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Effects of tramadol on immune responses and nociceptive thresholds in mice
Pain 72 (1997) 325–330
Effects of tramadol on immune responses and nociceptive thresholds in mice Paola Sacerdote*, Mauro Bianchi, Barbara Manfredi, Alberto E. Panerai Department of Pharmacology, University of Milan, via Vanvitelli 32, 20129 Milano, Italy Received 12 July 1996; revised version received 18 February 1997; accepted 8 May 1997
Abstract Tramadol is a centrally acting analgesic drug with a dual mechanism of action: binding to m-opioid receptors and potentiation of the monoaminergic systems. In this study, we evaluated the effects of the acute and chronic administration of tramadol on nociceptive thresholds (by the hot-plate test) and on immune responses (by measuring Concanavalin A-induced splenocyte proliferation, IL-2 production and natural killer activity) in the mouse. After acute subcutaneous administration, tramadol induced antinociception starting from a dose of 20 mg/kg, whereas it significantly enhanced natural killer activity and IL-2 production at doses as low as 1 mg/kg and splenocyte proliferation starting from a dose of 10 mg/kg. After the chronic administration, the antinociceptive effect of the drug was still present, whereas the immune modifications disappeared. Thus, the pharmacological profile of tramadol is totally different from that of other drugs which bind m-opioid receptors. Our results suggest that tramadol could be a good choice for the treatment of pain in patients where immunosuppression may be particularly contraindicated. 1997 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Hot-plate; Interleukin-2; Natural killer activity; Splenocyte proliferation; Immunity
1. Introduction Tramadol is a centrally acting analgesic drug, used mainly for the treatment of pain of intermediate or severe intensity (Kayser et al., 1992; Dayer et al., 1994). It was initially described as binding with low affinity to opioid receptors, showing higher selectivity for the m receptor (Raffa et al., 1992). However, the therapeutic use of tramadol was not associated with the classical side-effects of opiate drugs, such as respiratory depression, constipation, or sedation (Besson and Vickers, 1994; Dayer et al., 1994).The pharmacology of tramadol was therefore reevaluated, and it was demonstrated that the drug also exerted a modulatory effect on central monoaminergic pathways, inhibiting the neuronal uptake of noradrenaline and serotonin (Driessen and Reimann, 1992; Kayser et al., 1992; Raffa et al., 1992; Driessen et al., 1993). Tramadol, therefore, causes the activation of both systems mainly involved in the inhibition of pain: the opioid and the descending monoaminergic system. * Corresponding author. Tel.: +39 2 719750; fax: +39 2 730470; e-mail: [email protected]
Recent evidence has demonstrated that neurotransmitters play an important role in the regulation of the immune responses, and a number of pharmacological agents that modulate the serotoninergic or noradrenergic tones are known to directly or indirectly affect immune function (Peterson et al., 1993; Besedowsky and Del Rey, 1996). Moreover, the lymphoid organs are directly innervated by sympathetic fibers and it has been shown that central and peripheral catecholaminergic systems are deeply involved in the modulation of immune responses (Livnat et al., 1985). Similarly, the opioid endogenous system and most opiates, (e.g., morphine), have been reported to profoundly affect immune responses (Heijnen et al., 1991; Sacerdote et al., 1991; Manfredi et al., 1993; Peterson et al., 1993; Panerai et al., 1995). On considering the dual mechanism of action of tramadol, i.e., the activation of the opioid and of the monoaminergic systems, we considered it of interest to evaluate the potential immunological effects of this drug. We therefore studied, in the mouse, the effects of both acute and chronic tramadol administration on T lymphocyte function, by evaluating Concanavalin-A (ConA) induced proliferation and interleukin-2 (IL-2) production of spleno-
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cytes, and on natural killer (NK) cell activity. At the same time, we evaluated the antinociceptive action of this drug by the hot-plate test, in order to define whether the immunological effects were present at analgesic doses of tramadol.
