Effects of subacute oral warfarin administration on peripheral blood granulocytes in rats

Food and Chemical Toxicology 50 (2012) 1499–1507

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Effects of subacute oral warfarin administration on peripheral blood granulocytes in rats Sandra Belij a, Djordje Miljkovic´ b, Aleksandra Popov a, Vesna Subota c, Gordana Timotijevic´ d, Marija Slavic´ e, Ivana Mirkov a, Dragan Kataranovski a,f, Milena Kataranovski a,g,⇑ a

Department of Ecology, Institute for Biological Research ‘‘Siniša Stankovic´’’, University of Belgrade, Bulevar Despota Stefana 142, Belgrade, Serbia Department of Immunology, Institute for Biological Research ‘‘Siniša Stankovic´’’, University of Belgrade, Bulevar Despota Stefana 142, Belgrade, Serbia Institute for Medical Biochemistry, Military Medical Academy, Crnotravska 17, Belgrade, Serbia d Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, Belgrade, Serbia e Department of Physiology, Institute for Biological Research ‘‘Siniša Stankovic´’’, University of Belgrade, Bulevar Despota Stefana 142, Belgrade, Serbia f Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia g Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade Studentski trg 16, 11000 Belgrade, Serbia b c

a r t i c l e

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Article history: Received 13 December 2011 Accepted 31 January 2012 Available online 8 February 2012 Keywords: Oral warfarin intake Rats Peripheral blood granulocytes Inflammation

a b s t r a c t Warfarin affects mainly vitamin K dependent (VKD) processes, but the effects on some non-VKD-related activities such as tumor growth inhibition and mononuclear cell-mediated immune reactions were shown as well. In this study, the effect of subchronic (30 days) oral warfarin (0.35 mg/l and 3.5 mg/l) intake on peripheral blood granulocytes in rats was investigated. Increase in prothrombin and partial thromboplastin time at high warfarin dose reflected its basic activity. Priming effect for respiratory burst was noted at both warfarin doses, while only high warfarin dose resulted in priming for adhesion, the rise in intracellular myeloperoxidase content/release and stimulation of nitric oxide production. Differential effects of high warfarin dose were noted on granulocyte cytokines IL-6 (lack of the effect), TNF-a (decreased release and mRNA expression) and IL-12 (increase in mRNA for IL-12 subunits p35 and p40). Changes in granulocytes seems not to rely on mitogen activated kinases p38 and ERK. Warfarin intake was associated with an increase in circulating IL-6, fibrinogen and haptoglobin and with changes in the activity of erythrocyte antioxidant enzymes superoxide dismutase and catalase. The effects of oral warfarin intake on peripheral blood granulocytes demonstrated in this study might be relevant for oral anticoagulant therapy strategies in humans. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Warfarin (4-OH coumarin) and its analogs are vitamin K (VK) antagonists (Shearer, 1990). Their use in prophylactic medicine to prevent tromboembolic diseases in patients at risk is based on the inhibition of the vitamin K-dependent (VKD) step in the complete synthesis of a number of blood coagulation factors in the liver that are required for normal blood coagulation (Furie, 2000). Warfarin inhibits vitamin K epoxide reductase (VKOR). As a consequence, a rapid depletion of hydroquinone (K1H2), a cofactor of c-glutamyl carboxylase, which mediates carboxylation of glutamyl (Gla) residues on intracellular precursors of several VKD proteins involved in coagulation process, takes place. Reduced supply of cofactor form of VK results in accumulation of undercarboxylated (inactive) form of factor II (prothrombin), factor VII (FVII), factor IX (FIX) and factor ⇑ Corresponding author at: Department of Ecology, Institute for Biological Research ‘‘Siniša Stankovic´’’, University of Belgrade, Bulevar Despota Stefana 142, Belgrade, Serbia. Tel.: +381 11 2078 375; fax: +381 11 2761 433. E-mail address: [email protected] (M. Kataranovski). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2012.01.049

X (FX) (Furie, 2000). Impairment of production of these essential VKD blood clotting factors by warfarin results in an increase in clotting time up to the point where no clotting occurs. The inhibition of VKOR affects catalytic rate of VKD proteins required for biological processes other than hemostasis, including those which regulate bone growth and calcification (bone Gla protein, BGP/osteocalcin and matrix Gla protein, MGP) (Becker, 2007; Price, 1988). The effects of warfarin on BGP and MGP are considered responsible for ‘‘warfarin embryopathy’’, developmental defects associated with warfarin consumption during pregnancy as well as for bone mass loss in patients on long-term anticoagulant therapy (WHO, 1995). Suppression of MGP is considered as the one of the underlying mechanisms in arterial calcification in experimental warfarin consumption in rats (Howe and Webster, 2000) and a possible determinant of this process in humans on warfarin therapy (Becker, 2007). Warfarin inhibits production of growth arrest-specific gene 6 (Gas6), VKD-dependent growth-potentiating factor for vascular smooth muscle cells (Nakano et al., 1997) and for mesangial cells (Yanagita et al., 1999) what imply broader effects of this anticoagulant. Indeed, warfarin effects on processes unrelated to

