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Inhibition of cyclooxygenase-2 promotes the stimulatory action of adenosine A3 receptor agonist on hematopoiesis in sublethally γ-irradiated mice
Biomedicine & Pharmacotherapy 65 (2011) 427–431
Original article
Inhibition of cyclooxygenase-2 promotes the stimulatory action of adenosine A3 receptor agonist on hematopoiesis in sublethally g-irradiated mice Michal Hofer a,*, Milan Pospı´sˇil a, Ladislav Dusˇek b, Zuzana Hoferova´ a, Lenka Weiterova´ a a b
Research Group of Experimental Hematology, Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic Institute of Biostatistics and Analyses, Masaryk University, Brno, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 March 2011 Accepted 14 April 2011 Available online 12 June 2011
Mouse hematopoiesis, suppressed by a sublethal dose of ionizing radiation, was the target for combined therapy with a cyclooxygenase-2 (COX-2) inhibitor meloxicam and an adenosine A3 receptor agonist IBMECA. The drugs were administered in an early postirradiation treatment regimen: meloxicam was given in a single dose 1 hour after irradiation, IB-MECA in two doses 24 and 48 hours after irradiation. Treatment-induced changes in several compartments of hematopoietic progenitor and precursor cells of the bone marrow were evaluated on day 3 after irradiation. Values of hematopoietic progenitor cells for granulocytes/macrophages and erythrocytes (GM-CFC and BFU-E, respectively), as well as those of proliferative granulocytic cells were found to be significantly higher in the mice treated with the drug combination in comparison to irradiated controls and attained the highest increase factors of 1.6, 1.6, and 2.6, respectively. The study emphasizes the significance of the combined treatment of suppressed hematopoiesis with more agents. Mechanisms of the action of the individual compounds of the studied drug combination and of their joint operation are discussed. ß 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Hematopoiesis COX-2 inhibitor Adenosine A3 receptor agonist
1. Introduction Hematopoiesis is a complex system in which proliferation and differentiation of hematopoietic stem, progenitor, and precursor cells are regulated by many factors whose action is often pleiotropic and where final effects stem from interactions in the regulatory network. These factors are either produced endogenously or can be administered exogenously with the aim to modulate hematopoiesis. Many studies have been published on the topic of attempts to stimulate hematopoiesis suppressed by ionizing radiation or cytotoxic chemotherapy; combined treatment with two or more agents has been often accentuated because of the possibility of better outcomes and/or lower intensity and incidence of undesirable side effects [1–4]. Our laboratory has been engaged on a long-term basis in investigations of two untraditional approaches to stimulation of hematopoietic processes, namely in evaluating the effects of Abbreviations: COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; NSAIDs, nonsteroidal anti-inflammatory drugs; GM-CFC, granulocyte/macrophage colonyforming cells (hematopoietic progenitor cells for granulocytes and macrophages); BFU-E, burst-forming units (hematopoietic progenitor cells for erythrocytes); IBMECA, N6-(3-iodobenzyl)adenosine-5’-N-methyluronamide; G-CSF, granulocyte colony-stimulating factor. * Corresponding author. Tel.: +420 541517171; fax: +420 541211293. E-mail address: [email protected] (M. Hofer). 0753-3322/$ – see front matter ß 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2011.04.033
administration of inhibitors of prostaglandin production, acting on the principle of inhibition of cyclooxygenases and of adenosine receptor signaling (for summaries [5,6]). The mechanism of hematopoiesis-stimulating action of cyclooxygenase inhibitors, known also as non-steroidal anti-inflammatory drugs (NSAIDs), is based on the evidence that prostaglandins of the E series constitute a part of a negative control loop involved in the regulation of myelopoiesis and that the inhibition of prostaglandin production might strengthen the mechanisms of the positive control [7–9]. Prostaglandin synthesis is carried out by cyclooxygenases existing in two isoforms, namely cyclooxygenase-1 (COX-1), expressed constitutively in a variety of tissues, and cyclooxygenase-2 (COX-2), which is inducible and responsible for the production of prostaglandins during inflammatory states [10]. Classical NSAIDs (e.g., indomethacin, diclofenac, flurbiprofen) inhibit both COX-1 and COX-2 and their action is accompanied with an incidence of undesirable, mainly gastrointestinal effects attributed to the inhibition of COX-1 [11]. Therefore, the use of modern COX-2-selective NSAIDs is currently preferred [12]. It has been demonstrated that meloxicam, a selective COX-2 inhibitor, when administered before irradiation, acts positively on hematopoiesis, as well as on survival of lethally irradiated mice [13,14]. Adenosine cell surface receptors, denominated A1, A2a, A2b, and A3, serve as mediators of the paracrine regulatory action of extracellular adenosine. Receptor activation may be achieved either non-selectively, by adenosine itself, or selectively, using
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various adenosine analogs [15–17]. Hematological studies have been published revealing the significant role of non-selective activation of adenosine receptors in stimulation of hematopoiesis [2,18] and later it has been found that similar effects can be induced by selective agonists of adenosine A3 receptors [19–23]. Both the regulatory principles, i.e. COX-2 action and adenosine receptor signaling, have been reported to interact with the production and function of the important hematopoietic cytokine – granulocyte colony-stimulating factor (G-CSF). Inhibition of cyclooxygenase-2 has been found to increase production of G-CSF in mice [14,24]. Controversial findings have been described concerning the effectiveness of elevating G-CSF serum levels by treatment of mice with an agonist of adenosine A3 receptors [19,25]; however, the experimental conditions in these experiments have differed markedly. Non-selective as well as selective activation of adenosine A3 receptors have been demonstrated to support granulopoiesis-stimulating effects of exogenously administered G-CSF to mice [2,18,25]. Based on this knowledge we have investigated the effects of the combined therapeutic (postirradiation) administration of the inhibitor of COX-2, meloxicam, and the agonist of adenosine A3 receptors, IB-MECA, on hematopoietic indices in sublethally g-irradiated mice.