from the spleens using 20-gauge sterile needles through an incision made in the spleen cuticle (Manfredi et al., 1993). 2.5. Evaluation of splenocyte proliferation
2. Methods 2.1. Animals Male Swiss mice (Charles River, Calco, Italy) 20–25 g bwt were used. Animals were housed at 22 ± 2°C, with a light:dark cycle of 14:10 h, with food and water ad libitum. In all experiments, each experimental group consisted of eight animals. 2.2. Antinociception test Nociceptive thresholds were measured by the hot-plate method (Eddy et al., 1950) In our experimental conditions, the hot-plate was maintained at 54°C. An animal was placed on the heated surface, and the time interval (s) between placement and the simultaneous licking of both fore paws was recorded. Basal reaction times were between 8 and 12 s, and the cut-off time was 30 s. In the acute study, tramadol (Contramal, Formenti, Italy) was administered subcutaneously (s.c.) at doses of 10, 20, 40 and 80 mg/kg, and analgesic thresholds were evaluated 15, 30, 45, 60 and 90 min after treatment. In the chronic experiments, tramadol was administered s.c. at a dose of 20 mg/kg twice daily, for 2 weeks. Nociceptive thresholds were evaluated at the first day and the last day of treatment, 30 min after the administration of the drug. In all experiments, control animals received s.c. the same volume of saline (0.2 ml). 2.3. Immunological studies In the acute study, tramadol was administered s.c. at doses of 0.1, 1.0, 5.0, 10, 20, 40 and 80 mg/kg, and the animals were killed by cervical dislocation 60 min after treatment. In the chronic study, tramadol was administered s.c. at the dose of 20 mg/kg, twice daily for 2 weeks. One group of animals was sacrificed 1 h after the last injection of tramadol, while another group was killed 24 h after the last injection of the drug. Moreover, a group of animals received saline twice daily for 2 weeks, and was then acutely treated with tramadol (10 and 20 mg/kg, s.c.). In all the experiments, control animals received s.c. the same volume of saline (0.2 ml). 2.4. Collection of splenocytes Spleen was aseptically removed, and cells were teased
Microcultures of splenocytes were set up (4 × 106 mouse cells/ml) in RPMI 1640, 10% FCS ± ConA (2.5 mg/ml, 5 mg/ml, 10 mg/ml). After 48-h incubation at 37°C, 1.0 mCi of [3H]thymidine (specific activity 2 Ci/mmol; Amersham, UK) was added to all cultures. Cells were harvested 18 h later by an automated cell harvester (Skatron) and radioactivity was measured in a liquid scintillation counter (Packard, Downers Grove, IL, USA). Background values, i.e., thymidine incorporation of unstimulated cells, were subtracted from mitogen-induced proliferation. The three concentrations of ConA were selected in order to provide submaximal as well as maximal stimulation of proliferation. 2.6. Evaluation of natural killer activity NK activity was evaluated by a 4-h 51Cr release assay. Briefly, 5 × 106 YAC-1 cells were labeled by incubation with 100 mCi sodium chromate, (specific activity 250–500 mCi/mg chromium; Amersham) in 0.2 ml RPMI + 10% FCS for 60 min at 37°C in 5% CO2. After three washes, the YAC-1 cells were suspended in RPMI, 10% FCS at a concentration of 105/ml. 51Cr labeled YAC-1 cells (104/ well) were incubated with effector cells in 96-well microtiter plates, at effector:target (E:T) cell ratios of 200:1 and 100:1. Each E:T ratio was tested in triplicate. Following an incubation of 4 h at 37°C in 5% CO2, plates were centrifuged at 400 × g for 5 min, a 100-ml aliquot of supernatant was removed from each well, and counted in a Packard gamma counter. Maximum 51Cr release and spontaneous release were determined in wells containing 3 M HCl or medium, respectively. Specific 51Cr release was calculated according to the formula: 100 × [(experimental release − spontaneous release)= (maximal release − spontaneous release)]: 2.7. Measurement of IL-2 Spleen cells were adjusted to 107 cells/ml medium and incubated for 24 h with or without 10 mg/ml of ConA. The levels of IL-2 in the supernatants were determined by enzyme linked immunosorbent assay (ELISA) protocol as standardized by Pharmingen (San Diego, CA, USA). Briefly the anti IL-2 capture monoclonal antibody (mAb) was absorbed on a polystyrene 96-well plate and the IL-2 present in the sample was bound to the antibody coated wells. The biotinylated anti IL-2 detecting mAb, was
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Fig. 3. Effect of acute tramadol on natural killer activity (effector:target = 100:1, 200:1). Values are means ± SD. *P , 0.01 vs. saline group (0) (two-way ANOVA).
