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The effects of H1-antihistamines on the nitric oxide production by RAW 264.7 cells with respect to their lipophilicity
International Immunopharmacology 9 (2009) 990–995
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
The effects of H1-antihistamines on the nitric oxide production by RAW 264.7 cells with respect to their lipophilicity Jana Králová a, Lucia Račková b, Michaela Pekarová a, Lukáš Kubala a, Radomír Nosáľ b, Viera Jančinová b, Milan Číž a, Antonín Lojek a,⁎ a b
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovak Republic
a r t i c l e
i n f o
Article history: Received 18 December 2008 Received in revised form 17 March 2009 Accepted 6 April 2009 Keywords: H1-antihistamines Nitric oxide RAW 264.7 cells Inducible nitric oxide synthase Nitrites
a b s t r a c t H1-antihistamines are known to be important modulators of inflammatory response. However, the information about the influence of these drugs on reactive nitrogen species generation is still controversial. The main aim of the present study was to investigate the effects of selected H1-antihistamines on nitric oxide production by lipopolysaccharide-stimulated murine macrophages RAW 264.7, measured as changes in inducible nitric oxide synthase (iNOS) protein expression in cell lysates by Western blotting and nitrite formation in cell supernatants using the Griess reaction. In pharmacological non-toxic concentrations, H1antihistamines significantly inhibited nitrite accumulation that was not caused by the scavenging ability of drugs against nitric oxide, measured amperometrically. The degree of inhibition of nitrite accumulation positively correlated with the degree of tested lipophilicity, measured by reversed-phase thin layer chromatography. Furthermore, H1-antihistamines differentially modulated the iNOS protein expression. In conclusion, as was shown in this study, the modulation of nitric oxide production could be caused by the downregulation of iNOS protein expression and/or the iNOS protein activity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Histamine plays an important role in a variety of physiological processes. It regulates smooth muscle contraction, increases vascular permeability, stimulates gastric secretion, regulates functions of the central nervous system and modulates the regulation of cell proliferation and differentiation [1]. Nevertheless histamine is also a potent mediator of inflammation and tissue remodeling and serves as a modulator in various autoimmune diseases e.g. rheumatoid arthritis, osteoarthritis and especially allergic diseases [2,3]. The biological effects of histamine are mediated via binding to 4 types of histamine receptors (H1–H4) expressed on various cell types. The binding of histamine to an H1-receptor induces the progress of the allergic symptoms and these are prevented using histamine H1-receptor blockers in particular. This group of drugs, known as H1-antihistamines, is used clinically as anti-allergic and anti-emetic drugs [4,5]
Abbreviations: ATP, adenosine trisphosphate; DMEM, Dulbecco's Modified Eagle's Medium; ECL, enzymatic chemiluminescence; IgG, immunoglobulin G; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NF-κB, nuclear factor-kappa B; NO, nitric oxide; RP TLC, reversed phase thin layer chromatography; QSAR, quantitative structure– activity relationship; RNS, reactive nitrogen species; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; Tris, tris (hydroxy methyl) aminomethane. ⁎ Corresponding author. Tel.: +420 541 517 160; fax: +420 541 211 293. E-mail address: [email protected] (A. Lojek). 1567-5769/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2009.04.005
and possesses antiinflammatory and antiplatelet activities [6,7]. The suppresive effects of H1-antihistamines, on histamine secretion from mast cells and basophils are also well known. Results published by several investigators [6,8] suggests that the chemical structure of some H1-antihistamines, namely positively charged lipophilic molecules, allows them to associate with the cell membrane. Moreover, they are able to inhibit the activity of calcium-dependent enzymes, affect the calcium mobilization and the discharge of intracellular calcium stores as being responsible for various inflammatory reactions including histamine secretion, formation of eicosanoids, and reactive oxygen species (ROS). In addition to ROS, reactive nitrogen species (RNS), produced mainly by macrophages, are one of the important microbicidal tools in the process of inflammation during the fight against pathogenic microorganisms, bacteria and tumor cells. Nitric oxide (NO), a member of RNS, is an important molecule involved in the regulation of many physiological and microbicidal processes. However, the overproduction of NO is included in several chronic inflammatory diseases such as bronchitis, osteoarthritis and rheumatoid arthritis [9]. NO is generated by several types of cells in an L-arginine pathway in the presence of NO synthases [10,11]. Phagocytes express mainly inducible NO synthase (iNOS) in response to inflammatory stimuli, such as bacterial lipopolysaccharide (LPS) or proinflammatory cytokines (interleukin-1 or tumor necrosis factor-α). Nitrites are the main and stable oxidative end product of NO chemistry and they
J. Králová et al. / International Immunopharmacology 9 (2009) 990–995 Table 1 List of tested H1-antihistamines of the 1st and 2nd generations, manufacturers and abbreviations used in the text (see Materials). H1-antihistamines 1st generation
Abbreviation
Manufacturer
Antazoline-hydrochloride Bromadryl Brompheniramine-maleate Clemastine-fumarate Cyclizine-hydrochloride
ANT BRO BPH CLE CYC
Dithiaden Chlorcyclizine-hydrochloride Chlorpheniramine-maleate Oxatomide Pheniramine-maleate
DIT CHC CPH OXA PHE
Ciba–Geigy, Switzerland Zentiva, Czech Republic Wyeth, Austria SANDOZ GmbH, Austria The Welcome foundation LTD, Great Britain Zentiva, Czech Republic IDC ABBOTT Lab. LTD, Great Britain GLAXO Smithkline, Great Britain Janssen research foundation, Belgium Hoechst, Germany
2nd generation Acrivastine
ACR
Astemizole Ketotifen-fumarate Loratadine
AST KET LOR
The Welcome Foundation LTD, Great Britain Janssen Research Foundation, Belgium Zentiva, Slovak Republic Zentiva, Slovak Republic
sensitively reflect changes in iNOS activity. Detection of nitrite concentrations and iNOS protein expression are considered to be reliable methods for verifying the influence of drugs on the NO production by cells at various levels [12]. Whereas the effect of H1-antihistamines on the production of ROS is relatively well described [13,14], the information about the influence of these drugs on RNS generation is still insufficient. Therefore, the main aim of the present study was to investigate the effects of H1-antihistamines on NO production by phagocytes. The study measures the changes in iNOS protein expression and nitrite formation by murine macrophages RAW 264.7 stimulated by LPS. To describe the direct interactions of H1-antihistamines with NO, the scavenging properties of these drugs against NO in a cell free system were also detected. The second aim of this study was to explain the relationship between the effect of the drugs tested on the nitric oxide production and their lipophilicity. 2. Materials and methods 2.1. Materials The H1-antihistamines of the 1st generation and the 2nd generation are listed in Table 1. The stock solutions of drugs (3 × 10− 3 M) dissolved in distilled water were stored in − 20 °C. For the experiments, stock solutions were diluted in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with heat-inactivated 10% fetal bovine serum or in phosphate buffer solution (PBS) and the final concentrations (5 × 10− 5 M and 10− 4 M) were tested. LPS from Escherichia coli serotype 0111:B4 (Sigma-Aldrich, USA) was dissolved in PBS (1 mg/ml) and stored in −20 °C. Other chemicals were purchased from local distributors. 2.2. RAW 264.7 cells A murine leukaemic macrophage-like RAW 264.7 cells (ATCC, USA) were grown in plastic culture plates in DMEM supplemented with 10% fetal bovine serum, gentamycin, glucose and NaHCO3 in a CO2 incubator (5% CO2 and 95% of air humidity) at 37 °C. 2.3. Experimental procedure Cells were seeded at an initial density of 2.5 × 106 cells/1 ml/ well in a 6-well tissue culture plates and preincubated with H1-