2. Materials and methods 2.1. Animals B10CBAF1 male mice aged 3 months and weighing in average 30 g were obtained from the breeding facility of the Medical Faculty, Masaryk University, Brno, Czech Republic. The mice were kept under controlled conditions; standardized pelleted diet and HCl-treated tap water were available ad libitum. The use and treatment of the animals followed the European Community Guidelines as accepted principles for the use of experimental animals. The experiments were performed with the approval of the Institute’s Ethical Committee. 2.2. Irradiation The mice were whole-body irradiated at a dose rate of 0.15 Gy/ min using a g-ray source (60Co, Chisostat, Chirana, Prague, Czech Republic). A single sublethal dose of 4 Gy was used. 2.3. Drugs and their administration Meloxicam (Sigma, St. Louis, MO, USA) was dissolved in sterile saline and administered i.p. at a dose of 20 mg/kg per mouse in a volume of 0.2 mL 1 hour after irradiation. This dose was found to increase production of G-CSF in mice in our earlier experiments [14,25]. N6-(3-iodobenzyl)adenosine-5’-N-methyluronamide (IBMECA, Sigma) was dissolved initially in dimethyl sulfoxide, diluted by sterile saline and injected i.p. in doses of 105 mg/kg in a volume of 0.2 mL on hour 24 (day 1) and 48 (day 2) after irradiation. The choice of the dose of IB-MECA was based on our former experiments showing that this dose induced cycling of murine hematopoietic cells under in vivo conditions [20]. The final concentration of dimethyl sulfoxide in the injected volume was 2%. Pertinent solvents were used for control injections. 2.4. Hematological techniques Concentrations of granulocyte colony-stimulating factor (GCSF) in mouse serum were determined by an enzyme immunoassay using a mouse enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). The assay had the
sensitivity of 4.5 pg/mL. For the utilization of the ELISA technique, the animals were anesthetized by an i.p. injection of a Narcamon/ Rometar solution (5% Narcamon and 2% Rometar [both Spofa, Prague, Czech Republic] in the ratio of 2.63:1) and the peripheral blood was sampled by cardiac puncture. In mice sacrificed by cervical dislocation, femurs were removed, marrow cells were harvested by standard procedures, and numbers of nucleated cells of the femoral marrow were determined using a Coulter Counter (Model ZF, Coulter Electronics, Luton, UK). Standard procedures were used for the in vitro assays of the femoral clonogenic cells. Granulocyte-macrophage colonyforming cells (GM-CFC) were assayed using a semisolid plasma clot technique. The plasma clot contained Iscove’s modification of Dulbecco’s medium (Sigma), 20% fetal calf serum (PAN Biotech GmbH, Aidenbach, Germany), 10 ng/mL recombinant mouse interleukin-3 (rmIL-3) (Sigma), and 10% citrate bovine plasma (Veterinary Research Institute, Brno, Czech Republic). Erythroid progenitor cells (burst-forming unit, BFU-E) were cultivated on methylcellulose (Stem Cell Technologies, Inc., Vancouver, Canada) with 4 U/mL recombinant human erythropoetin (EPREX, JanssenCilag s.r.o., Prague, Czech Republic), 50 ng/mL mouse stem cell factor (Sigma), and 10 ng/mL rmIL-3 (Sigma). Femoral marrow cell suspensions were plated (1.5 105 and 1 105 nucleated bone marrow cells for GM-CFC and BFU-E, respectively) in triplicate for both assays and incubated at 37 8C in a humidified atmosphere containing 95% air and 5% CO2. GM-CFC were scored after a 7-day incubation as colonies containing 50 or more cells. Hemoglobinized colonies were counted as BFU-E after an 8-day incubation. For evaluation of the compartment of morphologically recognizable granulocytic and erythroid precursor cells of the femoral bone marrow, differential counts were performed on marrow smears stained by the May-Gru¨nwald-Giemsa method. In the granulocytic lineage myeloblasts through myelocytes were classified as proliferative cells, metamyelocytes through segmented stages as non-proliferative ones. In the erythroid lineage proerythroblasts through basophilic erythroblasts were classified as proliferative cells, polychromatic and orthochromatic erythroblasts as non-proliferative ones. Total counts of granulocytic or erythroid cells represent the sum of proliferative and nonproliferative cells, respectively. 2.5. Statistics Primary data are summarized as means standard error of the mean (SEM). One-way model of analysis of variance (Anova) was applied to test the differences among experimental variants. Investigated parameters were analysed separately. Mutual differences among experimental variants were evaluated by means of Tukey post hoc test. In the case of the experiment testing the effect of meloxicam on serum G-CSF levels, t-test for two independent samples was used to compare the variants. All analyses were performed using Statistica 8.0 software (StatSoft, Inc.). The significance level was set at P < 0.05.