3. Results Fig. 1. Antinociceptive effect of acute tramadol in the hot-plate test. Values are means ± SEM. *P , 0.01 vs. saline (one-way ANOVA).
added to bind the IL-2 captured by the first antibody. After washing, avidin-peroxidase (Sigma) was added to the wells to detect the biotinylated detecting antibody and finally 2,2′-azino-bis (3-ethylbenthiazoline-6-sulfonic acid) (ABTS; Sigma) substrate was added and a colored product was formed in proportion to the amount of IL-2 present in the sample, which was measured at OD 405 nM. 2.8. Statistical analysis Statistical analysis for NK activity and cell proliferation was performed by two-way ANOVA, followed by Bonferroni’s test. IL-2 production and analgesic responses were evaluated by one-way ANOVA, followed by Dunnet’s test for multiple comparison.
Fig. 2. Effect of acute tramadol on ConA-induced (2.5, 5, 10 mg/ml) splenocyte proliferation. Values are means ± SD. *P , 0.01 vs. saline group (0) (two-way ANOVA).
Fig. 1 shows that, as expected, tramadol induced a dose-related antinociceptive effect in the hot-plate test. The effect was evident starting from the dose of 20 mg/ kg, and the maximum attainable antinociceptive response was observed after the administration of 80 mg/kg. The peak of antinociceptive activity was obtained within 15 min. The effect of acute tramadol on splenocyte prolifer– ation induced by three different concentrations of the mitogen ConA is reported in Fig. 2. The drug induced a significant enhancement of splenocyte proliferation. This action of tramadol was evident starting at 10 mg/kg, and remained un-changed after the administration of higher doses. Also, splenic NK activity was significantly enhanced after acute administration of tramadol (Fig. 3). A significant stimulation of this immune parameter was already evident after the administration of 1 mg/kg of the drug. The immunostimulatory effect of tramadol was further confirmed by the observation that IL-2 production by splenocytes was increased after the administration of the drug; this effect was also already evident after the administration of 1 mg/kg (Fig. 4).
Fig. 4. Effect of acute tramadol on IL-2 production by ConA-stimulated splenocytes. Values are means ± SD. *P , 0.01 vs. saline group (0) (oneway ANOVA).
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4. Discussion
Fig. 5. Nociceptive thresholds measured before (A) and 30 min (g)after the injection of tramadol (20 mg/kg, s.c.), on the first day and after 14 days of drug administration (20 mg/kg s.c., twice daily). Values are means ± SEM. *P , 0.01 vs. saline.
In order to evaluate the development of tolerance to the analgesic or to the immune effects of tramadol, the nociceptive thresholds and the immune parameters were evaluated after chronic treatment. As shown in Fig. 5, no tolerance developed to the antinociceptive effect of tramadol. In fact, the analgesic response to the challenge with 20 mg/kg of tramadol was unchanged after 14 days of treatment. On the contrary, all the immune effects of tramadol disappeared after chronic treatment (Table 1). The loss of the stimulation of splenocyte proliferation, IL-2 production and NK activity was observed when immune function was evaluated either 1 h or 24 h after the last administration of the drug. In order to distinguish between immune tolerance to the drug versus a possible tolerance of the immune effects induced by mild stressors represented by handling procedures, the same parameters were measured in animals acutely treated with two doses of tramadol after 14 days of treatment with saline. As shown in Fig. 6, in these animals tramadol was still able to enhance ConA-induced proliferation (panel A), NK activity (panel B) and IL-2 production (panel C).