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antihistamine for 60 min. Cells were subsequently stimulated with LPS in the concentration 0.1 μg/ml and incubated 24 h/37 °C/5% CO2. Non stimulated cells incubated in the absence of H1-antihistamines served as a negative control. Cells stimulated with 0.1 μg/ml LPS and incubated in the absence of H1-antihistamines were used as a positive control. After 24 h supernatants were harvested and the nitrite accumulation was determined. The cells were lysed and used for the measurement of adenosine triphosphate (ATP) content and iNOS protein expression. 2.4. Toxicity of H1-antihistamines Toxicity was tested using bioluminescent bacteria Photorhabdus luminescens subsp. thracensis CCM 7295T according to [15]. Bacteria were cultivated in medium DSMZ 423. Bright bacterial cells collected from the culture in stationary phase (2.4 * 108 cells) were transferred to Hanks balanced salt solution with H1-antihistamines and bacterial bioluminescence was measured using luminometer LM-01T (Immunotech, Czech Republic). The inhibition of bacterial bioluminescence was evaluated 15 and 30 min after measuring began. Results obtained revealed that concentrations of drugs higher than 10− 4 M are potentially toxic. Therefore, 5 × 10− 5 M and 10− 4 M final concentrations of drugs were selected for further experiments. 2.5. ATP test of cell viability The viability of RAW 264.7 cells was tested by the commercial ATP cellular kit (Biothema, Sweden). Cells were incubated according to the experimental procedure, supernatant was removed and cells were lyzed by the Somatic cell ATP releasing reagent (Sigma Aldrich, USA). Then 50 μl of lysate were mixed with 20 μl of ATP reagent containing D-luciferin, luciferase and stabilizers. Intracellular ATP contents were determined luminometrically using luminometer Orion II (Berthold Detection Systems GmbH, Germany). 2.6. Determination of nitrite production by cells Detection of nitrites (NO− 2 ) accumulated in the cell supernatants was performed using the Griess reagent as described previously [16]. The volume of 150 μl of the cell supernatant was incubated with 150 μl Griess reagent (Sigma-Aldrich, USA) for 15 min in the dark at room temperature and the absorbance was measured at 546 nm using the Spectra Rainbow UV/Vis microplate reader (SLT Tecan, Germany). The concentrations of nitrites were derived from regression analysis using serial dilutions of sodium nitrite as a standard. The concentration values of each sample are expressed as a percentage of positive control. 2.7. Determination of iNOS protein expression in cells Cells were lysed with 1% SDS (sodium dodecyl sulfate) lysing buffer with the addition of 1% phenylmethanesulphonylfluoride. Protein volume in cell samples was determined using commercial bicinochoninic acid protein assay (Pierce, USA). The same concentration of proteins (22 μg) were separated by 7.5% SDS-PAGE (SDSpolyacrylamide gel electrophoresis) and then transferred to a polyvinylidene difluoride membrane (Millipore, USA) in a buffer containing Tris-glycine and 20% methanol. Membranes were incubated with 5% fat free milk in Tris buffer-Tween 20 (TBS-T) at room temperature for 1 h. The protein was labeled using a mouse antibody (1:1000) specific to iNOS (Anti-iNOS/NOS Type II mAb, BD Transduction Laboratories, USA) and a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) antibody (1:2000; ECL™ Antimouse IgG, Amersham, Biosciences, USA). After each incubation with both antibodies the membrane was washed three times in TBS-T buffer for 10 min. Subsequently immunoreactive bands were
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Fig. 1. The viability of RAW 264.7 cells (evaluated by luminometrical ATP-test) stimulated by 0.1 µg/ml LPS and incubated in the presence of 10− 4 M (blank bars) and 5 × 10− 5 M (full bars) H1-antihistamines for 24 h in 37 °C/5% CO2. Data are expressed as mean ± SEM of at least 3 independent experiments, which were run in duplicates. The symbol (*) shows significant differences (p b 0.05) as compared to control cells. The symbol (**) shows significant differences (p b 0.01) as compared to control cells.
visualised using ECL detection reagent (Detection reagents kit, Pierce, USA) and exposed to radiografic film (AGFA, Belgium). Relative levels of proteins were quantified by scanning densitometry using the ImageJ™ program and the optical density for each individual band was expressed as a percent of positive control. The equal loading of proteins was verified by β-actin immunoblotting.
reached the background current. The potential NO scavenger causes the rapid decrease of the NO-induced signal. The signal was measured for 280 s to obtain kinetic curves. Then the integral areas under the control curve and sample curves were calculated and the scavenging activity of drugs was evaluated. 2.9. Determination of RM values
2.8. Amperometrical detection of NO scavenging The scavenging properties of H1-antihistamines against NO were performed in a chemical system amperometrically using three electrode systems as described previously [17]. A porphyrinic microsensor working electrode, platinum wire counter electrode and a miniature saturated silver/silver chloride reference electrode were connected to the ISO-NO MARK II potentiostat (WPI, USA). The measurement was practised using distilled water saturated with pure NO gas (according to the WPI manual, [18]). The injection of 1 µl of the NO-saturated water into the glass vial (final concentration of NO in the vial = 595 nM) caused the rapid increase (peak time = 15 ± 5 s) with a subsequent gradual decrease of an NO-induced signal until it
The lipophilicity parameter represented by RM values was measured by the reversed-phase thin-layer chromatography technique (RP TLC). The mobile phase consisted of a PBS (0.1 M, pH 7.4) mixture with acetone (20:80, v/v). The stationary phase was obtained by impregnation of the layer of silica gel G F254 plates with a 5% solution of liquid paraffin in ether. The impregnation of plates was described previously [19,20]. H1-antihistamines were dissolved in methanol, and 1 μl of the solution was spotted onto the plates. The developed plates were dried, and the compounds were detected under UV light at 254 nm. The RM values were calculated by the formula RM = log (1/RF − 1). RF =a/b, where a is a distance from the start to the spot center of the sample and b is a distance from the start to the dissolvent forehead.
Fig. 2. The nitrite accumulation in cell supernatants (blank bars) and iNOS protein expression (full bars) in RAW 264.7 cells stimulated by 0.1 µg/ml LPS and incubated in the presence of 10− 4 M H1-antihistamines for 24 h. Representative Western blot of iNOS protein expression are also shown. Equal loading of proteins was verified by β-actin immunoblotting. Data are expressed as mean ± SEM of at least 5 independent experiments, which were run in duplicates. The symbol (*) shows significant differences (p b 0.05) as compared to control cells. The symbol (**) shows significant differences (p b 0.01) as compared to control cells.
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2.10. Statistical evaluation Data are expressed as the mean± standard error of the mean (SEM) of at least 3 independent experiments, that were run in duplicates. Results were analysed by Student's two-tailed t-test using Statistica software (StatSoft, USA), values below 0.05 (*) and 0.01 (**) were considered as statistically significant. 3. Results 3.1. Modulation of cell viability by H1-antihistamines The effect of drugs on cell viability was tested in two selected concentrations (5 × 10− 5 M and 10− 4 M). ANT, KET, CYC, CPH, PHE, ACR, and BPH did not significantly decrease cell viability in either concentration (Fig. 1). In a 10− 4 M concentration, LOR decreased the cell viability to 53.9 ± 2.1%, in comparison with the control cells. Despite that, LOR was tested in both concentrations. CHC, OXA, CLE, DIT, and BRO in a 10− 4 M concentration exerted a significant effect on cell viability. Thus, these drugs were only used in a 5 × 10− 5 M concentration in the following experiments. AST was the only drug that exerted a strong effect on cell viability in both tested concentrations; and, therefore, it was not used in further experiments.
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Table 3 Scavenging effect of 10− 4 M H1-antihistamines against NO evaluated amerometrically in cell-free system. H1-antihistamine
Integral under curves mean ± SEM
Control ACR ANT AST BRO CLE CYC CHC DIT CPH BPH PHE KET LOR OXA
20.2 ± 0.6 19.0 ± 0.3 24.7 ± 0.7 20.3 ± 3.4 21.3 ± 1.2 19.0 ± 1.6 17.4 ± 1.3 17.9 ± 1.0 24.9 ± 5.9 19.9 ± 2.9 21.7 ± 0.7 18.4 ± 4.3 24.0 ± 6.1 20.4 ± 0.5 18.5 ± 4.4
Data are expressed as mean ± SEM of at least 3 independent experiments, which were run in duplicates. (see NO scavenging by H1-antihistamines).
BPH, PHE, and KET) did not significantly affect the nitrite accumulation in cell supernatants.