3. Results The sublethal irradiation of mice with the dose of 4 Gy g-rays induced an expressive damage in the hematopoietic system. To illustrate the extent of the damage, the values of the hematopoietic parameters studied in control non-irradiated mice and in nontreated mice exposed to the dose of 4 Gy, sampled on day 3 after irradiation are summarized in Table 1. The percent decrease observed on day 3 after irradiation represented about 90%, and more than 50% in the compartments of bone marrow progenitor cells and bone marrow precursor cells, respectively.
M. Hofer et al. / Biomedicine & Pharmacotherapy 65 (2011) 427–431 Table 1 Hematopoietic parameters in control non-irradiated mice and 4 Gy-irradiated mice.
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Experimental protocol for investigating the combined effects of the two selected drugs utilized one dose of meloxicam administered 1 hour after irradiation and two doses of IB-MECA given 24 and 48 hours after irradiation (for details: Materials and methods). Four groups of animals, i.e. controls which did not obtain any drug and were treated only with solvents, mice treated with meloxicam or IB-MECA alone, and mice treated with the combination of both drugs were euthanized on day 3 after 4-Gy irradiation and effects of the different treatments on the hematopoietic indices in the bone marrow have been compared. These effects are illustrated in Figs. 1–6. One-way Anova test was used to define hematopoietic indices influenced significantly by the action of the treatment. Significant effects were found in indices of GM-CFC (Fig. 1), BFU-E (Fig. 4), and proliferative granulocytic cells (Fig. 2). As shown by Tukey post hoc test, IB-MECA per se, as compared to irradiated controls, induced a significant enhancement in the parameters of BFU-E (Fig. 4) and of proliferative granulocytic cells (Fig. 2). Meloxicam per se, in a similar comparison, did not elicit significant
effects in any of the investigated parameters. However, the most expressive stimulatory effects were observed when using the combined treatment. Here the values of GM-CFC (Fig. 1), BFU-E (Fig. 4), as well as those of proliferative granulocytic cells (Fig. 2) were significantly higher in comparison with irradiated controls and attained the highest increase factors of 1.6, 1.6, and 2.6, respectively. Significant statistical differences were observed also when comparing the values obtained after combined treatment with the values observed in the group treated with meloxicam alone in the indices of GM-CFC (Fig. 1), BFU-E (Fig. 4), and proliferative granulocytic cells (Fig. 2). A significant statistical difference was also found in comparison of the values in mice treated with the combination of the drugs and in those treated with IB-MECA alone in the parameter of proliferative granulocytic cells (Fig. 2). The fact that the effects of the drug treatment in the precursor cell compartments are evident only in the granulocytic line and not in the erythroid one is understandable because of the higher proliferation pressure in the granulocytic cell system and low proliferation pressure in the erythroid one. Proliferation pressures are determined by the life span of the end cells in a system, i.e. a long one in erythrocytes and a short one in granulocytes. Approximately similar effects of the treatment in the investigated progenitor cell compartments of GM-CFC (Fig. 1) and BFU-E (Fig. 4) can be ascribed to the action of the drugs at the level of progenitors of the common myeloid pathway [26]. These cells might be the targets of the drug action. The absence of differences between the values of total granulocytic cells (Fig. 3) in the femoral marrow of the investigated groups can be explained by the outflow of differentiated cells into the peripheral blood under the conditions of enhanced proliferation activity in this cell system. In our previous study performed with the same mouse strain, no effects of IB-MECA on the serum levels of granulocyte colonystimulating factor (G-CSF) under identical experimental conditions were observed [25]. Therefore, our experiments aimed at testing the ability of the treatment described above to modulate serum concentrations of G-CSF were targeted only to investigation of the effects of meloxicam. The results are shown in Table 2. The serum levels of G-CSF were found to be rather sensitive to non-specific stimuli like irradiation and saline injection and were increased
Fig. 1. GM-CFC per femur in 4 Gy-treated mice. Meloxicam or its solvent (saline) were adminstered 1 hour after irradiation. IB-MECA or its solvent (2% DMSO in saline) were administered 24 and 48 hours after irradiation. Control – mice treated only with solvents. Data are given as means SEM. Twelve to 15 animals per group were used. * P < 0.05 in comparison with Control. ++ P < 0.01 in comparison with meloxicam.
Fig. 2. Proliferative granulocytic cells per femur in 4 Gy-irradiated mice. Five animals per group were used. *, *** P < 0.05, P < 0.001, respectively, in comparison with Control. # P < 0.05 in comparison with IB-MECA. +++ P < 0.001 in comparison with meloxicam. For other explanations see legend to Fig. 1.
Parameter
Control non-irradiated mice
4 Gy-irradiated mice
GM-CFC per femur
17,983 820 n = 10
1,884 122 n = 12
Proliferative granulocytic cells per femur ( 103)
2,134 253 n = 10
809 48 n=5
Total granulocytic cells per femur ( 103)
11,198 534 n = 10
5,082 443 n=5
BFU-E per femur
23,736 1,580 n = 10
859 115 n = 15
Proliferative erythroid cells per femur ( 103)
1,807 216 n = 10
713 126 n=5
Total erythroid cells per femur ( 103)
6,232 653 n = 10
2,145 307 n=5
Values of hematopoietic parameters are given as arithmetic means standard errors of the means (SEM). Sampling in 4 Gy-irradiated mice was performed on day 3 after irradiation. n: numbers of animals.
