig. 2 shows the extension of NO production stimulated by several compounds in hepatocytes. Treatment with LPS (1000 pg/ml) or LPS + IFN-γ (5000 pg/ml) increased nitrite accumulation in hepatocytes
Fig. 1. Production of NO by rat peritoneal cells culture (A) and primary rat hepatocyte culture (B). The supernatant nitrite concentration was determined by Griess reagent after the 24 hours culture of cells in presence of acyclic nucleoside phosphonates (100 μM). For full chemical names see Table 1. Hepatocytes were screened according the increasing activity in macrophages. The bars represent means ± S.E.M. Statistical analysis was done using one-way ANOVA followed by Dunnett´s multiple comparison post-test (⁎P b 0.05, ⁎⁎P b 0.01). The results are representative of two identical experiments on macrophages (A) and seven experiments on hepatocytes (B).
to about 38 and 48 μM, respectively. The concentration of nitrites induced by the most potent compounds was higher than values induced by LPS or LPS + IFN-γ. Treatment with IFN-γ alone showed little effect on nitrite level (data not shown). The dose dependency of NO production is demonstrated in Fig. 3. The effects of active compounds were dose-dependent. The NO production was stimulated by one of the most potent representative of ANP, compound D2. In both macrophages (Fig. 3A) and hepatocytes, (Fig. 3B) the NO level was increasing dose-dependently in range of test concentration 0 to 100 μM. Already low concentration of D2 (10 μM) was able to stimulate the NO production in macrophages and hepatocytes. The basal level of nitrites in cultured hepatocytes was increased signiﬁcantly by IFN-γ + LPS as positive control more than twice, the increase of NO in macrophages was even more pronounced. The time dependency progress of NO production in hepatocyte culture is shown in Fig. 4. Nitrite concentration was measured in 4-h interval for 24 h. Detectable levels of nitrites appeared after 8 h of incubation with or without any stimulus. NO level in untreated cells raised steadily until 16th h and then leveled. Maximal stimulation provoked by the combination of LPS + IFN-γ strongly enhanced the NO production with an increasing tendency after 24 h. The compound D2
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Fig. 2. The extent of NO production in primary hepatocyte culture induced by ANP. Cells were cultured for 24 h in presence of test compounds (100 μM) or LPS (1000 pg/ml) or combination LPS + IFN-γ (5000 pg/ml) as positive controls. Each value is a mean for six experiments ± S.E.M.
Fig. 4. Time-dependent increase of NO production in primary hepatocyte culture. Nitrite concentration was measured in 4 h interval for 24 h. Combination LPS (1000 pg/ml)+ IFN-γ (5000 pg/ml) served as the positive control. Compound D2 in concentration 100 μM was chosen as representative of ANP. Each value is a mean for two independent experiments.
(100 μM) induced the NO synthesis very effectively, with maximum at 20th h of monitored 24-h interval. The toxicity of highest concentration of compounds (100 μM) was excluded using WST-1 viability assay after 24 h exposure to the test compounds (Fig. 5). Optical density of samples was measured 3 h after addition of WST-1. The values of samples were compared with data from untreated cells and dead cells. The values of cells treated with ANP were similar to data of untreated cells that indicates the presence of ANP did not cause substantial changes in cell viability. 3.2. Cytokines secretion in hepatocyte and macrophage cultures
Fig. 3. Dose-dependent effects of acyclic nucleoside phosphonate D2 on NO production in macrophages (A) and hepatocytes (B) in vitro. Concentration of nitrites was determined by Griess reagent after 24-h cultivation. Each value is a mean ± S.E.M. derived from six independent experiments. Statistical analysis was done using one-way ANOVA followed by Dunnett´s multiple comparison post-test (⁎P b 0.05, ⁎⁎P b 0.01).