3.2. Modulation of macrophage NO production by H1-antihistamines
3.3. Modulation of macrophage iNOS expression by H1-antihistamines
The nitrite accumulation in cell supernatants was dependent on the activation state of RAW 264.7 cells. While only very low concentrations of nitrites (0.5–2 μM) were detected in non-stimulated cells, the cells stimulated with 0.1 μg/ml LPS produced 28.2 ± 2.52 μM nitrites (mean ± SEM). Fig. 2 demonstrates that, in a 10− 4 M concentration, LOR, CPH, ACR, and CYC significantly affected nitrite accumulation in the LPS-stimulated cell supernatants. On the other hand, ANT significantly upregulated the nitrite concentration. KET, PHE, and BPH had no significant effect on nitrite concentration. The results described in Table 2 show that nitrite concentrations were significantly decreased by BRO, CLE, CHC, DIT, LOR, and OXA in the presence of 5 × 10− 5 M drugs. The other drugs (ACR, ANT, CYC, CPH,
The possibility that the changes in nitrite concentration were associated with iNOS protein expression was determined using Western blot analysis. Fig. 2 represents the effect of 10− 4 M H1antihistamines on iNOS protein expression in LPS-stimulated cells. In comparison with the iNOS protein level in the control sample, iNOS protein expression was significantly inhibited by LOR, CPH, ACR, CYC, KET, PHE, BPH, and ANT. The effects of 5 × 10− 5 M H1-antihistamines are shown in Table 2. In LPS-stimulated cells, iNOS protein expression was significantly inhibited by BRO, CLE, DIT, BPH, PHE, KET, and OXA. iNOS protein expression was also inhibited by ANT, CYC, CHC, CPH, and LOR-however, without statistical significance.
Table 2 The lipophilicity of 5 × 10− 5 M H1-antihistamines and their effects on the nitrite concentrations in cell supernatants and iNOS protein expression in RAW 264.7 cells stimulated by 0.1 µg/ml LPS. H1-Antihistamine
ACR ANT AST BRO CLE CYC CHC DIT CPH BPH PHE KET LOR OXA
Lipophilicity
NO-2 concentration
RM
% of inhibition
0.07 ± 0.18 1.36 ± 0.13 2.30 ± 0.0 1.66 ± 0.36 1.99 ± 0.0 0.97 ± 0.08 1.44 ± 0.0 1.49 ± 0.29 1.06 ± 0.2 1.12 ± 0.0 0.63 ± 0.08 0.95 ± 0.0 1.57 ± 0.1 2.30 ± 0.0
10.4 ± 0.6 2.8 ± 1.0 – 27.3 ± 6.3* 43.1 ± 4.6** 11.2 ± 0.8 48.5 ± 0.3** 43.9 ± 3.3** 12.7 ± 1.2 11.0 ± 4.9 4.1 ± 1.2 4.1 ± 0.5 46.0 ± 1.7** 69.4 ± 0.9**
Densitometric analysis of iNOS protein 1.6 ± 2.1 15.7 ± 2.6 – 50.5 ± 4.2** 59.3 ± 3.7** 15.6 ± 4.4 13.8 ± 3.0 40.1 ± 4.0* 19.0 ± 3.7 39.0 ± 6.4* 31.9 ± 2.5* 23.5 ± 2.3* 15.2 ± 6.4 44.3 ± 4.0**
Data are expressed as mean ± SEM of at least 3 independent experiments, which were run in duplicates. The symbol (*) shows significant differences (p b 0.05) as compared to control cells. The symbol (**) shows significant differences (p b 0.01) as compared to control cells. (see Modulation of macrophage NO production by H1-antihistamines, Modulation of macrophage iNOS expression by H1-antihistamines and Lipophilicity of studied H1antihistamines).
Fig. 3. The effect of 5 × 10− 5 M H1-antihistamines. The regression line, 95% confidence interval, data points and linear correlation equation for cell viability and lipophilicity (RM). Data are expressed as mean ± SEM of at least 3 independent experiments, which were run in duplicates.
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Fig. 4. The effect of 5 × 10− 5 M H1-antihistamines. The regression line, 95% confidence interval, data points and linear correlation equation for nitrite inhibition and lipophilicity (RM). Data are expressed as mean ± SEM of at least 3 independent experiments, which were run in duplicates.
3.4. NO scavenging by H1-antihistamines To verify whether the changes in nitrite concentration were induced by the scavenging ability of H1-antihistamines against NO, various drugs were tested, using direct amperometrical analysis. In a 10− 4 M concentration, none of the tested drugs exerted scavenging properties against NO. The integrals of the run curves for all the drugs tested are presented in Table 3. 3.5. Lipophilicity of studied H1-antihistamines The results describing the lipophilicity of all the drugs evaluated by RP TLC analysis are demonstrated in Table 2. ACR exerted the lowest lipophilicity, while the highest lipophilicity was recorded for AST and OXA. No linear correlation was detected between lipophilicity and the effect on the viability of cells incubated with 5× 10− 5 M H1-antihistamines (see Fig. 3). The degree of inhibition of nitrite accumulation positively correlated with the degree of tested H1-antihistamines lipophilicity (r =0.7786, p= 0.0017) (see Fig. 4). A less significant correlation (r =0.66, p =0.0143) was obtained between iNOS protein expression and lipophilicity. 4. Discussion H1-antihistamines represent an important class of pharmaceuticals and are currently the second-most-commonly-used medications (after antibiotics), with more than 40 varieties used in clinical practice worldwide. The volume of recently published evidence claims that H1-antihistamines play a very important role in the regulation of phagocyte-derived ROS production [7,13,14,21] as well as the regulation of myeloperoxidase activity [22]. But, the effects of H1-antihistamines on NO production remain to be elucidated. We studied this effect in in vitro experiments with a murine macrophage RAW 264.7 cell line, which is a convenient cell model for monitoring NO production after stimulation with LPS [23,24]. The most important finding of our study is that, corresponding to their pertinence to different groups of drugs, the selected H1antihistamines in a concentration 5 × 10− 5 M have a different ability
to modulate NO production by LPS-stimulated macrophages. The group of drugs—including CLE, BRO, and DIT—inhibited iNOS expression in LPS-stimulated cells. This inhibition was followed by a significant reduction in nitrite levels. In the presence of BPH, OXA, PHE, and KET, the LPS-induced iNOS protein expression was also inhibited; however, the decrease in nitrite accumulation was less pronounced. On the other hand, CHC and LOR did not affect iNOS protein expression, but they were able to decrease nitrite levels in the cell supernatants. Finally, ACR, ANT, CPH, and CYC had no effect on nitrite accumulation and iNOS protein expression. In our study, selected H1-antihistamines were also tested in a 10− 4 M concentration. In this concentration, KET, CPH, CYC, PHE, and BPH revealed an effect similar to that found in a 5 × 10− 5 M concentration. Further, 10− 4 M ACR and LOR significantly reduced the expression of iNOS protein and caused a similar decrease in nitrite levels. For ANT, very interesting effects on NO production were observed. Although, in a 10− 4 M concentration, it caused a significant increase in nitrite concentration and significantly inhibited the expression of iNOS, ANT did not induce any effect in a 5 × 10− 5 M concentration. AST, CHC, OXA, CLE, DIT, and BRO significantly affected macrophage viability; thus, their effects on nitrite accumulation and iNOS protein expression were not evaluated. With respect to the fact that all the drugs studied had no scavenging activity against NO in the chemical system, we suppose that H1-antihistamines modulate NO production by activated macrophages. The inhibitory effect on macrophage iNOS protein expression could be explained by the ability to affect the intracellular signaling pathways that lead to NO production. It was previously documented that the activation of histamine receptors leads to the activation of nuclear factor-κB, which is responsible for the regulation of iNOS expression [25–27]. Therefore, from our results, it can be assumed that the binding of H1-antihistamines could downregulate the activation of nuclear factor-κB. The effect of H1-antihistamines was probably mediated via the affection of iNOS enzyme activity in a case when the decrease in nitrite accumulation was not accompanied with the inhibition of iNOS protein expression. It is apparent from our results that there is a significant difference between the effects of various H1-antihistamines on NO production by RAW 264.7 cells stimulated by LPS. These findings lead us to look for some relationship between the physico-chemical properties of various H1-antihistamines and their effects on NO production by macrophages. RM values provide important