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Fig. 3. Total granulocytic cells per femur in 4 Gy-irradiated mice. Five animals per group were used. For other explanations see legend to Fig. 1.
Fig. 5. Proliferative erythroid cells per femur in 4 Gy-irradiated mice. Five animals per group were used. For other explanations see legend to Fig. 1.
compared to those observed in non-irradiated untreated controls. After the administration of meloxicam, the serum levels of G-CSF increased further to 167% compared to those in saline-treated controls, the difference being statistically significant.
4. Discussion The presented data indicate that the early postirradiation administration of the inhibitor of COX-2 meloxicam and the subsequent administration of the agonist of adenosine A3 receptor IB-MECA to g-irradiated mice represent a positively acting drug combination activating recovery of the damaged hematopoiesis. Effects of the combination of the drugs cannot be considered to be a result of a simple additivity of stimulatory actions. This is evident by the inability of meloxicam given alone to influence the indices of the bone marrow cellularity at the bone marrow sampling time interval of 3 days after irradiation. If the targets of the proliferation-inducing or curative action of meloxicam are some early progenitor or stem cells, these effects remain hidden because of the very low percentage of these cells in the cellular system of the bone
Fig. 6. Total erythroid cells per femur in 4 Gy-irradiated mice. Five animals per group were used. For other explanations see legend to Fig. 1.
marrow (less than 0.1%). From this point of view it seems to be logical that hematopoiesis-stimulating effects of meloxicam in parameters determined in our study manifest themselves on day 3 after irradiation only in connection with the activity of the agonist of adenosine A3 receptors which enhances proliferation in a wide spectrum of cells in the bone marrow cell renewal system, as has been found earlier [20–23]. As stated by Metcalf [27], stimulatory interactions of the two regulatory principles can be due to their
Table 2 Effects of meloxicam administration on serum concentrations of G-CSF in 4 Gyirradiated mice.
Fig. 4. BFU-E per femur in 4 Gy-irradiated mice. Fourteen to 15 animals per group were used. * P < 0.05 in comparison with Control. + P < 0.05 in comparison with meloxicam. For other explanations see legend to Fig. 1.
Group of mice
Concentration of G-CSF (pg/mL)
Non-irradiated untreated controls Saline-treated 4 Gy-irradiated mice Meloxicam-treated 4 Gy-irradiated mice
316.4 81.2 793.5 34.2* 1,330.0 41.4**
Values are given as means standard errors of the means (SEM). Five animals per group were used. * P < 0.05 vs. non-irradiated untreated controls. ** P < 0.001 vs. 4 Gy-irradiated saline-treated mice (t-test for two independent samples).
M. Hofer et al. / Biomedicine & Pharmacotherapy 65 (2011) 427–431
different operations at different levels of the age structure of the hematopoietic cell system. At the present time, it is possible only to speculate about the nature of the mechanisms inducing the meloxicam action. A certain role can be played by the ability of meloxicam to enhance the production of G-CSF, a phenomenon which has been observed in our earlier studies [14,24] as well as under conditions of the presented experiments. It is known that hematopoietic colonystimulating factors enhance cell survival by suppressing apoptosis [28]. If the effects of meloxicam occur at the level of the early progenitor cells and/or stem cells, the concomitant cell proliferation enhancing the action of IB-MECA can result in a distinct cell amplification in the hematopoietic cell system including its more differentiated compartments. It has to be mentioned that our interpretation of the curative effects of meloxicam is similar to the concept of the cytokine-based anti-apoptotic treatment of radiation injury proposed by He´rodin et al. [3,4]. Our experimental approach using a combination of a COX-2 inhibitor and an adenosine A3 receptor agonist administered early after irradiation can be also regarded as a contribution to the experimental efforts aimed at optimizing the postirradiation approach to the treatment of radiation injuries as proposed by Moulder [29]. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported by the Ministry of Defense (project ‘‘Receptor’’, project No. 1001 8 5090), by the Grant Agency of the Czech Republic (grants Nos. 305/08/0158 and P303/11/0128), and by the Academy of Sciences of the Czech Republic (grants Nos. AV0Z50050507 and AV0Z50040702). References [1] Weiss JF, Kumar KS, Walden TL, Neta R, Landauer MR, Clark EP. Advances in radioprotection through the use of combined agent regimens. Int J Radiat Biol 1990;57:709–22. [2] Pospı´sˇil M, Hofer M, Znojil V, Va´cha J, Netı´kova´ J, Hola´ J. Synergistic effects of granulocyte colony-stimulating factor and drugs elevating extracellular adenosine on neutrophil production in mice. Blood 1995;86:3692–7. [3] He´rodin F, Bourin P, Mayol JF, Lataillade JJ, Drouet M. Short-term injection of antiapoptotic cytokine combinations soon after lethal g-irradiation promotes survival. Blood 2003;101:2609–16. [4] He´rodin F, Drouet M. Cytokine-based treatment of accidentally irradiated victims and new approaches. Exp Hematol 2005;33:1071–80. [5] Hofer M, Pospı´sˇil M. Role of adenosine signaling in hematopoiesis – a short review. Med Hypotheses Res 2006;3:629–35. [6] Hofer M, Pospı´sˇil M. Stimulated recovery of perturbed haematopoiesis by inhibition of prostaglandin production – promising therapeutic strategy. Cent Eur J Biol 2006;1:584–93.