Stimulation of rat macrophages and hepatocytes by LPS + IFN-γ is shown at Fig. 7A. Only two cytokines, IL-6 and TNF-α were secreted strongly in macrophages while in hepatocytes the levels of IL-1α, IL-1β, IL-6, IL-10, TNF-α and GM-CSF were demonstrated. ANP, applied in 100 μM concentration, were tested for their effect on different cytokine production in hepatocyte and macrophage cultures in vitro. ANP, which proved their competence to increase the NO production in previous experiments, also demonstrated their effect on cytokine secretion. The two most effective compounds (B5 and D2) and the two inactive (A1 and B1) were chosen for cytokine analysis by Luminex. Cultured hepatocytes treated with active representative of ANP enhanced secretion of six cytokines IL-1α, IL-1β, IL6, IL-10, TNF-α and GM-CSF (Fig. 6C). Production of some of them was elevated signiﬁcantly. Compounds A1 and B1 did not affected concentrations of measured cytokines (data not shown). In macrophage culture four cytokine levels were increased including IL-1α, IL-1β, IL-6, and TNF-α (Fig. 6B). In contrast to hepatocytes, concentrations of cytokines IL-10 and GM-CSF were undetectable. The supernatant levels of other measured cytokines (IL-2, IL-4, IL13, IL-18, IFN-γ) in both, hepatocytes and macrophages, were below the detection limits or remained unaffected uninﬂuenced (data not shown). The data recorded by Luminex were contrasted to data gained by ELISA. Three cytokines were analyzed (Fig. 7). Similarly to LUMINEX, secretion of TNF-α and IL-10 were increased in the presence of ANP (B5, D2) in hepatocytes while only secretion of TNF-α but not IL-10 was manifested in macrophages. IFN-γ was detected neither in hepatocytes nor in macrophages (data not shown). We demonstrated a dose-dependent secretion for cytokines in macrophages and hepatocytes after 24-h treatment with D2 compound 0–100 μM (Fig. 8). Original results expressed as pg/ml were transformed to % of basal production (i.e. control = 100%) for individual cytokines. In rat macrophages, the large-scale rising of secretion
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Fig. 5. Cell viability after 24 h cultivation with ANP was determined using WST-1 assay on macrophages (A) and hepatocytes (B) culture. The cell cultures were incubated with test ANP (100 μM) for 24 h. The optical density was measured 3 h after addition of WST-1. The concentration of tested ANP was 100 μM. Each value is a mean for quadruplicate culture well ± S.E.M.
was observable for IL-1α and IL-6 to approximately 10000% at 100 μM level of compound D2. Secretion of IL-1β and TNF-α increased to 1000%. In hepatocytes, the most extensive secretion was reached for TNF-α (nearly 700%) at the highest concentration of D2 compound. Secretion of cytokines IL-10, IL-1α and IL-1β was increased maximally 4-times and secretion of IL-6 maximally increased 2-times in comparison to untreated hepatocytes. Signiﬁcant stimulation of TNF-α and IL-10 secretion was observed with 25 μM D2 compound and further continued. 4. Discussion Parent compounds of acyclic nucleoside phosphonates (ANP), PMEA and PMPA also known under the names adefovir and tenofovir, were approved by FDA for treatment of hepatitis B (Hepsera), and acquired immunodeﬁciency syndrome (AIDS) (Viread). In addition to antimetabolite mode of action, our recent experiments demonstrated that many acyclic nucleoside phosphonates exert immunostimulatory effects. They are able to affect immune system through its cytokine network regulation. This involves NO production and cytokine and chemokine secretion [3,12]. Immunomodulatory activity was conventionally tested on mice, rat or human macrophages [4,13]. Immune role of hepatocytes has recently been assessed by several authors
Fig. 6. Concentrations of cytokines measured after 24 h cultivation in presence of LPS and IFN-γ (A) or the most potent acyclic nucleoside phosphonates B5 and D2 (100 μM) in macrophages (B) and hepatocytes (C) were analyzed by Luminex. Concentration of LPS was 1000 pg/ml and of IFN-γ 5000 pg/ml. The bars present means ± S.E.M. (n= 3). Statistical analysis was provided by one-way ANOVA followed by Dunnett´s multiple comparison post-test (⁎P b 0.05, ⁎⁎P b 0.01). Levels of cytokines IL-10 and GM-CSF were not detected in macrophages culture.