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VK were demonstrated and include antitumor and immunomodulatory activity. Antiproliferative effects of warfarin on some tumor cells as well as increase in natural killer (NK) cell activity in patients on warfarin therapy (Bobek et al., 2005) explain its use as an adjuvant antitumor therapy in humans (Bobek and Kovarik, 2004; Nakchbandi et al., 2006). It should be noted, however, that early studies demonstrated hepatotoxicity and tumorigenic potential in livers of mice exposed to high doses of this agent (Cohen, 1979; Lake et al., 1994). More recent study showed no genotoxic potential of coumarin (Edwards et al., 2000) and authors suggested other mechanisms of tumor formation in rodents. Mechanisms of warfarin action on immune system are the least known. Data from the early studies in humans showed inhibition of the development of skin induration in the delayed hypersensitivity test by warfarin at doses which induce anticoagulant effect (Edwards and Rickles, 1978). In contrast, warfarin therapy resulted in an enhancement of patient’s lectin (PHA)-induced proliferation of peripheral blood mononuclear cells (Berkarda et al., 1983). Similar data were obtained in animals where both immunostimulatory (Berkarda et al., 1978) and immunosuppressive (Eichbaum et al., 1979; Kurohara et al., 2008; Perez et al., 1994) effects of warfarin were noted. There are also animal data which showed no effect of warfarin on the activity of some peripheral blood elements such as platelets (Takahashi, 1991). Certain clinical complications connected to warfarin therapy including thrombosis in patients with overcoagulation (Poli et al., 2003) as well as some adverse reactions associated with the presence of inflammatory cells in affected tissues (Ad-El et al., 2000; Hermes et al., 1997; Jo et al., 2011; Kapoor and Bekaii-Saab, 2008; Kuwahara et al., 1995) imply proinflammatory effects of warfarin. We have shown previously that acute administration of warfarin can prime neutrophils in rats for respiratory burst when applied in acute regime (for three consecutive days) intraperitoneally (Kataranovski et al., 2007) or epicutaneously (Kataranovski et al., 2008). To see if warfarin affects granulocytes in a more chronic regime of exposure, the effect of 30-day oral warfarin intake on peripheral blood granulocytes of rats was determined in this study. Changes in several granulocyte activity were examined including oxidants production, ability of adhesion, as well as production of proinflammatory cytokines. To get informations concerning the character of granulocyte microenvironment, several parameters of inflammation were determined in circulation.

conventionally housed at IBISS were used. Four to six animals were assigned to each treatment group in at least three independent experiments. Warfarin sodium was prepared in drinking water at concentration of 0.35 mg/l and 3.5 mg/l and was given to rats (four to six individuals for the low and the same numbers of individuals for higher warfarin dose, per experiment) for 30 days. Control rats (four to six individuals per experiment) were given drinking water solely. Warfarin and water were replaced with freshly prepared solution or water twice a week. All functional measurements were carried out 24 h following 30-day period in animals anesthetized by i.p. 40 mg/kg b.w. of thiopental sodium (Rotexmedica, Tritau, Germany). 2.3. Clinical biochemistry and leukocyte counts Prothrombin time (PT) and partial thromboplastin time (PTT) were determined in blood samples diluted in citrate buffer (1:5). PT was determined by one-stage method using citrate plasma and Thromborel S reagents (Behring Diagnostics GmbH, Marburg, Germany) with Siemens equipment. The caolin-activated PTT was determined by one stage method using Pathrombin (Behring). Plasma fibrinogen was measured by Siemens-Dade Behring-BCT analyzer using Multifibren U test for quantitative determination in plasma. Haptoglobin, ceruloplasmin and albumin were measured in serum by BN (Dade Behring) immunochemical system for human blood proteins measured by Siemens BNII (Dade Behring) BCT analyzer. Crossreactivity with rat blood proteins was checked using serum obtained from turpentineinduced inflammation in rat, known inflammatory model of acute phase reaction in these animals (Giffen et al., 2003). Changes in plasma or serum proteins are expressed as the relative changes, calculated as percentages of the value obtained in control (warfarin 0 mg/l) animals, which were considered as 100%. Total leukocyte counts were determined by improved Neubauer hemocytometer. Differential leukocyte counts were determined by differentiating at least 300 cells from air-dried whole blood smears stained according to the May-Grünwald-Giemsa (MGG) protocol. 2.4. Peripheral blood granulocyte isolation Peripheral blood granulocyte assays were performed with cells isolated from the heparinized blood by dextrane sedimentation and centrifugation on OptiPrep (Nycomed AS, Norway) density gradient. Following the lysis of erythrocytes from the pellet cell fraction with the isotonic NH4Cl solution, the remaining granulocytes were washed and resuspended in culture medium for functional studies. The purity of granulocytes was more than 95%, as determined morphologically by MayGrunwald-Giemsa staining. Granulocyte viability was determined by a quantitative colorimetric assay described for human granulocytes (Oez et al., 1990a) which is based on metabolical reduction of tetrazolium salt MTT to a colored end product, formazan. Cells were added to wells of a 96-well plate (0.25  106 cells/well) and incubated with 500 lg/ml of MTT (added immediately or following 24 h in culture) for 3 h. Formazan produced by the cells was dissolved by overnight incubation in 10% sodium dodecyl sulfate (SDS) – 0.01 N HCl and absorbance was then measured at 540/650 nm by ELISA 96-well plate reader (GDV EC, Roma, Italy). In some assays, peripheral blood mononuclear cells from the band formed at the interface were harvested as well.