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Original article
Inhibition of cyclooxygenase-2 promotes the stimulatory action of adenosine A3 receptor agonist on hematopoiesis in sublethally g-irradiated mice Michal Hofer a,*, Milan Pospı´sˇil a, Ladislav Dusˇek b, Zuzana Hoferova´ a, Lenka Weiterova´ a a b
Research Group of Experimental Hematology, Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic Institute of Biostatistics and Analyses, Masaryk University, Brno, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 March 2011 Accepted 14 April 2011 Available online 12 June 2011
Mouse hematopoiesis, suppressed by a sublethal dose of ionizing radiation, was the target for combined therapy with a cyclooxygenase-2 (COX-2) inhibitor meloxicam and an adenosine A3 receptor agonist IBMECA. The drugs were administered in an early postirradiation treatment regimen: meloxicam was given in a single dose 1 hour after irradiation, IB-MECA in two doses 24 and 48 hours after irradiation. Treatment-induced changes in several compartments of hematopoietic progenitor and precursor cells of the bone marrow were evaluated on day 3 after irradiation. Values of hematopoietic progenitor cells for granulocytes/macrophages and erythrocytes (GM-CFC and BFU-E, respectively), as well as those of proliferative granulocytic cells were found to be significantly higher in the mice treated with the drug combination in comparison to irradiated controls and attained the highest increase factors of 1.6, 1.6, and 2.6, respectively. The study emphasizes the significance of the combined treatment of suppressed hematopoiesis with more agents. Mechanisms of the action of the individual compounds of the studied drug combination and of their joint operation are discussed. ß 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Hematopoiesis COX-2 inhibitor Adenosine A3 receptor agonist
1. Introduction Hematopoiesis is a complex system in which proliferation and differentiation of hematopoietic stem, progenitor, and precursor cells are regulated by many factors whose action is often pleiotropic and where final effects stem from interactions in the regulatory network. These factors are either produced endogenously or can be administered exogenously with the aim to modulate hematopoiesis. Many studies have been published on the topic of attempts to stimulate hematopoiesis suppressed by ionizing radiation or cytotoxic chemotherapy; combined treatment with two or more agents has been often accentuated because of the possibility of better outcomes and/or lower intensity and incidence of undesirable side effects [1–4]. Our laboratory has been engaged on a long-term basis in investigations of two untraditional approaches to stimulation of hematopoietic processes, namely in evaluating the effects of Abbreviations: COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; NSAIDs, nonsteroidal anti-inflammatory drugs; GM-CFC, granulocyte/macrophage colonyforming cells (hematopoietic progenitor cells for granulocytes and macrophages); BFU-E, burst-forming units (hematopoietic progenitor cells for erythrocytes); IBMECA, N6-(3-iodobenzyl)adenosine-5’-N-methyluronamide; G-CSF, granulocyte colony-stimulating factor. * Corresponding author. Tel.: +420 541517171; fax: +420 541211293. E-mail address: [email protected] (M. Hofer). 0753-3322/$ – see front matter ß 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2011.04.033
administration of inhibitors of prostaglandin production, acting on the principle of inhibition of cyclooxygenases and of adenosine receptor signaling (for summaries [5,6]). The mechanism of hematopoiesis-stimulating action of cyclooxygenase inhibitors, known also as non-steroidal anti-inflammatory drugs (NSAIDs), is based on the evidence that prostaglandins of the E series constitute a part of a negative control loop involved in the regulation of myelopoiesis and that the inhibition of prostaglandin production might strengthen the mechanisms of the positive control [7–9]. Prostaglandin synthesis is carried out by cyclooxygenases existing in two isoforms, namely cyclooxygenase-1 (COX-1), expressed constitutively in a variety of tissues, and cyclooxygenase-2 (COX-2), which is inducible and responsible for the production of prostaglandins during inflammatory states [10]. Classical NSAIDs (e.g., indomethacin, diclofenac, flurbiprofen) inhibit both COX-1 and COX-2 and their action is accompanied with an incidence of undesirable, mainly gastrointestinal effects attributed to the inhibition of COX-1 [11]. Therefore, the use of modern COX-2-selective NSAIDs is currently preferred [12]. It has been demonstrated that meloxicam, a selective COX-2 inhibitor, when administered before irradiation, acts positively on hematopoiesis, as well as on survival of lethally irradiated mice [13,14]. Adenosine cell surface receptors, denominated A1, A2a, A2b, and A3, serve as mediators of the paracrine regulatory action of extracellular adenosine. Receptor activation may be achieved either non-selectively, by adenosine itself, or selectively, using
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various adenosine analogs [15–17]. Hematological studies have been published revealing the significant role of non-selective activation of adenosine receptors in stimulation of hematopoiesis [2,18] and later it has been found that similar effects can be induced by selective agonists of adenosine A3 receptors [19–23]. Both the regulatory principles, i.e. COX-2 action and adenosine receptor signaling, have been reported to interact with the production and function of the important hematopoietic cytokine – granulocyte colony-stimulating factor (G-CSF). Inhibition of cyclooxygenase-2 has been found to increase production of G-CSF in mice [14,24]. Controversial findings have been described concerning the effectiveness of elevating G-CSF serum levels by treatment of mice with an agonist of adenosine A3 receptors [19,25]; however, the experimental conditions in these experiments have differed markedly. Non-selective as well as selective activation of adenosine A3 receptors have been demonstrated to support granulopoiesis-stimulating effects of exogenously administered G-CSF to mice [2,18,25]. Based on this knowledge we have investigated the effects of the combined therapeutic (postirradiation) administration of the inhibitor of COX-2, meloxicam, and the agonist of adenosine A3 receptors, IB-MECA, on hematopoietic indices in sublethally g-irradiated mice.