evidence about the lipophilicity of the drugs studied. In comparison with PHE, increased lipophilicity predisposed CPH and BPH to be better incorporated into the lipid part of the membrane and its interaction with key proteins [28]. Actually, in comparison with PHE, CPH and BPH were able to suppress nitrite production by LPS-stimulated macrophages significantly. Analogously, in comparison with its non-halogenated analog CYC, the halogen substituent in CHC responsible for the increase of RM was associated with its more pronounced inhibition of nitrite formation. Furthermore, regardless of their general structural variability, the ability of H1-antihistamines to reduce nitrite concentration was dependent on lipophilicity. The increase in the effect on nitrite production by cells correlates with the increase in lipophilicity observed for several compounds (CYC vs CHC, PHE vs CPH and BPH). This was clearly documented by a good linear correlation between the percentual inhibition of nitrite concentration and lipophilicity. Despite Pauli [29] reporting that the toxicity of some compounds increases with higher lipophilicity, we did not prove any significant correlation between viability and lipophilicity. Nevertheless, a notable drop in viability was observed for those compounds with increased lipophilicity. In agreement with a range of QSAR (quantitative structure–activity relationship) studies [30], our results point to the dominant role of the lipophilicity of the agents tested in their biological activity in LPSstimulated RAW 264.7 macrophages. The Ferguson principle [31] is also quite applicable to our data, since the rate limiting step for the
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effect of H1-antihistamines on macrophage functions appeared to be directed by their ability to reach the site of action. Furthermore, during transportation through the membrane system, the entrapment of more hydrophobic compounds in the lipid phase could affect their inhibitory effect on iNOS expression. On the other hand, the observed effects of the tested H1-antihistamines did not appear to be limited by their higher hydrophobicity, thus pointing to the involvement of the membrane as their site of action. We can conclude from our results that the structural properties of H1-antihistamines, particularly lipophilicity, significantly affect the biological activity of macrophages. These drugs are able to modulate NO production in vitro by macrophage cell line RAW 264.7 stimulated by LPS. The modulation shown in this study could be caused by the downregulation of iNOS protein expression or by iNOS enzyme activity. Acknowledgements This study was conducted under the research plans AVOZ50040507 and AVOZ50040702 and supported by grants 1QS500040507 GA AS CR, GA CR 525/06/1196 and VEGA 2/7019/27. References [1] Leurs R, Smit MJ, Timmerman H. Molecular pharmacological aspects of histamine receptors. Pharmacol Ther 1995;66:413–63. [2] Buckley MG, Walters C, Wong WM, Cawley MI, Ren S, Schwartz LB, et al. Mast cell activation in arthritis: detection of alpha- and beta-tryptase, histamine and eosinophil cationic protein in synovial fluid. Clin Sci (Lond) 1997;93:363–70. [3] Renoux M, Hilliquin P, Galoppin L, Florentin I, Menkes CJ. Release of mast cell mediators and nitrites into knee joint fluid in osteoarthritis—comparison with articular chondrocalcinosis and rheumatoid arthritis. Osteoarthr Cartil 1996;4:175–9. [4] De Vos C. H1-receptor antagonists: effects on leukocytes, myth or reality? Clin Exp Allergy 1999;29(Suppl 3):60–3. [5] Berthon B, Taudou G, Combettes L, Czarlewski W, Carmi-Leroy A, Marchand F, et al. In vitro inhibition, by loratadine and descarboxyethoxyloratadine, of histamine release from human basophils, and of histamine release and intracellular calcium fluxes in rat basophilic leukemia cells (RBL-2H3). Biochem Pharmacol 1994;47:789–94. [6] Church MK. H(1)-antihistamines and inflammation. Clin Exp Allergy 2001;31:1341–3. [7] Nosal R, Drabikova K, Jancinova V, Petrikova M, Fabryova V. Antiplatelet and antiphagocyte activity of H1-antihistamines. Inflamm Res 2005;54(Suppl 1): S19–20. [8] Lullmann H, Plosch H, Ziegler A. Ca replacement by cationic amphiphilic drugs from lipid monolayers. Biochem Pharmacol 1980;29:2969–74. [9] McInnes IB, Leung BP, Field M, Wei XQ, Huang FP, Sturrock RD, et al. Production of nitric oxide in the synovial membrane of rheumatoid and osteoarthritis patients. J Exp Med 1996;184:1519–24. [10] Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329: 2002–12. [11] Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524–6.
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[12] Stuehr DJ, Marletta MA. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci U S A 1985;82:7738–42. [13] Kralova J, Ciz M, Nosal R, Drabikova K, Lojek A. Effect of H(1)-antihistamines on the oxidative burst of rat phagocytes. Inflamm Res 2006;55(Suppl 1):S15–16. [14] Nosal R, Drabikova K, Jancinova V, Macickova T, Pecivova J, Holomanova D. On the pharmacology and toxicology of neutrophils. Neuro Endocrinol Lett 2006;27 (Suppl 2):148–51. [15] Lojek A, Pecivova J, Macickova T, Nosal R, Papezikova I, Ciz M. Effect of carvedilol on the production of reactive oxygen species by HL-60 cells. Neuro Endocrinol Lett 2008;29:779–83. [16] Migliorini P, Corradin G, Corradin SB. Macrophage NO2-production as a sensitive and rapid assay for the quantitation of murine IFN-gamma. J Immunol Methods 1991;139:107–14. [17] Hrbac J, Gregor C, Machova M, Kralova J, Bystron T, Ciz M, et al. Nitric oxide sensor based on carbon fiber covered with nickel porphyrin layer deposited using optimized electropolymerization procedure. Bioelectrochemistry 2007;71:46–53. [18] Feelisch M, Kelm M. Biotransformation of organic nitrates to nitric oxide by vascular smooth muscle and endothelial cells. Biochem Biophys Res Commun 1991;180:286–93. [19] Devinsky F, Zamocka J, Lacko I, Polakovicova M. QSAR and CAMM study of amphiphilic antimicrobially active 2,2'-bipyridyl monoammonium salts. Pharmazie 1996;51:727–31. [20] Glavac D. RM values of some colchicines and colchiceinamides determined by reversed-phase thin-layer chromatography. J Chromatogr 1992;591:367–70. [21] Jancinova V, Drabikova K, Nosal R, Holomanova D. Extra- and intracellular formation of reactive oxygen species by human neutrophils in the presence of pheniramine, chlorpheniramine and brompheniramine. Neuro Endocrinol Lett 2006;27(Suppl 2):141–3. [22] Kralova J, Nosal R, Drabikova K, Jancinova V, Denev P, Moravcova A, et al. Comparative investigations of the influence of H1-antihistamines on the generation of reactive oxygen species by phagocytes. Inflamm Res 2008;57 (Suppl 1):S49–50. [23] Raschke WC, Baird S, Ralph P, Nakoinz I. Functional macrophage cell lines transformed by Abelson leukemia virus. Cell 1978;15:261–7. [24] Hofer M, Vacek A, Lojek A, Hola J, Streitova D. Ultrafiltered pig leukocyte extract (IMUNOR®) decreases nitric oxide formation and hematopoiesis-stimulating cytokine production in lipopolysaccharide-stimulated RAW 264.7 macrophages. Int Immunopharmacol 2007;7:1369–74. [25] Bakker RA, Schoonus SB, Smit MJ, Timmerman H, Leurs R. Histamine H(1)receptor activation of nuclear factor-kappa B: roles for G beta gamma- and G alpha (q/11)-subunits in constitutive and agonist-mediated signaling. Mol Pharmacol 2001;60:1133–42. [26] Aoki Y, Qiu D, Zhao GH, Kao PN. Leukotriene B4 mediates histamine induction of NF-kappaB and IL-8 in human bronchial epithelial cells. Am J Physiol 1998;274: L1030–1039. [27] Hu Q, Deshpande S, Irani K, Ziegelstein RC. [Ca(2+)](i) oscillation frequency regulates agonist-stimulated NF-kappaB transcriptional activity. J Biol Chem 1999;274:33995–8. [28] Jancinova V, Drabikova K, Nosal R, Majekova M, Rackova L, Holomanova D. Antiradical effects of antihistamines in human blood. Structure–activity relationship. Inflamm Res 2006;55(Suppl 1):S85–86. [29] Pauli A. Relationship between lipophilicity and toxicity of essential oils. Int J Ess Oil Ther 2008;2:60–8 (9). [30] Dearden JC. Partitioning and lipophilicity in quantitative structure–activity relationships. Environ Health Perspect 1985;61:203–28. [31] Ferguson J. The use of chemical potentials as indices of toxicity. Proc Roy Soc London 1939;B127:387–403.