[6,14]. The aim of our investigations was to observe the immune response of rat hepatocytes upon ANP stimulation and to compare eventual effects to peritoneal macrophages. The results presented in this
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Fig. 7. Concentration of cytokines IL-10 and TNF-α. Macrophage and hepatocyte supernatant samples were analysed using ELISA after 24 h cultivation with ANP (100 μM). Presented data are representative of two independent experiments. The bars represent means ± S.E.M.
work demonstrate that ANP with immunostimulatory effects on macrophages induce cytokine secretion and NO production in rat hepatocytes. Included in the study were 23 ANP derivatives differing by substitution of the amino group at C-6 of the heterocyclic base, i.e. adenine (A)
Fig. 8. Dose-dependent increase of cytokine levels measured by Luminex after 24 h in vitro cultivation in presence of acyclic nucleoside phosphonate D2 in macrophages (A) and hepatocytes (B). Each presented value is a mean ± S.E.M. derived from three independent experiments.
and 2,6-diaminopurine (DAP), and at the N9-side chain moieties, i.e. 9(PME; i.e. PMEA, and PMEDAP) and (R)- or (S)-enantiomers of 9-[2(phosphonomethoxy)propyl] (PMP; i.e. (R)-PMPA, (S)-PMPA, (R)PMPDAP, and (S)-PMPDAP). They were tested for immunobiological properties on rat hepatocytes and macrophages in vitro. The results showed that mainly N6-substituted derivatives of test groups 9-[2-hydroxy-3-(phosphonomethoxy)ethyl]2,6-diaminopurine (PMEDAP), and 9-[2-hydroxy-3-(phosphonomethoxy)propyl]2,6diaminopurine ((R)-PMPDAP) are the potent compounds for activation of rat macrophages and hepatocytes as well. In general, the data are in agreement with ﬁndings on mouse model of peritoneal macrophages  to induce NO production by some of ANP including those compounds used in the present study with rats. We have previously shown that NO-stimulatory abilities of ANP occurred only in the presence of exogenous IFN-γ in mice . However, rat peritoneal cells responded to the stimulation of the same compounds without the need for IFN-γ . Species differences were discussed in our previous study . The effect of ANP on NO production in rat hepatocytes has not been studied, yet. We present for the ﬁrst time the ability of hepatocytes to produce NO under the treatment with ANP. Despite of the fact that basal level of NO in untreated isolated hepatocytes was measurable, NO synthesis was triggered signiﬁcantly by several ANP (B5, B7, D2, D3) when applied alone without any need of IFN-γ + LPS. Interestingly, the same ANP were also the most potent compounds in macrophages to produce NO. Increasing of NO synthesis was manifested in a dosedependent manner in range of test concentration 0–100 μM. On the other hand, PMEA (adefovir) with its derivatives, (S)-PMPA derivatives and (S)-PMPDAP did not show any inﬂuence on NO production in macrophages and hepatocytes. Tenofovir (C1) stimulated NO synthesis non-signiﬁcantly in hepatocytes in comparison to macrophages. Experiments, where time dependent production of NO in hepatocytes were analyzed in untreated cells and with ANP or LPS + IFN-γ further conﬁrmed the ability of ANP to induce synthesis of NO. In our experimental conditions, the measurable level of NO produced in unstimulated hepatocytes appeared in 8th h and remained so toward 24 h. The increase of NO stimulated by compound D2 paralleled to 20th h with stimulation by LPS + IFN-γ, which were further continued in monitored 24-h interval. The possibility that NO derived from iNOS is formed by other type of liver cells seems to be negligible. Hepatocytes constitute the major cell population of the liver (70–80%). We have not found any cNOS (neuronal type) expression in hepatocyte cultures, which is normally detectable in Kupffer cells . Stellate cells, eventual source of NO (iNOS), create less than 1% of nonparenchymal cells . Moreover, in cultured hepatocytes, isolated hepatocytes represented more than 99% of cell population. The purity of our cultures was checked routinely by light microscopy and in addition was assessed by ﬂow cytometry (FACS ARIA 2, used ﬂuorescent antibodies from BD Bioscience, Erembodegem, Belgium). We registered constitutive eNOS mRNA expression in hepatocyte cultures but no difference in expression levels was detected between untreated group and group cultured in the presence of LPS + IFN-γ or ANP. Our results are supported by the ﬁndings of Alexander et al.  on eNOS mRNA expression in rat hepatocytes. The role of iNOS in liver injury is