2. Materials and methods 2.1. Animals B10CBAF1 male mice aged 3 months and weighing in average 30 g were obtained from the breeding facility of the Medical Faculty, Masaryk University, Brno, Czech Republic. The mice were kept under controlled conditions; standardized pelleted diet and HCl-treated tap water were available ad libitum. The use and treatment of the animals followed the European Community Guidelines as accepted principles for the use of experimental animals. The experiments were performed with the approval of the Institute’s Ethical Committee. 2.2. Irradiation The mice were whole-body irradiated at a dose rate of 0.15 Gy/ min using a g-ray source (60Co, Chisostat, Chirana, Prague, Czech Republic). A single sublethal dose of 4 Gy was used. 2.3. Drugs and their administration Meloxicam (Sigma, St. Louis, MO, USA) was dissolved in sterile saline and administered i.p. at a dose of 20 mg/kg per mouse in a volume of 0.2 mL 1 hour after irradiation. This dose was found to increase production of G-CSF in mice in our earlier experiments [14,25]. N6-(3-iodobenzyl)adenosine-5’-N-methyluronamide (IBMECA, Sigma) was dissolved initially in dimethyl sulfoxide, diluted by sterile saline and injected i.p. in doses of 105 mg/kg in a volume of 0.2 mL on hour 24 (day 1) and 48 (day 2) after irradiation. The choice of the dose of IB-MECA was based on our former experiments showing that this dose induced cycling of murine hematopoietic cells under in vivo conditions [20]. The final concentration of dimethyl sulfoxide in the injected volume was 2%. Pertinent solvents were used for control injections. 2.4. Hematological techniques Concentrations of granulocyte colony-stimulating factor (GCSF) in mouse serum were determined by an enzyme immunoassay using a mouse enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). The assay had the
sensitivity of 4.5 pg/mL. For the utilization of the ELISA technique, the animals were anesthetized by an i.p. injection of a Narcamon/ Rometar solution (5% Narcamon and 2% Rometar [both Spofa, Prague, Czech Republic] in the ratio of 2.63:1) and the peripheral blood was sampled by cardiac puncture. In mice sacrificed by cervical dislocation, femurs were removed, marrow cells were harvested by standard procedures, and numbers of nucleated cells of the femoral marrow were determined using a Coulter Counter (Model ZF, Coulter Electronics, Luton, UK). Standard procedures were used for the in vitro assays of the femoral clonogenic cells. Granulocyte-macrophage colonyforming cells (GM-CFC) were assayed using a semisolid plasma clot technique. The plasma clot contained Iscove’s modification of Dulbecco’s medium (Sigma), 20% fetal calf serum (PAN Biotech GmbH, Aidenbach, Germany), 10 ng/mL recombinant mouse interleukin-3 (rmIL-3) (Sigma), and 10% citrate bovine plasma (Veterinary Research Institute, Brno, Czech Republic). Erythroid progenitor cells (burst-forming unit, BFU-E) were cultivated on methylcellulose (Stem Cell Technologies, Inc., Vancouver, Canada) with 4 U/mL recombinant human erythropoetin (EPREX, JanssenCilag s.r.o., Prague, Czech Republic), 50 ng/mL mouse stem cell factor (Sigma), and 10 ng/mL rmIL-3 (Sigma). Femoral marrow cell suspensions were plated (1.5 105 and 1 105 nucleated bone marrow cells for GM-CFC and BFU-E, respectively) in triplicate for both assays and incubated at 37 8C in a humidified atmosphere containing 95% air and 5% CO2. GM-CFC were scored after a 7-day incubation as colonies containing 50 or more cells. Hemoglobinized colonies were counted as BFU-E after an 8-day incubation. For evaluation of the compartment of morphologically recognizable granulocytic and erythroid precursor cells of the femoral bone marrow, differential counts were performed on marrow smears stained by the May-Gru¨nwald-Giemsa method. In the granulocytic lineage myeloblasts through myelocytes were classified as proliferative cells, metamyelocytes through segmented stages as non-proliferative ones. In the erythroid lineage proerythroblasts through basophilic erythroblasts were classified as proliferative cells, polychromatic and orthochromatic erythroblasts as non-proliferative ones. Total counts of granulocytic or erythroid cells represent the sum of proliferative and nonproliferative cells, respectively. 2.5. Statistics Primary data are summarized as means standard error of the mean (SEM). One-way model of analysis of variance (Anova) was applied to test the differences among experimental variants. Investigated parameters were analysed separately. Mutual differences among experimental variants were evaluated by means of Tukey post hoc test. In the case of the experiment testing the effect of meloxicam on serum G-CSF levels, t-test for two independent samples was used to compare the variants. All analyses were performed using Statistica 8.0 software (StatSoft, Inc.). The significance level was set at P < 0.05.