2. Materials and methods

2.5. Peripheral blood granulocyte activity assays

2.1. Chemicals

Cytochemical NBT reduction assay for the respiratory burst based upon spontaneous or PMA-stimulated capacity of granulocytes to reduce NBT (Choi et al., 2006) was used. NBT (10 ll, 5 mg/ml) was added to granulocyte suspension (5  105 cells/ well of 96-well plate, in 100 ll) and incubated for 30 min. Formazan produced by granulocytes was extracted overnight in 10% SDS – 0.01 N HCl and was measured at 540/650 nm by ELISA 96-well plate reader. Granulocyte adhesion was assessed by using a modified adhesion assay initially described by Oez et al. (1990b), based upon their spontaneous or PMA-stimulated capacity to adhere to plastic. Cells were cultured at 5  105 cells/well of 96-well plate, in 100 ll for 60 min. After incubation, nonadherent cells were carefully removed by washing with prewarmed culture medium. Cells adhering to plastic were stained with 0.1% methylene blue. The absorbance of dissolved dye was measured at 650/540 nm by ELISA 96-well plate reader. Granulocyte MPO activity was assesssed on the basis of the oxidation of o-dianisidine dihydrochloride by cells or by medium conditioned by granulocytes in culture (Bozeman et al., 1990). MPO was evaluated by the addition of 33 ll of granulocyte lysate, obtained by repeated freezing and thawing, or the conditioned medium (CM) (see bellow) to 966 ll of substrate solution (0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% H2O2 in 50 mM potassium phosphate buffer, pH 6.0). Apsorbance was read at 450 nm at three-minute intervals up to ten minutes against the standard of MPO. Values are expressed as MPO units per 106 cells or MPO U/ml of granulocyte CM. As an indicator of nitric oxide (NO) formation, the concentration of the stable NO oxidation product, nitrite, was measured by using Griess assay (Hibbs et al., 1988) in 48-h CM harvested from cultures of peripheral blood granulocytes (0.5  106 cells/well in 96 well plate) cultured in medium only (spontaneous production) or with 100 ng/ml of LPS or 100 lg/ml of aminoguanidine. Briefly, 50 ll

Warfarin sodium was purchased from Serva Fein Biochemica (Heidelberg, Germany). Hexadecyltrimethylammonium bromide (HTAB), o-dianisidine dihydro chloride, myeloperoxidase (MPO), three-(4,5-dimethyl-thiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), lipopolysaccharide (LPS), phorbol-12-myristate 13acetate (PMA), N-(1-naphtyl) ethylenediamine dihydrochloride, sulfanilamide (paminobenzenesulfonoamide) and aminoguanidine (bicarbonate salt) were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA). Nitroblue tetrazolium (NBT), sodium nitrite and hydrogen peroxide (H2O2) were purchased from ICN Pharmaceutical (Costa Mesa, CA, USA), Fluka Chemika (Buchs, Switzerland) and from Zorka Farma (Šabac, Serbia) respectively. LPS was dissolved in culture medium under sterile condition. PMA was dissolved in dimethylsulfoxide (DMSO) at 1000 times greater concentration and diluted before the use in cell culture medium. MPO and NBT were used dissolved in water. All solutions for cell culture experiments were either prepared under sterile conditions or were sterile filtered (Flowpore, pore size 0.22 lm) before use. Culture medium RPMI-1640 (PAA laboratories, Austria) supplemented with 2 mM glutamine, 20 lg/ml gentamycine (Galenika a.d., Serbia), 5% (v/v) heat inactivated fetal calf serum (PAA laboratories, Austria) was used in cell culture experiments.

2.2. Animals and warfarin treatment Animal treatment was carried out in adherence to the guidelines of the Ethical Committee of the Institute for Biological Research ‘‘Siniša Stankovic´’’ (IBISS), Belgrade, Serbia. Male Dark Agouti (DA) rats 12–16 weeks old, weighing 200–240 g,

S. Belij et al. / Food and Chemical Toxicology 50 (2012) 1499–1507 aliquots of CM were mixed with an equal volume of Griess reagent (a mixture of 0.1% naphtylenediamine dihydrochloride in water and 1% sulfanilamide in 5% phosphoric acid) and incubated for 10 min at room temperature. The absorbance was measured at dual wavelength 540/670 nm by an ELISA 96-well plate reader. 2.6. Cytokine determination by ELISA Tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) concentration in plasma and the levels of these cytokines and interleukin-10 (IL-10) in granulocyte CM were evaluated using enzyme-linked immunosorbent assays (ELISA) for rat TNF-a (eBioscience Inc., San Diego, CA, USA), rat IL-6 and rat IL-10 (R&D systems, Minneapolis, USA) according to manufacturer instructions. Cytokine titer was calculated by the reference to a standard curve constructed with known amounts of recombinant TNF-a, IL-6 or IL-10. 2.7. Reverse transcription - real time polymerase chain reaction (RT-PCR) Total RNA was isolated from the granulocytes immediately after isolation with an RNA Isolator (Metabion, Martinsried, Germany) following the manufacturer’s instructions. The isolated RNA was reverse transcribed using random hexamer primers and MMLV (Moloney Murine Leukemia Virus) reverse transcriptase, according to manufacturer’s instructions (Fermentas, Vilnius, Lithuania). Prepared cDNAs were amplified by using Power SYBRÒ Green PCR Master Mix (Applied Biosystems, Foster City, CA) according to the recommendations of the manufacturer in a total volume of 20 ll in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Thermocycler conditions comprised an initial step at 50 °C for 5 min, followed by a step at 95 °C for 10 min and subsequent 2-step PCR program at 95 °C for 15 s and 60 °C for 60 s for 40 cycles. The PCR primers were as follows: b-actin forward 50 -CCC TGG CTC CTA GCA CCA T-30 , b-actin backward 50 -GAG CCA CCA ATC CAC ACA GA-30 ; TNF-a forward 50 -TCG AGT GAC AAG CCC GTA GC-30 , TNF-a backward: 50 -CTC AGC CAC TCC AGC TGC TC-30 ; indicuble nitric oxide synthase (iNOS) forward 50 -TTC CCA TCG CTC CGC TG-30 , iNOS backward 50 -CCG GAG CTG TAG CAC TGC A-30 ; p35 forward 50 -CCG GTC CAG CAT GTG TCA-30 , p35 backward 50 -GCC GAA GTG AGG TGG TTT AGG-30 ; p40 forward 50 -ACG GAC TTG AAG TTT AAC ATC AAG AG-30 , p40 backward 50 -AGA GAT GCT CGT CCA CAT GTC A-30 . Accumulation of PCR products was detected in real time and the results were analyzed with 7500 System Software (AB) and calculated as 2dCt, where dCt was difference between Ct values of specific gene and endogenous control (b-actin). 2.8. Immunoblot Granulocyte lysates were prepared in a solution containing 62.5 mM Tris–HCl (pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol (DTT), 0.01% w/v bromophenol blue, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 g/ml aprotinin, 2 mM EDTA and were electrophoresed on a 12% SDS–polyacrylamide gel. The samples were electro-transferred to polyvinylidene difluoride membranes at 5 mA/cm2,