complex and depends particularly on redox status and/or experimental settings. On one hand, beneﬁcial effects of iNOS have been described. Suppression of apoptosis through S-nitrosylation of caspases is mediated by iNOS and is well documented either in both isolated rodent hepatocytes  and in vivo . iNOS is established as one of survival factors in liver regeneration after partial hepatectomy as demonstrated in iNOS knock-out models of mice . After partial hepatectomy, iNOS expression increased within 4–6 hours  and caspase 3 was inhibited by S-nitrosylation. Another protective role of iNOS is in the ﬁeld of microbial infection. Its inhibitory effects on viral replication have been proved in many viruses including hepatitis B virus  and HIV virus . In this respect, ANP that
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produce NO can contribute by this mechanism to overall antiviral therapy including viral infection in the liver. On the other hand, detrimental role of NO in liver injury is largely associated with severe oxidative stress. Subsequent activation of inﬂammatory cascades leads to liver injury presented as hepatitis, ischemia-reperfusion injury or hemorrhagic shock [24,25]. In LPStreated hepatocytes we found that inhibition of iNOS induced by cyclosporin A enhanced hepatic injury , while other study showed attenuation of endotoxemia in the presence of iNOS selective inhibitor aminothylisothiourea . In the pathogenesis of liver cirrhosis, the activity of iNOS was unaltered . In the present study, we observed ANP-mediated iNOS expression in hepatocytes. We should take into account an action of such compounds as N6-substituted derivatives of 9-[2-hydroxy-3-(phosphonomethoxy) ethyl]2,6-diaminopurine (PMEDAP), and 9-[2-hydroxy-3-(phosphonomethoxy)propyl]2,6-diaminopurine ((R)-PMPDAP) on hepatic metabolism and immune processes. In this step, we can speculate about hepatoprotective employment of NO induced by ANP in a regulation of apoptosis during liver regeneration or in the treatment of hepatic infections by nonspeciﬁc immune defence mechanism. Based on our unpublished data (showing no LDH release during treatment with ANP), and on clinically proved safety of ANP (adefovir and tenofovir), these drugs are not toxic for liver. It should be mentioned that primary structure of hepatic iNOS is very similar between rat and humans [29,30]. A promising idea to treat viral hepatitis might be the use of ANP which possess dual mode of action, i.e. antiviral and immunomodulatory. ANP compounds that proved to augment NO production were found to stimulate secretion of cytokines in macrophages and hepatocytes. In this study the cytokine-stimulatory potential is presented by 2 compounds, B5 and D2 from PMEDAP and (R)-PMPDAP series, respectively. The cytokines were produced in a concentration-dependent manner in test compounds concentration 0–100 μM. Secretion of cytokines was elevated signiﬁcantly and reached the magnitude of positive control. The screening for 10 cytokines in rat cell models was performed by multiplex analysis (Luminex). In rat macrophages ANP-induced secretion of four cytokines (IL-1α and IL-1β, IL-6 and TNF-α) was observed. In rat hepatocyte cultures six cytokines were found in supernatants in high amounts in comparison to untreated cells. In addition to cytokines found in macrophages, IL-10 and GM-CSF were identiﬁed in hepatocytes. IFN-γ, which was analyzed in macrophages and hepatocytes by ELISA, was not detected under ANP treatment. With regard to macrophages, our previous results  showed that secretion of IFN-γ remained uninﬂuenced by PMEDAP with its N6- derivatives including B5, irrespective of the species origin of macrophages (mouse, rat, human). Derivatives of PMEDAP and (R)-PMPDAP included active compounds (B5, D2) were found to stimulate secretion of cytokines TNF-α, IL-10 and chemokines MIP-1α, RANTES in mouse resident peritoneal macrophages . Dose-dependent activation of cytokine secretion induced by 9-[2-hydroxy-3-(phosphonomethoxy)propyl]2,6diaminopurine is documented in murine splenocytes . Lately we observed  no secretion of IL-10 in rat peritoneal macrophages activated by ANP in spite of murine and human cells. In the present study we conﬁrmed by Luminex that IL-10 is not expressed in rat peritoneal macrophages. However hepatocytes were highly responsive to ANP treatment with respect to IL-10. Recently, clinically approved acyclic nucleoside phosphonates are used for the treatment of HIV infection and chronic hepatitis B and the mechanism of action is based on inhibition of replication of retroviruses and DNA viruses. Speciﬁcally, tenofovir has been found to have potential in the prevention of antiviral infection . Currently, adefovir dipivoxil was found to increase immunity in patients with chronic hepatitis B. They determined cytokine expression (IL-2, IFN-γ, TNF-α, IL-4,IL-6, IL-10) and their correlation to with liver functions . Immunomodulatory mode of action of these ANP might explain observed prophylactic effect of tenofovir in HIV infection and treatment efﬁcacy