The present study describes, for the first time, some immunomodulatory properties of tramadol. When administered acutely, this drug enhances the responses of different lymphocyte populations. In fact, it significantly stimulates T cell functions, as indicated by the enhancement of ConAinduced proliferation and of ConA-induced production of IL-2; moreover, it augments the activity of NK cells, a population of lymphocytes which plays a significant role in tumor surveillance and in the defense against viral infections (Herberman and Hortaldo, 1981). The immune effects of tramadol are observed at extremely low doses; in fact, the effects on IL-2 production and NK activity are already evident at the dose of 1 mg/kg. However, the sensitivity to the drug of the immune parameters evaluated is different: whereas NK activity and IL-2 production are increased by the dose of 1 mg/kg of tramadol, the potentiation of ConAinduced proliferation is evident only after the administration of higher doses. This observation is not surprising, since a different sensitivity of NK cells and T lymphocytes to pharmacological treatments has been reported also for opiate induced immunosuppression (Lysle et al., 1992). Moreover, experimental models of stress have been shown to affect different immune cell populations depending on stress intensity, duration, frequency, etc. (Labeur et al., 1995). T lymphocyte proliferation is dependent on the IL-2 pathways; in fact, many reports have demonstrated the critical requirements of IL-2 production as well as of IL-2 receptor (IL-2R) induction for proliferation (Toribio et al., 1989). Thus an increase of IL-2R could also be necessary in order to measure an increase of T lymphocyte proliferation. It is therefore feasible to suppose that the induction of IL-2R is achieved only after the administration of 20 mg/kg of tramadol (when increased proliferation is also measurable). However, it is interesting to observe that the doses effective in the hot-plate test are always much higher. We observed, in fact, a significant enhancement of nociceptive thresholds only after the administration of the dose of 20 mg/kg. The immune actions of tramadol were unexpected since the activation of central m-opiate receptors has classically been associated with immunosuppression (Taub et al., 1991;
Table 1 Effects of chronic treatment (2 weeks) with tramadol (20 mg/kg) on different immune parameters
Splenocyte proliferation (cpm [3H]thymidine) ConA 2.5 mg/ml ConA 5.0 mg/ml ConA 10 mg /ml NK activity (% specific lysis) E:T 200:1 E:T 100:1 IL-2 production (units/ml) Values are mean ± SD.
Saline
Tramadol (last injection 24 h earlier)
Tramadol (last injection 1 h earlier)
55600 ± 16230 83050 ± 28100 61000 ± 21500
56890 ± 12563 87000 ± 30500 66450 ± 25750
53874 ± 19856 82500 ± 25200 59400 ± 18000
16.2 ± 7.2 15.4 ± 7.1 202.5 ± 57.7
17.8 ± 9 16.8 ± 5 227 ± 108
21.2 ± 9.6 19.4 ± 4.8 188.5 ± 59.68
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Fig. 6. Effects on immune parameters of tramadol (10 and 20 mg/kg s.c.) after 2 weeks of treatment with saline. Panel A: ConA-induced splenocyte proliferation; panel B: NK activity; panel C: IL-2 production. Values are means ± SD. *P , 0.01 vs. saline group (0).
Band et al., 1992). A wide literature has described the suppressive effects of morphine and endogenous opioids on many immune functions, including lymphocyte proliferation, IL-2 production and NK activity (Band et al., 1992; Manfredi et al., 1993; Peterson et al., 1993; Panerai et al., 1995). However, in very recent work, the opiate derived compound OHM3295 has been shown to potentiate splenic NK activity in vivo throughout the activation of m- and kopioid receptors (Baker et al., 1995). Therefore, we cannot rule out the possibility that the enhancement of immune function induced by tramadol could be mediated via opiate receptors. Further experiments in order to elucidate this point are particularly difficult since, in our hands, classical opioid antagonists such as naloxone and naltrexone exert immunostimulatory activity by themselves (Manfredi et al., 1993). It is well known that the antinociceptive effects of tramadol are mediated not only via an opioid mechanism, but also via a separate, non-opioid, mechanism, due to the inhibition of neuronal uptake of noradrenaline and serotonin (Kayser et al., 1992; Raffa et al., 1992). It is therefore also possible that the immune effects of this drug on the immune system could be mediated by the activation of the monoaminergic pathways. The involvement of catecholamine and serotonin systems in neural-immune interactions has been studied using different experimental models. Both enhancement and reduction of immune response have been related to the activation of noradrenergic system, providing evidence for the heterogeneity of sympathetic nervous system regulation of lymphocyte function (Livnat et al., 1985; Madden et al., 1994). On the other hand, the increase of serotoninergic tone has been frequently associated with stimulation of NK activity and lymphocyte proliferation (Steplewsky and Vogel, 1985; Gabrilovac et al., 1992). Similarly, drugs which increase serotoninergic tone such as d-fenfluoramine (Petrovic et al., 1990) and fluoxetine (Sacerdote, unpublished results), stimulate immune function in rodents.