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using semi-dry blotting system (Fastblot B43, Biorad, Munich, Germany). The blots were blocked with 5% w/v nonfat dry milk in PBS with 0.1% Tween-20 and probed with specific antibodies to p38 mitogen activated protein kinase (MAPK), phosphorylated-p38 MAPK, p44/42 MAPK (Erk1/2) and phosphorylated-p44/42 MAPK at 1:1000 dilution (all from Cell Signaling Technology, Boston, MA, USA), followed by incubation with secondary antibody at 1:10000 dilution (ECL donkey anti-rabbit horseradish peroxidase (HRP)-linked, GE Healthcare, Buckinghamshire, England, UK). Detection was conducted by chemiluminescence (ECL, GE Healthcare). 2.9. Erythrocyte isolation and determination of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) activity Heparinized blood was centrifuged at 400g for 20 min to separate the plasma. The remaining pellet was washed with cold physiological saline and haemolyzed by ultrapure water until original volume was restored. SOD activity was determined by the adrenalin method (Misra and Fridovich, 1972). One unit of activity was defined as the amount of enzyme necessary to decrease by 50% the rate of adrenalin autooxidation at pH 10.2. The activity of CAT was determined by the rate of H2O2 disappearance measured at 240 nm as described (Beutler, 1982). One unit of CAT activity is defined as the amount of enzyme that decomposes 1 mmol H2O2 per minute at 25 °C and pH 7.0. 2.10. Data display and statistical analysis Results are expressed as means ± standard deviation (S.D.). Statistical analysis was performed by using STATISTICA 7.0 (StatSoft Inc., Tulsa, Oklahoma, USA). Statistical significance was defined by Mann–Whitney U test (functional granulocyte assays and erythrocyte SOD and CAT) and t-test (gene expression and Western blot). p - values less than 0.05 were considered significant.

3. Results 3.1. Anticoagulant effects of oral warfarin intake Rats were given warfarin in drinking water at 0.35 mg/l (low dose) and 3.5 mg/l (high dose) during 30-day period. In this way rats consumed 35.0 ± 9.0 lg of warfarin/kg or 360 ± 50.0 lg/kg daily, the amount of warfarin within the range of doses previously shown to modulate granulocyte activity in an acute regime of administration (Kataranovski et al., 2007, 2008). Consumption of 3.5 mg/l of warfarin was associated with the increase in the mean PT and PTT (Fig. 1A). External (skin) (Fig. 1B) and internal (gastrointestinal) (Fig. 1C) hemorrhage was noted during the last week of

Fig. 1. Pathophysiological effects of oral warfarin intake. (A) Prothrombin (PT) and partial thromboplastin time (PTT) following warfarin consumption. (B) Skin hemorrhagic lesions (arrow). (C) Intestinal hemorrhage (arrows). Data are presented as mean values ± S.D. from three or more experiments with six animals per group per experiment. Significance at ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001 vs control (warfarin 0 mg/l).

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intake of high warfarin dose in 9.5% (4/42) of rats and these animals eventually died. 3.2. Lack of the effect of warfarin intake on peripheral blood granulocyte numbers and cell toxicity The effect of warfarin intake on granulocytes was first examined by measuring changes in their numbers and viability. No changes were noted in total or differential blood leukocyte numbers and the relative numbers of granulocytes following warfarin administration were 29.6 ± 4.1% at lower and 26.8 ± 2.0% at higher warfarin dose compared to 28.1 ± 5.2% in control animals. There were no differences between MTT reducing capacity (A540 nm) of freshly isolated granulocytes from control (0.31 ± 0.03) or warfarin-treated animals (0.35 ± 0.03 and 0.32 ± 0.02, at 0.35 mg/l and 3.5 mg/l, respectively). Similar levels of MTT reduction were noted following 24-h in culture (0.19 ± 0.01 in control and 0.19 ± 0.01 and 0.17 ± 0.01 at low and high warfarin doses, respectively).

determination of their capacity to reduce NBT salt (as the cytochemical measure of oxygen consumptions for respiratory burst), the MPO intracellular content and release and NO production (Fig. 2). There were no changes in spontaneous capacity of NBT reduction by granulocytes from rats which consumed 0.35 mg/l of warfarin, with a tendency (p = 0.06) of an increase at 3.5 mg/l of warfarin. Stimulation with PMA resulted in an increase in NBT reduction by granulocytes from rats which consumed high warfarin dose. Consumption of 3.5 mg/l of warfarin resulted in increased intracellular levels of MPO as well as in MPO release. Rise in spontaneous production of NO was noted at high warfarin dose solely, while stimulation with LPS resulted in higher levels of granulocyte NO production (as compared to controls) at both warfain doses (p = 0.052 at 0.35 mg/l of warfarin). The addition of aminoguanidine, which inhibits preferentially iNOS (Southan and Szabo, 1996) reduced the spontaneous accumulation of nitrites in medium conditioned by granulocytes of rats which consumed high warfarin dose. Examination of mRNA levels for iNOS revealed increased levels of message for the enzyme at this warfarin dose.