3. Results The sublethal irradiation of mice with the dose of 4 Gy g-rays induced an expressive damage in the hematopoietic system. To illustrate the extent of the damage, the values of the hematopoietic parameters studied in control non-irradiated mice and in nontreated mice exposed to the dose of 4 Gy, sampled on day 3 after irradiation are summarized in Table 1. The percent decrease observed on day 3 after irradiation represented about 90%, and more than 50% in the compartments of bone marrow progenitor cells and bone marrow precursor cells, respectively.
M. Hofer et al. / Biomedicine & Pharmacotherapy 65 (2011) 427–431 Table 1 Hematopoietic parameters in control non-irradiated mice and 4 Gy-irradiated mice.
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Experimental protocol for investigating the combined effects of the two selected drugs utilized one dose of meloxicam administered 1 hour after irradiation and two doses of IB-MECA given 24 and 48 hours after irradiation (for details: Materials and methods). Four groups of animals, i.e. controls which did not obtain any drug and were treated only with solvents, mice treated with meloxicam or IB-MECA alone, and mice treated with the combination of both drugs were euthanized on day 3 after 4-Gy irradiation and effects of the different treatments on the hematopoietic indices in the bone marrow have been compared. These effects are illustrated in Figs. 1–6. One-way Anova test was used to define hematopoietic indices influenced significantly by the action of the treatment. Significant effects were found in indices of GM-CFC (Fig. 1), BFU-E (Fig. 4), and proliferative granulocytic cells (Fig. 2). As shown by Tukey post hoc test, IB-MECA per se, as compared to irradiated controls, induced a significant enhancement in the parameters of BFU-E (Fig. 4) and of proliferative granulocytic cells (Fig. 2). Meloxicam per se, in a similar comparison, did not elicit significant
effects in any of the investigated parameters. However, the most expressive stimulatory effects were observed when using the combined treatment. Here the values of GM-CFC (Fig. 1), BFU-E (Fig. 4), as well as those of proliferative granulocytic cells (Fig. 2) were significantly higher in comparison with irradiated controls and attained the highest increase factors of 1.6, 1.6, and 2.6, respectively. Significant statistical differences were observed also when comparing the values obtained after combined treatment with the values observed in the group treated with meloxicam alone in the indices of GM-CFC (Fig. 1), BFU-E (Fig. 4), and proliferative granulocytic cells (Fig. 2). A significant statistical difference was also found in comparison of the values in mice treated with the combination of the drugs and in those treated with IB-MECA alone in the parameter of proliferative granulocytic cells (Fig. 2). The fact that the effects of the drug treatment in the precursor cell compartments are evident only in the granulocytic line and not in the erythroid one is understandable because of the higher proliferation pressure in the granulocytic cell system and low proliferation pressure in the erythroid one. Proliferation pressures are determined by the life span of the end cells in a system, i.e. a long one in erythrocytes and a short one in granulocytes. Approximately similar effects of the treatment in the investigated progenitor cell compartments of GM-CFC (Fig. 1) and BFU-E (Fig. 4) can be ascribed to the action of the drugs at the level of progenitors of the common myeloid pathway [26]. These cells might be the targets of the drug action. The absence of differences between the values of total granulocytic cells (Fig. 3) in the femoral marrow of the investigated groups can be explained by the outflow of differentiated cells into the peripheral blood under the conditions of enhanced proliferation activity in this cell system. In our previous study performed with the same mouse strain, no effects of IB-MECA on the serum levels of granulocyte colonystimulating factor (G-CSF) under identical experimental conditions were observed [25]. Therefore, our experiments aimed at testing the ability of the treatment described above to modulate serum concentrations of G-CSF were targeted only to investigation of the effects of meloxicam. The results are shown in Table 2. The serum levels of G-CSF were found to be rather sensitive to non-specific stimuli like irradiation and saline injection and were increased
Fig. 1. GM-CFC per femur in 4 Gy-treated mice. Meloxicam or its solvent (saline) were adminstered 1 hour after irradiation. IB-MECA or its solvent (2% DMSO in saline) were administered 24 and 48 hours after irradiation. Control – mice treated only with solvents. Data are given as means SEM. Twelve to 15 animals per group were used. * P < 0.05 in comparison with Control. ++ P < 0.01 in comparison with meloxicam.
Fig. 2. Proliferative granulocytic cells per femur in 4 Gy-irradiated mice. Five animals per group were used. *, *** P < 0.05, P < 0.001, respectively, in comparison with Control. # P < 0.05 in comparison with IB-MECA. +++ P < 0.001 in comparison with meloxicam. For other explanations see legend to Fig. 1.
Parameter
Control non-irradiated mice
4 Gy-irradiated mice
GM-CFC per femur
17,983 820 n = 10
1,884 122 n = 12
Proliferative granulocytic cells per femur ( 103)
2,134 253 n = 10
809 48 n=5
Total granulocytic cells per femur ( 103)
11,198 534 n = 10
5,082 443 n=5
BFU-E per femur
23,736 1,580 n = 10
859 115 n = 15
Proliferative erythroid cells per femur ( 103)
1,807 216 n = 10
713 126 n=5
Total erythroid cells per femur ( 103)
6,232 653 n = 10
2,145 307 n=5
Values of hematopoietic parameters are given as arithmetic means standard errors of the means (SEM). Sampling in 4 Gy-irradiated mice was performed on day 3 after irradiation. n: numbers of animals.