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Therefore, the activation of the serotoninergic system might be involved in the immune effects induced by the acute administration of tramadol. Consistent with the results obtained by other authors (Dayer et al., 1994), we did not observe any tolerance to the antinociceptive activity of tramadol. On the contrary, after 14 days of treatment, the immune effects of the drug were lost. This observation suggests that a dissociation exists between the mechanism of action of tramadol on nociceptive thresholds and on immune responses. At present, we do not have an explanation for the loss of the immunological effects induced by tramadol after chronic administration. However, it is important to note that an adaptation of the immune system has been reported to the effects of pharmacological (Bryant et al., 1988) as well as physiological, e.g., stress (Khansari et al., 1990; Sacerdote et al., 1994) stimuli. Tolerance also develops to the immunosuppressive effects of morphine (Bryant et al., 1988). Some types of mild stressors, such as handling and novelty, have been shown to enhance some immune responses (Wood et al., 1993; Dhabhar and McEwen, 1996).However, since we observed that the immune responses were also increased by acute injections of tramadol after 2 weeks of treatment with saline, during which potential tolerance to stress should have developed, we can exclude that we were actually observing the development of tolerance to these stressors rather than to the drug administration. The clinical value of our observations might be relevant. Indeed, the use of classical narcotic drugs with immunosuppressive effects is contraindicated in many situations where the immune system is compromised, such as in cancer patients (Provinciali et al., 1991) or in the postoperative period (Hammer et al., 1992; Saito et al., 1992). In this respect, the availability of a drug, like tramadol, which lacks immunosuppressive side-effects may represent a favorable therapeutic alternative to opiates for the treatment of acute pain.
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Effects of tramadol on immune responses and nociceptive thresholds in mice Paola Sacerdote*, Mauro Bianchi, Barbara Manfredi, Alberto E. Panerai Department of Pharmacology, University of Milan, via Vanvitelli 32, 20129 Milano, Italy Received 12 July 1996; revised version received 18 February 1997; accepted 8 May 1997
Abstract Tramadol is a centrally acting analgesic drug with a dual mechanism of action: binding to m-opioid receptors and potentiation of the monoaminergic systems. In this study, we evaluated the effects of the acute and chronic administration of tramadol on nociceptive thresholds (by the hot-plate test) and on immune responses (by measuring Concanavalin A-induced splenocyte proliferation, IL-2 production and natural killer activity) in the mouse. After acute subcutaneous administration, tramadol induced antinociception starting from a dose of 20 mg/kg, whereas it significantly enhanced natural killer activity and IL-2 production at doses as low as 1 mg/kg and splenocyte proliferation starting from a dose of 10 mg/kg. After the chronic administration, the antinociceptive effect of the drug was still present, whereas the immune modifications disappeared. Thus, the pharmacological profile of tramadol is totally different from that of other drugs which bind m-opioid receptors. Our results suggest that tramadol could be a good choice for the treatment of pain in patients where immunosuppression may be particularly contraindicated. 1997 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Hot-plate; Interleukin-2; Natural killer activity; Splenocyte proliferation; Immunity