of adefovir and tenofovir at all. Our previous studies on human mononuclear cells show that several N6 – substituted derivatives of ANP stimulate the anti-HIV chemokines even higher than tenofovir itself . Hepatocytes are mainly considered as cells with metabolic, biosynthetic and detoxifying functions. Their place in immunoregulation remains underestimated. Overwhelming majority of ﬁndings estimates the effects of cytokines on hepatocyte toxicity and functionality. In turn, little is known about potential of hepatocytes to secrete cytokines under various conditions. The knowledge of alterations in intrahepatic cytokine microenvironment could contribute to the treatment viral, inﬂammatory diseases or anticancer strategy. We found elevated levels of TNF-α and IL-6 and also IL-10 in hepatocytes treated with ANP. TNF-α and IL-10 are recognized as antiviral agents in many viral infections. Further, despite the general concept that TNF-α and IL-6 are pro-inﬂammatory mediators, all three cytokines are involved in liver regeneration after partial hepatectomy. TNF-α acts as survival factor via NF-κB signalling in hepatocytes , IL-6 is required for hepatocyte proliferation and IL-10 downregulates TNF-α-initiated inﬂammatory responses . GM-CSF is a recognized regulator of cell proliferation and differentiation. In our study, we revealed the ability of rat hepatocytes to secrete GM-CSF. We did not ﬁnd an augmented production of GM-CSF in macrophages. Previously, Sakamoto et al.  demonstrated production of GM-CSF in murine hepatocytes. GM-CSF administration has been shown to enhance the proliferative capacity in hepatocytes in normal and thioacetamideinduced cirrhotic liver . ANP-induced production of this factor in hepatocytes encourages further studies on liver hepatectomy and regeneration. Healthy liver has well-developed defense mechanisms that permit hepatocytes to adapt to cytokine-initiated stress. We suggest that ANP-derived cytokines can delicately regulate drug metabolism by cytochrome P450 enzymes, as well as protein and steroid syntheses or proliferation. During sustained stress however, the same compounds could modulate reactions to prime signals, provided by microbes or toxins. In that case, it can be anticipated that ANP will either ameliorate or aggravate the liver cellular responses, such as inﬂammation, cell deaths and proliferation. It might be speculated that instead of LPS, synthetically prepared ANP could be used as a diagnostic means in testing of idiosyncratic drug hepatotoxicity . Such considerations however, need future experiments. In summary, we have demonstrated NO induction and secretion of cytokines in parenchymal liver cells. Both effects occurred with stimulation of exogenous substances i.e. selected compounds of ANP, namely their N6- derivatives of PMEDAP and (R)-PMPDAP related to adefovir and tenofovir, respectively. Our results support the hypothesis that hepatocytes may play an active immunomodulatory role in the liver. Our pilot data on the ability of hepatocytes to secrete cytokines even qualitatively more than macrophages open further research on signalling pathway, functionality and pharmacotherapeutic implications in the liver. Acknowledgments Authors thank Mgr. Jana Křížková and Mrs. Vlasta Krejčová for their excellent assistance. The work was supported by grant no. 1M6138896301 from the Centre for New Antivirals and Antineoplastics. References  Holy A, Votruba I, Merta A, Cerny J, Vesely J, Vlach J, et al. Acyclic nucleotide analogues: synthesis, antiviral activity and inhibitory effects on some cellular and virus-encoded enzymes in vitro. Antiviral Res 1990;13:295–311.  Cesnek M, Holy A, Masojidkova M, Kmonickova E, Zidek Z. Synthesis of guanidino analogues of PMPDAP and their immunobiological activity. Bioorg Med Chem 2008;16:965–80.  Potmesil P, Krecmerova M, Kmonickova E, Holy A, Zidek Z. Nucleotide analogues with immunobiological properties: 9-[2-Hydroxy-3-(phosphonomethoxy)propyl]-
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