3.3. Peripheral blood granulocyte oxidative activity following warfarin intake

3.4. Peripheral blood granulocyte adhesion following warfarin intake

The effect of warfarin intake on granulocyte activity was first explored by changes in cell oxidative activity and included

Effect of warfarin consumption on granulocytes was further explored by changes in their capacity to adhere to plastic. No

Fig. 2. Peripheral blood granulocyte oxidative activity. (A) Spontaneous and PMA-stimulated NBT reduction by granulocytes (up) and intracellular MPO content and release in granulocyte conditioned medium (down). (B) Spontaneous and LPS-stimulated NO production, measured by nitrite levels (lM) in conditioned medium of peripheral blood granulocytes (up), spontaneous NO production in the presence of 100 lM of aminoguanidine (AG) (middle), relative expression of iNOS gene analyzed by RT-PCR (down). Data are expressed as mean values ± S.D. or as mean values ± S.E. (cytokine mRNA expression) from three (nitric oxide production and iNOS mRNA expression) or four experiments (NBT reduction and MPO), each with four to six animals per group. Significance at ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001 vs control (warfarin 0 mg/l).

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Fig. 3. Spontaneous and PMA-stimulated adhesion of peripheral blood granulocytes. Data are expressed as mean values ± S.D. from three experiments each with five animals per group. Significance at ⁄⁄p < 0.01 vs control (warfarin 0 mg/l).

increase in spontaneous granulocyte adhesion was noted at either of warfarin doses. However, consumption of high warfarin dose resulted in increased responsiveness of granulocytes to PMA stimulation of adhesion (Fig. 3). 3.5. The effect of warfarin intake on peripheral blood granulocyte cytokines As peripheral blood granulocyte proinflammatory cytokine production was shown in settings of inflammation (Kasama et al., 2005), TNF-a and IL-6 production by granulocytes from rats which consumed warfarin was further explored. There was a tendency (p = 0.06) of a decrease in TNF-a production following consumption of 0.35 mg/l of warfarin and a significant decrease in TNF-a production at 3.5 mg/l of warfarin (Fig. 4A). Stimulation with LPS resulted in a similar pattern of TNF-a production by granulocytes from rats which consumed warfarin. To correlate these findings with the gene expression, mRNA levels for TNF-a were measured in granulocytes of rats which consumed 3.5 mg/l of warfarin (Fig. 4B). As revealed by RT-PCR decreased levels of a message for the TNF-a were noted. As IL-10 was shown to inhibit granulocyte TNF-a production and expression (Cassatella et al., 1993), production of this cytokine was determined next. Variable spontaneous granulocyte IL-10 production was noted with no differences between control (89.0 ± 62.2 pg/ml) and animals which consumed high warfarin dose (78.9 ± 63.2 pg/ml). Stimulation with LPS resulted in a similar levels of production of this cytokine (109.0 ± 58.9 pg/ml vs 160.6 ± 97.1 pg/ml) in rats which consumed 3.5 mg/l of warfarin and control rats, respectively. When IL-6 production at this dose was examined, no differences were found between control and animals which consumed warfarin in both spontaneous (38.2 ± 31.0 pg/ml vs 25.4 ± 10.0 pg/ml in controls) and LPS-stimulated (153.3 ± 4.4 pg/ml vs 146.2 ± 52.5 pg/ml in controls) IL-6 production at high warfarin dose. In a preliminary experiment, no differences were noted between the levels of mRNA for IL-6 in control and granulocytes of rats which consumed warfarin. To see whether warfarin affects other cytokines, the expression of interleukin-12 (IL-12), proinflammatory cytokine for which neutrophils are, along with macrophages and dendritic cells, a significant source in humans and mice (Trinchieri, 2003) was measured. As shown in Fig. 4C, an increase of mRNA for IL-12-specific subunits p35 and p40 was noted in granulocytes of rats which consumed this warfarin dose. 3.6. Lack of activation of p38 and extracellular signal-related kinases in granulocytes following warfarin intake To get some insight into underlying mechanisms of changes in peripheral blood granulocyte activity in rats which consumed warfarin, the levels of MAPK p38 and extracellular signal-related

Fig. 4. Peripheral blood granulocyte (A) TNF-a production, (B) TNF-a mRNA expression and (C) mRNA expression of p35 and p40 following warfarin consumption. Data are expressed as mean values ± S.D. (TNF-a production) or as mean values ± S.E. (cytokine mRNA expression) from three experiments, each with four to six animals per group. Significance at ⁄p < 0.05; ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 vs control (warfarin 0 mg/l).

kinase (ERK), shown to be important for orchestration of a variety of neutrophil functions (Condliffe et al., 1998), were determined next. Analysis of the levels of activated (phosphorylated) forms of MAPKs in granulocytes isolated from the rats after the treatment with 3.5 mg/l of warfarin showed that there were no differences in activation levels of either p-38 MAPK or ERK between granulocytes from control and warfarin-treated animals (not shown). 3.7. The levels of TNF-a, IL-6 and acute phase proteins in the blood of rats which consumed warfarin Given the effect of warfarin on granulocytes, a cellular component of inflammation, the presence of soluble mediators of inflammation including TNF-a and IL-6, as well as several acute phase proteins were examined next (Fig. 5). Low and unchanged levels of TNF-a were detected in plasma of rats administered orally with warfarin (3.8 ± 3.6 pg/ml at low or 2.6 ± 1.6 pg/ml at high warfarin dose compared to 3.4 ± 2.1 pg/ml in control animals). Numerical and a significant increase of IL-6 was observed at 0.35 mg/l and 3.5 mg/l of warfarin, respectively. Warfarin consumption was associated with an increase in serum levels of haptoglobin (at both warfarin doses), fibrinogen (at high warfarin dose) and in a decrease in albumin levels.