1. Introduction Tramadol is a centrally acting analgesic drug, used mainly for the treatment of pain of intermediate or severe intensity (Kayser et al., 1992; Dayer et al., 1994). It was initially described as binding with low affinity to opioid receptors, showing higher selectivity for the m receptor (Raffa et al., 1992). However, the therapeutic use of tramadol was not associated with the classical side-effects of opiate drugs, such as respiratory depression, constipation, or sedation (Besson and Vickers, 1994; Dayer et al., 1994).The pharmacology of tramadol was therefore reevaluated, and it was demonstrated that the drug also exerted a modulatory effect on central monoaminergic pathways, inhibiting the neuronal uptake of noradrenaline and serotonin (Driessen and Reimann, 1992; Kayser et al., 1992; Raffa et al., 1992; Driessen et al., 1993). Tramadol, therefore, causes the activation of both systems mainly involved in the inhibition of pain: the opioid and the descending monoaminergic system. * Corresponding author. Tel.: +39 2 719750; fax: +39 2 730470; e-mail: [email protected]
Recent evidence has demonstrated that neurotransmitters play an important role in the regulation of the immune responses, and a number of pharmacological agents that modulate the serotoninergic or noradrenergic tones are known to directly or indirectly affect immune function (Peterson et al., 1993; Besedowsky and Del Rey, 1996). Moreover, the lymphoid organs are directly innervated by sympathetic fibers and it has been shown that central and peripheral catecholaminergic systems are deeply involved in the modulation of immune responses (Livnat et al., 1985). Similarly, the opioid endogenous system and most opiates, (e.g., morphine), have been reported to profoundly affect immune responses (Heijnen et al., 1991; Sacerdote et al., 1991; Manfredi et al., 1993; Peterson et al., 1993; Panerai et al., 1995). On considering the dual mechanism of action of tramadol, i.e., the activation of the opioid and of the monoaminergic systems, we considered it of interest to evaluate the potential immunological effects of this drug. We therefore studied, in the mouse, the effects of both acute and chronic tramadol administration on T lymphocyte function, by evaluating Concanavalin-A (ConA) induced proliferation and interleukin-2 (IL-2) production of spleno-
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cytes, and on natural killer (NK) cell activity. At the same time, we evaluated the antinociceptive action of this drug by the hot-plate test, in order to define whether the immunological effects were present at analgesic doses of tramadol.
from the spleens using 20-gauge sterile needles through an incision made in the spleen cuticle (Manfredi et al., 1993). 2.5. Evaluation of splenocyte proliferation
2. Methods 2.1. Animals Male Swiss mice (Charles River, Calco, Italy) 20–25 g bwt were used. Animals were housed at 22 ± 2°C, with a light:dark cycle of 14:10 h, with food and water ad libitum. In all experiments, each experimental group consisted of eight animals. 2.2. Antinociception test Nociceptive thresholds were measured by the hot-plate method (Eddy et al., 1950) In our experimental conditions, the hot-plate was maintained at 54°C. An animal was placed on the heated surface, and the time interval (s) between placement and the simultaneous licking of both fore paws was recorded. Basal reaction times were between 8 and 12 s, and the cut-off time was 30 s. In the acute study, tramadol (Contramal, Formenti, Italy) was administered subcutaneously (s.c.) at doses of 10, 20, 40 and 80 mg/kg, and analgesic thresholds were evaluated 15, 30, 45, 60 and 90 min after treatment. In the chronic experiments, tramadol was administered s.c. at a dose of 20 mg/kg twice daily, for 2 weeks. Nociceptive thresholds were evaluated at the first day and the last day of treatment, 30 min after the administration of the drug. In all experiments, control animals received s.c. the same volume of saline (0.2 ml). 2.3. Immunological studies In the acute study, tramadol was administered s.c. at doses of 0.1, 1.0, 5.0, 10, 20, 40 and 80 mg/kg, and the animals were killed by cervical dislocation 60 min after treatment. In the chronic study, tramadol was administered s.c. at the dose of 20 mg/kg, twice daily for 2 weeks. One group of animals was sacrificed 1 h after the last injection of tramadol, while another group was killed 24 h after the last injection of the drug. Moreover, a group of animals received saline twice daily for 2 weeks, and was then acutely treated with tramadol (10 and 20 mg/kg, s.c.). In all the experiments, control animals received s.c. the same volume of saline (0.2 ml). 2.4. Collection of splenocytes Spleen was aseptically removed, and cells were teased