Contents lists available at ScienceDirect
International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
The effects of H1-antihistamines on the nitric oxide production by RAW 264.7 cells with respect to their lipophilicity Jana Králová a, Lucia Račková b, Michaela Pekarová a, Lukáš Kubala a, Radomír Nosáľ b, Viera Jančinová b, Milan Číž a, Antonín Lojek a,⁎ a b
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovak Republic
a r t i c l e
i n f o
Article history: Received 18 December 2008 Received in revised form 17 March 2009 Accepted 6 April 2009 Keywords: H1-antihistamines Nitric oxide RAW 264.7 cells Inducible nitric oxide synthase Nitrites
a b s t r a c t H1-antihistamines are known to be important modulators of inflammatory response. However, the information about the influence of these drugs on reactive nitrogen species generation is still controversial. The main aim of the present study was to investigate the effects of selected H1-antihistamines on nitric oxide production by lipopolysaccharide-stimulated murine macrophages RAW 264.7, measured as changes in inducible nitric oxide synthase (iNOS) protein expression in cell lysates by Western blotting and nitrite formation in cell supernatants using the Griess reaction. In pharmacological non-toxic concentrations, H1antihistamines significantly inhibited nitrite accumulation that was not caused by the scavenging ability of drugs against nitric oxide, measured amperometrically. The degree of inhibition of nitrite accumulation positively correlated with the degree of tested lipophilicity, measured by reversed-phase thin layer chromatography. Furthermore, H1-antihistamines differentially modulated the iNOS protein expression. In conclusion, as was shown in this study, the modulation of nitric oxide production could be caused by the downregulation of iNOS protein expression and/or the iNOS protein activity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Histamine plays an important role in a variety of physiological processes. It regulates smooth muscle contraction, increases vascular permeability, stimulates gastric secretion, regulates functions of the central nervous system and modulates the regulation of cell proliferation and differentiation [1]. Nevertheless histamine is also a potent mediator of inflammation and tissue remodeling and serves as a modulator in various autoimmune diseases e.g. rheumatoid arthritis, osteoarthritis and especially allergic diseases [2,3]. The biological effects of histamine are mediated via binding to 4 types of histamine receptors (H1–H4) expressed on various cell types. The binding of histamine to an H1-receptor induces the progress of the allergic symptoms and these are prevented using histamine H1-receptor blockers in particular. This group of drugs, known as H1-antihistamines, is used clinically as anti-allergic and anti-emetic drugs [4,5]
Abbreviations: ATP, adenosine trisphosphate; DMEM, Dulbecco's Modified Eagle's Medium; ECL, enzymatic chemiluminescence; IgG, immunoglobulin G; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NF-κB, nuclear factor-kappa B; NO, nitric oxide; RP TLC, reversed phase thin layer chromatography; QSAR, quantitative structure– activity relationship; RNS, reactive nitrogen species; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; Tris, tris (hydroxy methyl) aminomethane. ⁎ Corresponding author. Tel.: +420 541 517 160; fax: +420 541 211 293. E-mail address: [email protected] (A. Lojek). 1567-5769/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2009.04.005
and possesses antiinflammatory and antiplatelet activities [6,7]. The suppresive effects of H1-antihistamines, on histamine secretion from mast cells and basophils are also well known. Results published by several investigators [6,8] suggests that the chemical structure of some H1-antihistamines, namely positively charged lipophilic molecules, allows them to associate with the cell membrane. Moreover, they are able to inhibit the activity of calcium-dependent enzymes, affect the calcium mobilization and the discharge of intracellular calcium stores as being responsible for various inflammatory reactions including histamine secretion, formation of eicosanoids, and reactive oxygen species (ROS). In addition to ROS, reactive nitrogen species (RNS), produced mainly by macrophages, are one of the important microbicidal tools in the process of inflammation during the fight against pathogenic microorganisms, bacteria and tumor cells. Nitric oxide (NO), a member of RNS, is an important molecule involved in the regulation of many physiological and microbicidal processes. However, the overproduction of NO is included in several chronic inflammatory diseases such as bronchitis, osteoarthritis and rheumatoid arthritis [9]. NO is generated by several types of cells in an L-arginine pathway in the presence of NO synthases [10,11]. Phagocytes express mainly inducible NO synthase (iNOS) in response to inflammatory stimuli, such as bacterial lipopolysaccharide (LPS) or proinflammatory cytokines (interleukin-1 or tumor necrosis factor-α). Nitrites are the main and stable oxidative end product of NO chemistry and they
J. Králová et al. / International Immunopharmacology 9 (2009) 990–995 Table 1 List of tested H1-antihistamines of the 1st and 2nd generations, manufacturers and abbreviations used in the text (see Materials). H1-antihistamines 1st generation
Abbreviation
Manufacturer
Antazoline-hydrochloride Bromadryl Brompheniramine-maleate Clemastine-fumarate Cyclizine-hydrochloride
ANT BRO BPH CLE CYC
Dithiaden Chlorcyclizine-hydrochloride Chlorpheniramine-maleate Oxatomide Pheniramine-maleate
DIT CHC CPH OXA PHE
Ciba–Geigy, Switzerland Zentiva, Czech Republic Wyeth, Austria SANDOZ GmbH, Austria The Welcome foundation LTD, Great Britain Zentiva, Czech Republic IDC ABBOTT Lab. LTD, Great Britain GLAXO Smithkline, Great Britain Janssen research foundation, Belgium Hoechst, Germany
2nd generation Acrivastine
ACR
Astemizole Ketotifen-fumarate Loratadine
AST KET LOR
The Welcome Foundation LTD, Great Britain Janssen Research Foundation, Belgium Zentiva, Slovak Republic Zentiva, Slovak Republic
sensitively reflect changes in iNOS activity. Detection of nitrite concentrations and iNOS protein expression are considered to be reliable methods for verifying the influence of drugs on the NO production by cells at various levels [12]. Whereas the effect of H1-antihistamines on the production of ROS is relatively well described [13,14], the information about the influence of these drugs on RNS generation is still insufficient. Therefore, the main aim of the present study was to investigate the effects of H1-antihistamines on NO production by phagocytes. The study measures the changes in iNOS protein expression and nitrite formation by murine macrophages RAW 264.7 stimulated by LPS. To describe the direct interactions of H1-antihistamines with NO, the scavenging properties of these drugs against NO in a cell free system were also detected. The second aim of this study was to explain the relationship between the effect of the drugs tested on the nitric oxide production and their lipophilicity. 2. Materials and methods 2.1. Materials The H1-antihistamines of the 1st generation and the 2nd generation are listed in Table 1. The stock solutions of drugs (3 × 10− 3 M) dissolved in distilled water were stored in − 20 °C. For the experiments, stock solutions were diluted in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with heat-inactivated 10% fetal bovine serum or in phosphate buffer solution (PBS) and the final concentrations (5 × 10− 5 M and 10− 4 M) were tested. LPS from Escherichia coli serotype 0111:B4 (Sigma-Aldrich, USA) was dissolved in PBS (1 mg/ml) and stored in −20 °C. Other chemicals were purchased from local distributors. 2.2. RAW 264.7 cells A murine leukaemic macrophage-like RAW 264.7 cells (ATCC, USA) were grown in plastic culture plates in DMEM supplemented with 10% fetal bovine serum, gentamycin, glucose and NaHCO3 in a CO2 incubator (5% CO2 and 95% of air humidity) at 37 °C. 2.3. Experimental procedure Cells were seeded at an initial density of 2.5 × 106 cells/1 ml/ well in a 6-well tissue culture plates and preincubated with H1-