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no changes in SOD activity at 0.35 mg/l warfarin (though a tendency, p = 0.086 was noted), while a decrease at 3.5 mg/l of warfarin was observed. Increase in CAT was noted in animals which consumed high warfarin dose. As warfarin have prooxidant properties (Fasco et al., 1983; Wallin and Martin, 1985) the effect of exogenous warfarin (3.5 lg/ml) on SOD activity was tested. No changes in SOD activity were noted in the presence of warfarin (825.5 ± 92.5 U/g Hb) compared to activity without warfarin added (657.0 ± 12.6 U/ g Hb), thus demonstrating the lack of inhibition of SOD activity by warfarin itself. No detectable levels of ceruloplasmin (which according to DiSilvestro and Marten (1990) might interfere with SOD activity by a competition for copper) were noted in sera of both control as well rats which consumed warfarin.

4. Discussion

Fig. 5. Changes in (A) IL-6, (B) fibrinogen, haptoglobin and albumin in blood following warfarin intake. Data are expressed as mean values ± S.D. from three experiments each with four to six animals per group. Significance at ⁄p < 0.05; and ⁄⁄ p < 0.01 vs control (warfarin 0 mg/l).

3.8. Erythrocyte SOD and CAT activity following warfarin intake As anticoagulant therapy or blood treatment with anticoagulants were shown to exert effects associated with changes in erythrocytes (Duncan et al., 1983; Hofbauer et al., 1999; Rosenblum, 1968) we further tested whether warfarin intake is associated with alterations in these cells. Measurements of basic oxygen free radical enzyme scavengers, SOD and CAT conducted in erythrocytes from peripheral blood of rats following warfarin intake (Fig. 6) revealed

Fig. 6. Erythrocyte antioxidant enzyme activity. (A) erythrocyte superoxide dismutase (SOD) and (B) catalase (CAT) activity following warfarin intake. Data are expressed as mean values ± S.D. from samples pooled from three independent experiments, each with six animals per group. Significance at ⁄p < 0.05 vs control (warfarin 0 mg/l).

In this study, the effect of subchronic warfarin intake on peripheral blood granulocytes was examined by analysis of several aspects of their activity including those related to oxidant production, adhesion and cytokine expression. Proinflammatory activity of warfarin was noted at high (3.5 mg/l) warfarin dose (judging on an increase of all of the examined aspects of granulocyte activity), while intake of 0.35 mg/l of warfarin resulted in priming for respiratory burst solely. Differential effects of high warfarin dose were noted on granulocyte cytokines (a decrease in TNF-a, the lack of effect on IL-6 and an increase in IL-12). Beside changes in granulocytes, increase in soluble inflammatory mediators were noted as well. Increase in PT and PTT noted at 3.5 mg/l of warfarin, demonstrated anticoagulant effect, the basic biological activity of warfarin. Internal as well as external hemorrhage might be a cause of death in less than 10% of rats, in line with data which showed that impaired coagulation, along with the hemorrhage, generally results in a death of rodents (Lund, 1988). Lack of changes in peripheral blood granulocyte numbers as well as viability and survival, imply that warfarin intake was not cytotoxic. It have resulted, however, in qualitative changes in these cells. Significantly higher responsiveness of granulocytes from rats administered with high warfarin dose to activation by PMA, a potent granulocyte activator, reflect their priming state, i.e. the state in which the functional responses to an activating stimulus are potentiated/amplified by a prior exposure to a priming stimuli (Hallett and Lloyds, 1995). This is in line with our previous data which demonstrated peripheral blood granulocyte priming for respiratory burst and adhesion following acute intraperitoneal or epicutaneous exposure of rats to similar doses of warfarin (Kataranovski et al., 2007, 2008). Increase in intracellular MPO content and release, which is along with phagocyte oxidase a source of oxidant activity in phagocytes (Finkel, 2003) stresses the effect of warfarin intake on peripheral blood granulocyte oxidative activities. Peripheral blood granulocyte priming for respiratory burst as well as increase in intracellular MPO content might rely on the increase of IL-6 in plasma, as high and significant correlation between plasma IL-6 and these activities was noted (r = 0.85, p < 0.001, y = 0.0003 + 0.0035  x for IL-6 and NBT, and r = 0.83, p < 0.001, y = 0.18 + 0.01  x for IL-6 and MPO). In corroboration, neutrophil priming for superoxide release by IL-6 was noted in humans in settings of systemic inflammation (Biffl et al., 1994) and IL-6 stimulated secretion of granulocyte primary/ azurophilic granule (which contain MPO) was observed (Borish et al., 1989). Priming of granulocytes for respiratory burst and adhesion to plastic might possibly be ascribed to oxidatively stressed erythrocytes, as reactive oxygen species (ROS) are known priming stimuli for granulocytes (Swain et al., 2002). In this regard, high and significant correlation between erythrocyte CAT and