Microcultures of splenocytes were set up (4 × 106 mouse cells/ml) in RPMI 1640, 10% FCS ± ConA (2.5 mg/ml, 5 mg/ml, 10 mg/ml). After 48-h incubation at 37°C, 1.0 mCi of [3H]thymidine (specific activity 2 Ci/mmol; Amersham, UK) was added to all cultures. Cells were harvested 18 h later by an automated cell harvester (Skatron) and radioactivity was measured in a liquid scintillation counter (Packard, Downers Grove, IL, USA). Background values, i.e., thymidine incorporation of unstimulated cells, were subtracted from mitogen-induced proliferation. The three concentrations of ConA were selected in order to provide submaximal as well as maximal stimulation of proliferation. 2.6. Evaluation of natural killer activity NK activity was evaluated by a 4-h 51Cr release assay. Briefly, 5 × 106 YAC-1 cells were labeled by incubation with 100 mCi sodium chromate, (specific activity 250–500 mCi/mg chromium; Amersham) in 0.2 ml RPMI + 10% FCS for 60 min at 37°C in 5% CO2. After three washes, the YAC-1 cells were suspended in RPMI, 10% FCS at a concentration of 105/ml. 51Cr labeled YAC-1 cells (104/ well) were incubated with effector cells in 96-well microtiter plates, at effector:target (E:T) cell ratios of 200:1 and 100:1. Each E:T ratio was tested in triplicate. Following an incubation of 4 h at 37°C in 5% CO2, plates were centrifuged at 400 × g for 5 min, a 100-ml aliquot of supernatant was removed from each well, and counted in a Packard gamma counter. Maximum 51Cr release and spontaneous release were determined in wells containing 3 M HCl or medium, respectively. Specific 51Cr release was calculated according to the formula: 100 × [(experimental release − spontaneous release)= (maximal release − spontaneous release)]: 2.7. Measurement of IL-2 Spleen cells were adjusted to 107 cells/ml medium and incubated for 24 h with or without 10 mg/ml of ConA. The levels of IL-2 in the supernatants were determined by enzyme linked immunosorbent assay (ELISA) protocol as standardized by Pharmingen (San Diego, CA, USA). Briefly the anti IL-2 capture monoclonal antibody (mAb) was absorbed on a polystyrene 96-well plate and the IL-2 present in the sample was bound to the antibody coated wells. The biotinylated anti IL-2 detecting mAb, was
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Fig. 3. Effect of acute tramadol on natural killer activity (effector:target = 100:1, 200:1). Values are means ± SD. *P , 0.01 vs. saline group (0) (two-way ANOVA).
3. Results Fig. 1. Antinociceptive effect of acute tramadol in the hot-plate test. Values are means ± SEM. *P , 0.01 vs. saline (one-way ANOVA).
added to bind the IL-2 captured by the first antibody. After washing, avidin-peroxidase (Sigma) was added to the wells to detect the biotinylated detecting antibody and finally 2,2′-azino-bis (3-ethylbenthiazoline-6-sulfonic acid) (ABTS; Sigma) substrate was added and a colored product was formed in proportion to the amount of IL-2 present in the sample, which was measured at OD 405 nM. 2.8. Statistical analysis Statistical analysis for NK activity and cell proliferation was performed by two-way ANOVA, followed by Bonferroni’s test. IL-2 production and analgesic responses were evaluated by one-way ANOVA, followed by Dunnet’s test for multiple comparison.
Fig. 2. Effect of acute tramadol on ConA-induced (2.5, 5, 10 mg/ml) splenocyte proliferation. Values are means ± SD. *P , 0.01 vs. saline group (0) (two-way ANOVA).