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antihistamine for 60 min. Cells were subsequently stimulated with LPS in the concentration 0.1 μg/ml and incubated 24 h/37 °C/5% CO2. Non stimulated cells incubated in the absence of H1-antihistamines served as a negative control. Cells stimulated with 0.1 μg/ml LPS and incubated in the absence of H1-antihistamines were used as a positive control. After 24 h supernatants were harvested and the nitrite accumulation was determined. The cells were lysed and used for the measurement of adenosine triphosphate (ATP) content and iNOS protein expression. 2.4. Toxicity of H1-antihistamines Toxicity was tested using bioluminescent bacteria Photorhabdus luminescens subsp. thracensis CCM 7295T according to [15]. Bacteria were cultivated in medium DSMZ 423. Bright bacterial cells collected from the culture in stationary phase (2.4 * 108 cells) were transferred to Hanks balanced salt solution with H1-antihistamines and bacterial bioluminescence was measured using luminometer LM-01T (Immunotech, Czech Republic). The inhibition of bacterial bioluminescence was evaluated 15 and 30 min after measuring began. Results obtained revealed that concentrations of drugs higher than 10− 4 M are potentially toxic. Therefore, 5 × 10− 5 M and 10− 4 M final concentrations of drugs were selected for further experiments. 2.5. ATP test of cell viability The viability of RAW 264.7 cells was tested by the commercial ATP cellular kit (Biothema, Sweden). Cells were incubated according to the experimental procedure, supernatant was removed and cells were lyzed by the Somatic cell ATP releasing reagent (Sigma Aldrich, USA). Then 50 μl of lysate were mixed with 20 μl of ATP reagent containing D-luciferin, luciferase and stabilizers. Intracellular ATP contents were determined luminometrically using luminometer Orion II (Berthold Detection Systems GmbH, Germany). 2.6. Determination of nitrite production by cells Detection of nitrites (NO− 2 ) accumulated in the cell supernatants was performed using the Griess reagent as described previously [16]. The volume of 150 μl of the cell supernatant was incubated with 150 μl Griess reagent (Sigma-Aldrich, USA) for 15 min in the dark at room temperature and the absorbance was measured at 546 nm using the Spectra Rainbow UV/Vis microplate reader (SLT Tecan, Germany). The concentrations of nitrites were derived from regression analysis using serial dilutions of sodium nitrite as a standard. The concentration values of each sample are expressed as a percentage of positive control. 2.7. Determination of iNOS protein expression in cells Cells were lysed with 1% SDS (sodium dodecyl sulfate) lysing buffer with the addition of 1% phenylmethanesulphonylfluoride. Protein volume in cell samples was determined using commercial bicinochoninic acid protein assay (Pierce, USA). The same concentration of proteins (22 μg) were separated by 7.5% SDS-PAGE (SDSpolyacrylamide gel electrophoresis) and then transferred to a polyvinylidene difluoride membrane (Millipore, USA) in a buffer containing Tris-glycine and 20% methanol. Membranes were incubated with 5% fat free milk in Tris buffer-Tween 20 (TBS-T) at room temperature for 1 h. The protein was labeled using a mouse antibody (1:1000) specific to iNOS (Anti-iNOS/NOS Type II mAb, BD Transduction Laboratories, USA) and a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) antibody (1:2000; ECL™ Antimouse IgG, Amersham, Biosciences, USA). After each incubation with both antibodies the membrane was washed three times in TBS-T buffer for 10 min. Subsequently immunoreactive bands were
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Fig. 1. The viability of RAW 264.7 cells (evaluated by luminometrical ATP-test) stimulated by 0.1 µg/ml LPS and incubated in the presence of 10− 4 M (blank bars) and 5 × 10− 5 M (full bars) H1-antihistamines for 24 h in 37 °C/5% CO2. Data are expressed as mean ± SEM of at least 3 independent experiments, which were run in duplicates. The symbol (*) shows significant differences (p b 0.05) as compared to control cells. The symbol (**) shows significant differences (p b 0.01) as compared to control cells.
visualised using ECL detection reagent (Detection reagents kit, Pierce, USA) and exposed to radiografic film (AGFA, Belgium). Relative levels of proteins were quantified by scanning densitometry using the ImageJ™ program and the optical density for each individual band was expressed as a percent of positive control. The equal loading of proteins was verified by β-actin immunoblotting.
reached the background current. The potential NO scavenger causes the rapid decrease of the NO-induced signal. The signal was measured for 280 s to obtain kinetic curves. Then the integral areas under the control curve and sample curves were calculated and the scavenging activity of drugs was evaluated. 2.9. Determination of RM values
2.8. Amperometrical detection of NO scavenging The scavenging properties of H1-antihistamines against NO were performed in a chemical system amperometrically using three electrode systems as described previously [17]. A porphyrinic microsensor working electrode, platinum wire counter electrode and a miniature saturated silver/silver chloride reference electrode were connected to the ISO-NO MARK II potentiostat (WPI, USA). The measurement was practised using distilled water saturated with pure NO gas (according to the WPI manual, [18]). The injection of 1 µl of the NO-saturated water into the glass vial (final concentration of NO in the vial = 595 nM) caused the rapid increase (peak time = 15 ± 5 s) with a subsequent gradual decrease of an NO-induced signal until it
The lipophilicity parameter represented by RM values was measured by the reversed-phase thin-layer chromatography technique (RP TLC). The mobile phase consisted of a PBS (0.1 M, pH 7.4) mixture with acetone (20:80, v/v). The stationary phase was obtained by impregnation of the layer of silica gel G F254 plates with a 5% solution of liquid paraffin in ether. The impregnation of plates was described previously [19,20]. H1-antihistamines were dissolved in methanol, and 1 μl of the solution was spotted onto the plates. The developed plates were dried, and the compounds were detected under UV light at 254 nm. The RM values were calculated by the formula RM = log (1/RF − 1). RF =a/b, where a is a distance from the start to the spot center of the sample and b is a distance from the start to the dissolvent forehead.
Fig. 2. The nitrite accumulation in cell supernatants (blank bars) and iNOS protein expression (full bars) in RAW 264.7 cells stimulated by 0.1 µg/ml LPS and incubated in the presence of 10− 4 M H1-antihistamines for 24 h. Representative Western blot of iNOS protein expression are also shown. Equal loading of proteins was verified by β-actin immunoblotting. Data are expressed as mean ± SEM of at least 5 independent experiments, which were run in duplicates. The symbol (*) shows significant differences (p b 0.05) as compared to control cells. The symbol (**) shows significant differences (p b 0.01) as compared to control cells.
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2.10. Statistical evaluation Data are expressed as the mean± standard error of the mean (SEM) of at least 3 independent experiments, that were run in duplicates. Results were analysed by Student's two-tailed t-test using Statistica software (StatSoft, USA), values below 0.05 (*) and 0.01 (**) were considered as statistically significant. 3. Results 3.1. Modulation of cell viability by H1-antihistamines The effect of drugs on cell viability was tested in two selected concentrations (5 × 10− 5 M and 10− 4 M). ANT, KET, CYC, CPH, PHE, ACR, and BPH did not significantly decrease cell viability in either concentration (Fig. 1). In a 10− 4 M concentration, LOR decreased the cell viability to 53.9 ± 2.1%, in comparison with the control cells. Despite that, LOR was tested in both concentrations. CHC, OXA, CLE, DIT, and BRO in a 10− 4 M concentration exerted a significant effect on cell viability. Thus, these drugs were only used in a 5 × 10− 5 M concentration in the following experiments. AST was the only drug that exerted a strong effect on cell viability in both tested concentrations; and, therefore, it was not used in further experiments.
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Table 3 Scavenging effect of 10− 4 M H1-antihistamines against NO evaluated amerometrically in cell-free system. H1-antihistamine
Integral under curves mean ± SEM
Control ACR ANT AST BRO CLE CYC CHC DIT CPH BPH PHE KET LOR OXA
20.2 ± 0.6 19.0 ± 0.3 24.7 ± 0.7 20.3 ± 3.4 21.3 ± 1.2 19.0 ± 1.6 17.4 ± 1.3 17.9 ± 1.0 24.9 ± 5.9 19.9 ± 2.9 21.7 ± 0.7 18.4 ± 4.3 24.0 ± 6.1 20.4 ± 0.5 18.5 ± 4.4
Data are expressed as mean ± SEM of at least 3 independent experiments, which were run in duplicates. (see NO scavenging by H1-antihistamines).
BPH, PHE, and KET) did not significantly affect the nitrite accumulation in cell supernatants.