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granulocyte priming for NBT reduction (r = 0.88, p < 0.001, y = 0.0009 + 0.000004  x) was noted. Priming of granulocytes is, however, a complicated process which depend on a variety of stimuli and cell receptor agonists (Condliffe et al., 1998) and the effect of other inflammation-relevant stimuli in circulation of orally administered rats might be presumed. Changes in the expression of NO demonstrated proinflammatory effect of warfarin consumption on peripheral blood granulocytes as well. Warfarin intake resulted in stimulation of NO production judging on the effect of aminoguanidine and increase in mRNA for iNOS. Increased NO production in response to LPS, known stimulator of iNOS, supports the contribution of iNOS to granulocyte production of NO in rats which consumed warfarin. Resting peripheral blood rat neutrophils contain no iNOS and produce NO following stimulation (Fierro et al., 1999; Miles et al., 1995). Vast array of inflammation-related and other mediators were reported to induce cell expression of NO (Bogdan et al., 2000). Increase in NO production and expression might be ascribed to increase in MPO, as high positive correlation was found between these granulocyte activities (r = 0.93, p < 0.001, y = 1.46 + 0.037  x). In the view of the recently described capacity of MPO to increase catalytic activity of iNOS at sites of inflammation (Galijasevic et al., 2003), the relevance of MPO for stimulation of NO in peripheral blood granulocytes might be assumed. However, no simple relationship between these molecules in granulocytes would be expected, as complicated interactions between NO and MPO were reported (Brovkovych et al., 2008). Warfarin intake exerted differential effects on granulocyte cytokines. A decrease in TNF-a production by granulocytes from rats which consumed warfarin resulted from inhibition at transcriptional level, as lower levels of TNF-a mRNA were noted in these cells. IL-10, a regulatory cytokine which transcriptionally regulate granulocyte TNF-a production (Cassatella et al., 1993) seems not responsible for the observed decrease, as similar granulocyte IL10 production was noted in control and experimental animals. Negative correlation between granulocyte NO and TNF-a production (r = 0.74, p < 0.001, y = 2348–22  x for LPS-stimulated) imply that increase in NO might, possibly, account for a decrease in granulocyte TNF-a. Negative feedback of endogenous NO on TNFa synthesis (Eigler et al., 1995), as well as inhibition of TNF-a production by exogenous NO (Thomassen et al., 1997) was shown in murine and human macrophages, respectively. A decrease in TNF-a expression and production is in line with previously observed inhibition of transduction of signal generated by TNF-a in macrophage cell line by warfarin in vitro (Kater et al., 2002). Collectively, these and our data indicate that TNF-a is a target of negative regulation by warfarin. Absence of the effect of warfarin intake on granulocyte IL-6 production is at variance with data which demonstrated that this agent might affect LPS-stimulated IL-6 production in murine macrophage cell line (Kater et al., 2002) and showed that warfarin effect might be cell-specific. Increase in mRNAs for subunits of IL-12 demonstrated that proinflammatory cytokines might be a target for positive regulation by warfarin. The absence of changes in activation of p38 and ERK might imply the lack of the relevance of MAP kinase pathways for peripheral blood neutrophil activity in rats administered orally with warfarin. Indeed, warfarin was shown to be without effect on SAPK/JNK pathway in human cells and cell lines (Cross et al., 1999). In addition, species-dependent differences in the expression of signaling kinases might have accounted for the lack of the effect of warfarin on MAP kinases in our study, as MAP (p38 and ERK) kinase-dependent failure of rat neutrophil priming with some agonists in vitro was demonstrated (Yaffe et al., 1999). Alternatively, inflammation following warfarin consumption might be of insufficient intensity to impinge on these signaling pathways, as it was shown that

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p38 MAP kinase activation is required for maximal neutrophil priming by some inflammatory stimuli (Partrick et al., 2000). It is also possible that desensitization/inhibition of p38 and ERK pathways might have contributed to the lack of their activation. In this sense, inhibition of inflammatory signal transduction by warfarin via inhibition of IjB phosphorylation in murine macrophage cell line observed in vitro (Kater et al., 2002) should be mentioned. Taking into account that priming/activation of neutrophils is a complicated event which depends on the nature of stimuli and in which several interacting signal pathways are involved in the overall response (Condliffe et al., 1998) the relevance of other pathways might be expected. Increase in circulating IL-6 and acute phase proteins imply that warfarin intake created a proinflammatory milieu for peripheral blood granulocytes of rats. Rise in plasma IL-6 observed following warfarin intake in rats is in line with data which demonstrated higher levels of this cytokine in patients on high warfarin therapy (Saminathan et al., 2010). Relative increase in haptoglobin and fibrinogen, moderate/major and moderate acute phase protein respectively in rats (Baumann et al., 1990), point to a systemic inflammation in rats. High and significant correlation between IL6 and these proteins (r = 0.70, p < 0.05, y = 0.13 + 0.003  x, for IL6 and haptoglobin; r = 0.83, p < 0.001, y = 0.065 + 0.007  x, for IL-6 and fibrinogen) imply involvement of this cytokine in their production, as shown in settings of acute (Giffen et al., 2003) or more chronic inflammation in rats (Mayot et al., 2008). Low levels of increase in haptoglobin (less than two times) as compared to an increase of up to seven times in acute inflammation (Giffen et al., 2003) suggest a presence of a low-grade inflammation following warfarin intake in rats. This is in line with a recent report which demonstrated low-level increase of CRP in patients receiving warfarin therapy (MacCallum et al., 2004). A decrease in albumin levels is in accordance with data which showed that elevation of positive acute phase reactants (haptoglobin and fibrinogen) in systemic low-grade inflammation in certain states in rats, is associated with a decrease in albumin levels (Mayot et al., 2008). Increase in erythrocyte CAT activity reflect enzyme engagement which resulted probably from the need for protection of hemoglobin from peroxidation (Halliwell et al., 2000). As there is no direct effect of warfarin on the activity of SOD and no detectable changes in ceruloplasmin, a decrease in the activity of SOD indicate enzyme expenditure (in converting O2 to H2O) in rats which consumed warfarin. Changes in both CAT and SOD activity might have resulted from the need for the activation of protective mechanisms necessary for scavenging ROS produced in plasma (Oishi et al., 1999; Toth et al., 1984). Given the interrelation of oxidative activity and inflammation (Halliwell et al., 1988) changes in antioxidant enzyme activity observed in erythrocytes of rats administered orally with warfarin might be considered as an indirect indicator of inflammation at systemic level in these animals. These changes might be a source for inflammation observed in settings of warfarin intake, as a decrease in SOD was shown to increase the sensitivity of erythrocytes to oxidative stress (Bartoli et al., 1992) and erythrocytes injured by antioxidant depletion were shown to impinge on inflammation (Lang et al., 2006). Circulating blood granulocytes are resting cells, acquiring a state of preactivation (priming) or become activated under appropriate stimulation to exert various effectors functions (phagocytosis, release of granule enzymes and proteins, production of reactive oxygen and nitrogen species and cytokines), activities essential for host defense against (noxious) external stimuli (Baggiolini, 1995). Increased propensity of neutrophils to exert these activities might, however, represent a risk of adverse intraluminal effects of these cells (Babior, 2000). Neutrophil priming/activation toward oxidant species production might result in endothelial cell injury, while modulation of adhesive/migratory potential of neutrophils might