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Fig. 3. Total granulocytic cells per femur in 4 Gy-irradiated mice. Five animals per group were used. For other explanations see legend to Fig. 1.
Fig. 5. Proliferative erythroid cells per femur in 4 Gy-irradiated mice. Five animals per group were used. For other explanations see legend to Fig. 1.
compared to those observed in non-irradiated untreated controls. After the administration of meloxicam, the serum levels of G-CSF increased further to 167% compared to those in saline-treated controls, the difference being statistically significant.
4. Discussion The presented data indicate that the early postirradiation administration of the inhibitor of COX-2 meloxicam and the subsequent administration of the agonist of adenosine A3 receptor IB-MECA to g-irradiated mice represent a positively acting drug combination activating recovery of the damaged hematopoiesis. Effects of the combination of the drugs cannot be considered to be a result of a simple additivity of stimulatory actions. This is evident by the inability of meloxicam given alone to influence the indices of the bone marrow cellularity at the bone marrow sampling time interval of 3 days after irradiation. If the targets of the proliferation-inducing or curative action of meloxicam are some early progenitor or stem cells, these effects remain hidden because of the very low percentage of these cells in the cellular system of the bone
Fig. 6. Total erythroid cells per femur in 4 Gy-irradiated mice. Five animals per group were used. For other explanations see legend to Fig. 1.
marrow (less than 0.1%). From this point of view it seems to be logical that hematopoiesis-stimulating effects of meloxicam in parameters determined in our study manifest themselves on day 3 after irradiation only in connection with the activity of the agonist of adenosine A3 receptors which enhances proliferation in a wide spectrum of cells in the bone marrow cell renewal system, as has been found earlier [20–23]. As stated by Metcalf [27], stimulatory interactions of the two regulatory principles can be due to their
Table 2 Effects of meloxicam administration on serum concentrations of G-CSF in 4 Gyirradiated mice.
Fig. 4. BFU-E per femur in 4 Gy-irradiated mice. Fourteen to 15 animals per group were used. * P < 0.05 in comparison with Control. + P < 0.05 in comparison with meloxicam. For other explanations see legend to Fig. 1.
Group of mice
Concentration of G-CSF (pg/mL)
Non-irradiated untreated controls Saline-treated 4 Gy-irradiated mice Meloxicam-treated 4 Gy-irradiated mice
316.4 81.2 793.5 34.2* 1,330.0 41.4**
Values are given as means standard errors of the means (SEM). Five animals per group were used. * P < 0.05 vs. non-irradiated untreated controls. ** P < 0.001 vs. 4 Gy-irradiated saline-treated mice (t-test for two independent samples).
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different operations at different levels of the age structure of the hematopoietic cell system. At the present time, it is possible only to speculate about the nature of the mechanisms inducing the meloxicam action. A certain role can be played by the ability of meloxicam to enhance the production of G-CSF, a phenomenon which has been observed in our earlier studies [14,24] as well as under conditions of the presented experiments. It is known that hematopoietic colonystimulating factors enhance cell survival by suppressing apoptosis [28]. If the effects of meloxicam occur at the level of the early progenitor cells and/or stem cells, the concomitant cell proliferation enhancing the action of IB-MECA can result in a distinct cell amplification in the hematopoietic cell system including its more differentiated compartments. It has to be mentioned that our interpretation of the curative effects of meloxicam is similar to the concept of the cytokine-based anti-apoptotic treatment of radiation injury proposed by He´rodin et al. [3,4]. Our experimental approach using a combination of a COX-2 inhibitor and an adenosine A3 receptor agonist administered early after irradiation can be also regarded as a contribution to the experimental efforts aimed at optimizing the postirradiation approach to the treatment of radiation injuries as proposed by Moulder [29]. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported by the Ministry of Defense (project ‘‘Receptor’’, project No. 1001 8 5090), by the Grant Agency of the Czech Republic (grants Nos. 305/08/0158 and P303/11/0128), and by the Academy of Sciences of the Czech Republic (grants Nos. AV0Z50050507 and AV0Z50040702). References [1] Weiss JF, Kumar KS, Walden TL, Neta R, Landauer MR, Clark EP. Advances in radioprotection through the use of combined agent regimens. Int J Radiat Biol 1990;57:709–22. [2] Pospı´sˇil M, Hofer M, Znojil V, Va´cha J, Netı´kova´ J, Hola´ J. Synergistic effects of granulocyte colony-stimulating factor and drugs elevating extracellular adenosine on neutrophil production in mice. Blood 1995;86:3692–7. [3] He´rodin F, Bourin P, Mayol JF, Lataillade JJ, Drouet M. Short-term injection of antiapoptotic cytokine combinations soon after lethal g-irradiation promotes survival. Blood 2003;101:2609–16. [4] He´rodin F, Drouet M. Cytokine-based treatment of accidentally irradiated victims and new approaches. Exp Hematol 2005;33:1071–80. [5] Hofer M, Pospı´sˇil M. Role of adenosine signaling in hematopoiesis – a short review. Med Hypotheses Res 2006;3:629–35. [6] Hofer M, Pospı´sˇil M. Stimulated recovery of perturbed haematopoiesis by inhibition of prostaglandin production – promising therapeutic strategy. Cent Eur J Biol 2006;1:584–93.
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