3.2. Modulation of macrophage NO production by H1-antihistamines
3.3. Modulation of macrophage iNOS expression by H1-antihistamines
The nitrite accumulation in cell supernatants was dependent on the activation state of RAW 264.7 cells. While only very low concentrations of nitrites (0.5–2 μM) were detected in non-stimulated cells, the cells stimulated with 0.1 μg/ml LPS produced 28.2 ± 2.52 μM nitrites (mean ± SEM). Fig. 2 demonstrates that, in a 10− 4 M concentration, LOR, CPH, ACR, and CYC significantly affected nitrite accumulation in the LPS-stimulated cell supernatants. On the other hand, ANT significantly upregulated the nitrite concentration. KET, PHE, and BPH had no significant effect on nitrite concentration. The results described in Table 2 show that nitrite concentrations were significantly decreased by BRO, CLE, CHC, DIT, LOR, and OXA in the presence of 5 × 10− 5 M drugs. The other drugs (ACR, ANT, CYC, CPH,
The possibility that the changes in nitrite concentration were associated with iNOS protein expression was determined using Western blot analysis. Fig. 2 represents the effect of 10− 4 M H1antihistamines on iNOS protein expression in LPS-stimulated cells. In comparison with the iNOS protein level in the control sample, iNOS protein expression was significantly inhibited by LOR, CPH, ACR, CYC, KET, PHE, BPH, and ANT. The effects of 5 × 10− 5 M H1-antihistamines are shown in Table 2. In LPS-stimulated cells, iNOS protein expression was significantly inhibited by BRO, CLE, DIT, BPH, PHE, KET, and OXA. iNOS protein expression was also inhibited by ANT, CYC, CHC, CPH, and LOR-however, without statistical significance.
Table 2 The lipophilicity of 5 × 10− 5 M H1-antihistamines and their effects on the nitrite concentrations in cell supernatants and iNOS protein expression in RAW 264.7 cells stimulated by 0.1 µg/ml LPS. H1-Antihistamine
ACR ANT AST BRO CLE CYC CHC DIT CPH BPH PHE KET LOR OXA
Lipophilicity
NO-2 concentration
RM
% of inhibition
0.07 ± 0.18 1.36 ± 0.13 2.30 ± 0.0 1.66 ± 0.36 1.99 ± 0.0 0.97 ± 0.08 1.44 ± 0.0 1.49 ± 0.29 1.06 ± 0.2 1.12 ± 0.0 0.63 ± 0.08 0.95 ± 0.0 1.57 ± 0.1 2.30 ± 0.0
10.4 ± 0.6 2.8 ± 1.0 – 27.3 ± 6.3* 43.1 ± 4.6** 11.2 ± 0.8 48.5 ± 0.3** 43.9 ± 3.3** 12.7 ± 1.2 11.0 ± 4.9 4.1 ± 1.2 4.1 ± 0.5 46.0 ± 1.7** 69.4 ± 0.9**
Densitometric analysis of iNOS protein 1.6 ± 2.1 15.7 ± 2.6 – 50.5 ± 4.2** 59.3 ± 3.7** 15.6 ± 4.4 13.8 ± 3.0 40.1 ± 4.0* 19.0 ± 3.7 39.0 ± 6.4* 31.9 ± 2.5* 23.5 ± 2.3* 15.2 ± 6.4 44.3 ± 4.0**
Data are expressed as mean ± SEM of at least 3 independent experiments, which were run in duplicates. The symbol (*) shows significant differences (p b 0.05) as compared to control cells. The symbol (**) shows significant differences (p b 0.01) as compared to control cells. (see Modulation of macrophage NO production by H1-antihistamines, Modulation of macrophage iNOS expression by H1-antihistamines and Lipophilicity of studied H1antihistamines).
Fig. 3. The effect of 5 × 10− 5 M H1-antihistamines. The regression line, 95% confidence interval, data points and linear correlation equation for cell viability and lipophilicity (RM). Data are expressed as mean ± SEM of at least 3 independent experiments, which were run in duplicates.
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Fig. 4. The effect of 5 × 10− 5 M H1-antihistamines. The regression line, 95% confidence interval, data points and linear correlation equation for nitrite inhibition and lipophilicity (RM). Data are expressed as mean ± SEM of at least 3 independent experiments, which were run in duplicates.
3.4. NO scavenging by H1-antihistamines To verify whether the changes in nitrite concentration were induced by the scavenging ability of H1-antihistamines against NO, various drugs were tested, using direct amperometrical analysis. In a 10− 4 M concentration, none of the tested drugs exerted scavenging properties against NO. The integrals of the run curves for all the drugs tested are presented in Table 3. 3.5. Lipophilicity of studied H1-antihistamines The results describing the lipophilicity of all the drugs evaluated by RP TLC analysis are demonstrated in Table 2. ACR exerted the lowest lipophilicity, while the highest lipophilicity was recorded for AST and OXA. No linear correlation was detected between lipophilicity and the effect on the viability of cells incubated with 5× 10− 5 M H1-antihistamines (see Fig. 3). The degree of inhibition of nitrite accumulation positively correlated with the degree of tested H1-antihistamines lipophilicity (r =0.7786, p= 0.0017) (see Fig. 4). A less significant correlation (r =0.66, p =0.0143) was obtained between iNOS protein expression and lipophilicity. 4. Discussion H1-antihistamines represent an important class of pharmaceuticals and are currently the second-most-commonly-used medications (after antibiotics), with more than 40 varieties used in clinical practice worldwide. The volume of recently published evidence claims that H1-antihistamines play a very important role in the regulation of phagocyte-derived ROS production [7,13,14,21] as well as the regulation of myeloperoxidase activity [22]. But, the effects of H1-antihistamines on NO production remain to be elucidated. We studied this effect in in vitro experiments with a murine macrophage RAW 264.7 cell line, which is a convenient cell model for monitoring NO production after stimulation with LPS [23,24]. The most important finding of our study is that, corresponding to their pertinence to different groups of drugs, the selected H1antihistamines in a concentration 5 × 10− 5 M have a different ability
to modulate NO production by LPS-stimulated macrophages. The group of drugs—including CLE, BRO, and DIT—inhibited iNOS expression in LPS-stimulated cells. This inhibition was followed by a significant reduction in nitrite levels. In the presence of BPH, OXA, PHE, and KET, the LPS-induced iNOS protein expression was also inhibited; however, the decrease in nitrite accumulation was less pronounced. On the other hand, CHC and LOR did not affect iNOS protein expression, but they were able to decrease nitrite levels in the cell supernatants. Finally, ACR, ANT, CPH, and CYC had no effect on nitrite accumulation and iNOS protein expression. In our study, selected H1-antihistamines were also tested in a 10− 4 M concentration. In this concentration, KET, CPH, CYC, PHE, and BPH revealed an effect similar to that found in a 5 × 10− 5 M concentration. Further, 10− 4 M ACR and LOR significantly reduced the expression of iNOS protein and caused a similar decrease in nitrite levels. For ANT, very interesting effects on NO production were observed. Although, in a 10− 4 M concentration, it caused a significant increase in nitrite concentration and significantly inhibited the expression of iNOS, ANT did not induce any effect in a 5 × 10− 5 M concentration. AST, CHC, OXA, CLE, DIT, and BRO significantly affected macrophage viability; thus, their effects on nitrite accumulation and iNOS protein expression were not evaluated. With respect to the fact that all the drugs studied had no scavenging activity against NO in the chemical system, we suppose that H1-antihistamines modulate NO production by activated macrophages. The inhibitory effect on macrophage iNOS protein expression could be explained by the ability to affect the intracellular signaling pathways that lead to NO production. It was previously documented that the activation of histamine receptors leads to the activation of nuclear factor-κB, which is responsible for the regulation of iNOS expression [25–27]. Therefore, from our results, it can be assumed that the binding of H1-antihistamines could downregulate the activation of nuclear factor-κB. The effect of H1-antihistamines was probably mediated via the affection of iNOS enzyme activity in a case when the decrease in nitrite accumulation was not accompanied with the inhibition of iNOS protein expression. It is apparent from our results that there is a significant difference between the effects of various H1-antihistamines on NO production by RAW 264.7 cells stimulated by LPS. These findings lead us to look for some relationship between the physico-chemical properties of various H1-antihistamines and their effects on NO production by macrophages. RM values