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contribute to the formation of microtrombi as well as undesired tissue recruitment and surrounding tissue injury. 5. Conclusion Data presented in this study, demonstrated pro-inflammatory effects of warfarin intake on granulocytes, judging on priming/activation of several of their effectors functions. Inhibition of granulocyte TNF-a generation, imply suppression of granulocyte activity. Inflammatory milieu generated by warfarin intake might have contributed to the observed granulocyte activities. These findings clearly present immunomodulatory effects of warfarin in vivo and contribute to the list of biological activities of anticoagulant warfarin, other than those affecting hemostasis. Presented data might also be relevant for anticoagulant therapy strategies in humans. 6. Conflict of Interest The authors declare that they have no conflict of interest. Acknowledgements This study was supported by the Ministry of Education and Science of Republic of Serbia, Grants #173039 and # 173035. The authors would like to thank Jelena Vrankovic´ and Jelena Stošic´ for their engagement in preliminary experiments and Jelena Djokic for the help in the final phase of manuscript preparation. We thank Duško Blagojevic´ for its help in determination of antioxidant enzyme activity. References Ad-El, D.D., Meirovitz, A., Weinberg, A., Kogan, L., Arieli, D., Neuman, A., Linton, D., 2000. Warfarin skin necrosis: local and systemic factors. Br. J. Plast. Surg. 53, 624–626. Babior, B.M., 2000. Phagocytes and oxidative stress. Am. J. Med. 109, 33–44. Baggiolini, M., 1995. Activation and recruitment of neutrophil leukocytes. Clin. Exp. Immunol. 101, 5–6. Baumann, H., Morella, K.K., Jahreis, G.P., Marinkovic´, S., 1990. Distinct regulation of the interleukin-1 and interleukin-6 response elements of the rat haptoglobin gene in rat and human hepatoma cells. Mol. Cell Biol. 10, 5967–5976. Bartoli, G.M., Palozza, P., Piccioni, E., 1992. Enhanced sensitivity to oxidative stress in Cu, ZnSOD depleted rat erythrocytes. Biochim. Biophys. Acta. 1123, 291–295. Becker, R.C., 2007. Warfarin-induced vasculopathy. J. Thromb. Thrombolysis 23, 79– 81. Berkarda, B., Marrack, P., Kappler, J.W., Bakemeier, R.F., 1978. Effects of warfarin administration on the immune response of mice. Arzneimittelforschung 28, 1407–1410. Berkarda, B., Bouffard-Eyüboglu, H., Derman, U., 1983. The effect of coumarin derivatives on the immunological system of man. Agents Actions 13, 50–52. Beutler, E., 1982. Catalase. In: Beutler, E. (Ed.), Red Cell Metabolism: A Manual of Biochemical Methods. Grune and Stratton, New York, pp. 105–106. Biffl, W.L., Moore, E.E., Moore, F.A., Carl, V.S., Kim, F.J., Franciose, R.J., 1994. Interleukin-6 potentiates neutrophil priming with platelet-activating factor. Arch. Surg. 129, 1131–1136. Bobek, V., Kovarik, J., 2004. Antitumor and antimetastatic effect of warfarin and heparins. Biomed. Pharmacother. 58, 213–219. Bobek, V., Boubelik, M., Fiserova, A., L’uptovcova, M., Vannucci, L., Kacprzak, G., Kolodzej, J., Majewski, A.M., Hoffman, R.M., 2005. Anticoagulant drugs increase natural killer cell activity in lung cancer. Lung cancer 47, 215–223. Bogdan, C., Röllinghoff, M., Diefenbach, A., 2000. The role of nitric oxide in innate immunity. Immunol. Rev. 173, 17–26. Borish, L., Rosenbaum, R., Albury, L., Clark, S., 1989. Activation of neutrophils by recombinant interleukin 6. Cell Immunol. 121, 280–289. Bozeman, P.M., Learn, D.B., Thomas, E.L., 1990. Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase. J. Immunol. Methods 126, 125–133. Brovkovych, V., Gao, X.P., Ong, E., Brovkovych, S., Brennan, M.L., Su, X., Hazen, S.L., Malik, A.B., Skidgel, R.A., 2008. Augmented inducible nitric oxide synthase expression and increased NO production reduce sepsis-induced lung injury and mortality in myeloperoxidase-null mice. Am. J. Physiol. Lung Cell Mol. Physiol. 295, L96–L103. Cassatella, M.A., Meda, L., Bonora, S., Ceska, M., Constantin, G., 1993. Interleukin 10 (IL-10) inhibits the release of proinflammatory cytokines from human

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