provide important evidence about the lipophilicity of the drugs studied. In comparison with PHE, increased lipophilicity predisposed CPH and BPH to be better incorporated into the lipid part of the membrane and its interaction with key proteins [28]. Actually, in comparison with PHE, CPH and BPH were able to suppress nitrite production by LPS-stimulated macrophages significantly. Analogously, in comparison with its non-halogenated analog CYC, the halogen substituent in CHC responsible for the increase of RM was associated with its more pronounced inhibition of nitrite formation. Furthermore, regardless of their general structural variability, the ability of H1-antihistamines to reduce nitrite concentration was dependent on lipophilicity. The increase in the effect on nitrite production by cells correlates with the increase in lipophilicity observed for several compounds (CYC vs CHC, PHE vs CPH and BPH). This was clearly documented by a good linear correlation between the percentual inhibition of nitrite concentration and lipophilicity. Despite Pauli [29] reporting that the toxicity of some compounds increases with higher lipophilicity, we did not prove any significant correlation between viability and lipophilicity. Nevertheless, a notable drop in viability was observed for those compounds with increased lipophilicity. In agreement with a range of QSAR (quantitative structure–activity relationship) studies [30], our results point to the dominant role of the lipophilicity of the agents tested in their biological activity in LPSstimulated RAW 264.7 macrophages. The Ferguson principle [31] is also quite applicable to our data, since the rate limiting step for the
J. Králová et al. / International Immunopharmacology 9 (2009) 990–995
effect of H1-antihistamines on macrophage functions appeared to be directed by their ability to reach the site of action. Furthermore, during transportation through the membrane system, the entrapment of more hydrophobic compounds in the lipid phase could affect their inhibitory effect on iNOS expression. On the other hand, the observed effects of the tested H1-antihistamines did not appear to be limited by their higher hydrophobicity, thus pointing to the involvement of the membrane as their site of action. We can conclude from our results that the structural properties of H1-antihistamines, particularly lipophilicity, significantly affect the biological activity of macrophages. These drugs are able to modulate NO production in vitro by macrophage cell line RAW 264.7 stimulated by LPS. The modulation shown in this study could be caused by the downregulation of iNOS protein expression or by iNOS enzyme activity. Acknowledgements This study was conducted under the research plans AVOZ50040507 and AVOZ50040702 and supported by grants 1QS500040507 GA AS CR, GA CR 525/06/1196 and VEGA 2/7019/27. References [1] Leurs R, Smit MJ, Timmerman H. Molecular pharmacological aspects of histamine receptors. Pharmacol Ther 1995;66:413–63. [2] Buckley MG, Walters C, Wong WM, Cawley MI, Ren S, Schwartz LB, et al. Mast cell activation in arthritis: detection of alpha- and beta-tryptase, histamine and eosinophil cationic protein in synovial fluid. Clin Sci (Lond) 1997;93:363–70. [3] Renoux M, Hilliquin P, Galoppin L, Florentin I, Menkes CJ. Release of mast cell mediators and nitrites into knee joint fluid in osteoarthritis—comparison with articular chondrocalcinosis and rheumatoid arthritis. Osteoarthr Cartil 1996;4:175–9. [4] De Vos C. H1-receptor antagonists: effects on leukocytes, myth or reality? Clin Exp Allergy 1999;29(Suppl 3):60–3. [5] Berthon B, Taudou G, Combettes L, Czarlewski W, Carmi-Leroy A, Marchand F, et al. In vitro inhibition, by loratadine and descarboxyethoxyloratadine, of histamine release from human basophils, and of histamine release and intracellular calcium fluxes in rat basophilic leukemia cells (RBL-2H3). Biochem Pharmacol 1994;47:789–94. [6] Church MK. H(1)-antihistamines and inflammation. Clin Exp Allergy 2001;31:1341–3. [7] Nosal R, Drabikova K, Jancinova V, Petrikova M, Fabryova V. Antiplatelet and antiphagocyte activity of H1-antihistamines. Inflamm Res 2005;54(Suppl 1): S19–20. [8] Lullmann H, Plosch H, Ziegler A. Ca replacement by cationic amphiphilic drugs from lipid monolayers. Biochem Pharmacol 1980;29:2969–74. [9] McInnes IB, Leung BP, Field M, Wei XQ, Huang FP, Sturrock RD, et al. Production of nitric oxide in the synovial membrane of rheumatoid and osteoarthritis patients. J Exp Med 1996;184:1519–24. [10] Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329: 2002–12. [11] Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524–6.
995
[12] Stuehr DJ, Marletta MA. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci U S A 1985;82:7738–42. [13] Kralova J, Ciz M, Nosal R, Drabikova K, Lojek A. Effect of H(1)-antihistamines on the oxidative burst of rat phagocytes. Inflamm Res 2006;55(Suppl 1):S15–16. [14] Nosal R, Drabikova K, Jancinova V, Macickova T, Pecivova J, Holomanova D. On the pharmacology and toxicology of neutrophils. Neuro Endocrinol Lett 2006;27 (Suppl 2):148–51. [15] Lojek A, Pecivova J, Macickova T, Nosal R, Papezikova I, Ciz M. Effect of carvedilol on the production of reactive oxygen species by HL-60 cells. Neuro Endocrinol Lett 2008;29:779–83. [16] Migliorini P, Corradin G, Corradin SB. Macrophage NO2-production as a sensitive and rapid assay for the quantitation of murine IFN-gamma. J Immunol Methods 1991;139:107–14. [17] Hrbac J, Gregor C, Machova M, Kralova J, Bystron T, Ciz M, et al. Nitric oxide sensor based on carbon fiber covered with nickel porphyrin layer deposited using optimized electropolymerization procedure. Bioelectrochemistry 2007;71:46–53. [18] Feelisch M, Kelm M. Biotransformation of organic nitrates to nitric oxide by vascular smooth muscle and endothelial cells. Biochem Biophys Res Commun 1991;180:286–93. [19] Devinsky F, Zamocka J, Lacko I, Polakovicova M. QSAR and CAMM study of amphiphilic antimicrobially active 2,2'-bipyridyl monoammonium salts. Pharmazie 1996;51:727–31. [20] Glavac D. RM values of some colchicines and colchiceinamides determined by reversed-phase thin-layer chromatography. J Chromatogr 1992;591:367–70. [21] Jancinova V, Drabikova K, Nosal R, Holomanova D. Extra- and intracellular formation of reactive oxygen species by human neutrophils in the presence of pheniramine, chlorpheniramine and brompheniramine. Neuro Endocrinol Lett 2006;27(Suppl 2):141–3. [22] Kralova J, Nosal R, Drabikova K, Jancinova V, Denev P, Moravcova A, et al. Comparative investigations of the influence of H1-antihistamines on the generation of reactive oxygen species by phagocytes. Inflamm Res 2008;57 (Suppl 1):S49–50. [23] Raschke WC, Baird S, Ralph P, Nakoinz I. Functional macrophage cell lines transformed by Abelson leukemia virus. Cell 1978;15:261–7. [24] Hofer M, Vacek A, Lojek A, Hola J, Streitova D. Ultrafiltered pig leukocyte extract (IMUNOR®) decreases nitric oxide formation and hematopoiesis-stimulating cytokine production in lipopolysaccharide-stimulated RAW 264.7 macrophages. Int Immunopharmacol 2007;7:1369–74. [25] Bakker RA, Schoonus SB, Smit MJ, Timmerman H, Leurs R. Histamine H(1)receptor activation of nuclear factor-kappa B: roles for G beta gamma- and G alpha (q/11)-subunits in constitutive and agonist-mediated signaling. Mol Pharmacol 2001;60:1133–42. [26] Aoki Y, Qiu D, Zhao GH, Kao PN. Leukotriene B4 mediates histamine induction of NF-kappaB and IL-8 in human bronchial epithelial cells. Am J Physiol 1998;274: L1030–1039. [27] Hu Q, Deshpande S, Irani K, Ziegelstein RC. [Ca(2+)](i) oscillation frequency regulates agonist-stimulated NF-kappaB transcriptional activity. J Biol Chem 1999;274:33995–8. [28] Jancinova V, Drabikova K, Nosal R, Majekova M, Rackova L, Holomanova D. Antiradical effects of antihistamines in human blood. Structure–activity relationship. Inflamm Res 2006;55(Suppl 1):S85–86. [29] Pauli A. Relationship between lipophilicity and toxicity of essential oils. Int J Ess Oil Ther 2008;2:60–8 (9). [30] Dearden JC. Partitioning and lipophilicity in quantitative structure–activity relationships. Environ Health Perspect 1985;61:203–28. [31] Ferguson J. The use of chemical potentials as indices of toxicity. Proc Roy Soc London 1939;B127:387–403.