Fig. 1 shows that, as expected, tramadol induced a dose-related antinociceptive effect in the hot-plate test. The effect was evident starting from the dose of 20 mg/ kg, and the maximum attainable antinociceptive response was observed after the administration of 80 mg/kg. The peak of antinociceptive activity was obtained within 15 min. The effect of acute tramadol on splenocyte prolifer– ation induced by three different concentrations of the mitogen ConA is reported in Fig. 2. The drug induced a significant enhancement of splenocyte proliferation. This action of tramadol was evident starting at 10 mg/kg, and remained un-changed after the administration of higher doses. Also, splenic NK activity was significantly enhanced after acute administration of tramadol (Fig. 3). A significant stimulation of this immune parameter was already evident after the administration of 1 mg/kg of the drug. The immunostimulatory effect of tramadol was further confirmed by the observation that IL-2 production by splenocytes was increased after the administration of the drug; this effect was also already evident after the administration of 1 mg/kg (Fig. 4).
Fig. 4. Effect of acute tramadol on IL-2 production by ConA-stimulated splenocytes. Values are means ± SD. *P , 0.01 vs. saline group (0) (oneway ANOVA).
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4. Discussion
Fig. 5. Nociceptive thresholds measured before (A) and 30 min (g)after the injection of tramadol (20 mg/kg, s.c.), on the first day and after 14 days of drug administration (20 mg/kg s.c., twice daily). Values are means ± SEM. *P , 0.01 vs. saline.
In order to evaluate the development of tolerance to the analgesic or to the immune effects of tramadol, the nociceptive thresholds and the immune parameters were evaluated after chronic treatment. As shown in Fig. 5, no tolerance developed to the antinociceptive effect of tramadol. In fact, the analgesic response to the challenge with 20 mg/kg of tramadol was unchanged after 14 days of treatment. On the contrary, all the immune effects of tramadol disappeared after chronic treatment (Table 1). The loss of the stimulation of splenocyte proliferation, IL-2 production and NK activity was observed when immune function was evaluated either 1 h or 24 h after the last administration of the drug. In order to distinguish between immune tolerance to the drug versus a possible tolerance of the immune effects induced by mild stressors represented by handling procedures, the same parameters were measured in animals acutely treated with two doses of tramadol after 14 days of treatment with saline. As shown in Fig. 6, in these animals tramadol was still able to enhance ConA-induced proliferation (panel A), NK activity (panel B) and IL-2 production (panel C).
The present study describes, for the first time, some immunomodulatory properties of tramadol. When administered acutely, this drug enhances the responses of different lymphocyte populations. In fact, it significantly stimulates T cell functions, as indicated by the enhancement of ConAinduced proliferation and of ConA-induced production of IL-2; moreover, it augments the activity of NK cells, a population of lymphocytes which plays a significant role in tumor surveillance and in the defense against viral infections (Herberman and Hortaldo, 1981). The immune effects of tramadol are observed at extremely low doses; in fact, the effects on IL-2 production and NK activity are already evident at the dose of 1 mg/kg. However, the sensitivity to the drug of the immune parameters evaluated is different: whereas NK activity and IL-2 production are increased by the dose of 1 mg/kg of tramadol, the potentiation of ConAinduced proliferation is evident only after the administration of higher doses. This observation is not surprising, since a different sensitivity of NK cells and T lymphocytes to pharmacological treatments has been reported also for opiate induced immunosuppression (Lysle et al., 1992). Moreover, experimental models of stress have been shown to affect different immune cell populations depending on stress intensity, duration, frequency, etc. (Labeur et al., 1995). T lymphocyte proliferation is dependent on the IL-2 pathways; in fact, many reports have demonstrated the critical requirements of IL-2 production as well as of IL-2 receptor (IL-2R) induction for proliferation (Toribio et al., 1989). Thus an increase of IL-2R could also be necessary in order to measure an increase of T lymphocyte proliferation. It is therefore feasible to suppose that the induction of IL-2R is achieved only after the administration of 20 mg/kg of tramadol (when increased proliferation is also measurable). However, it is interesting to observe that the doses effective in the hot-plate test are always much higher. We observed, in fact, a significant enhancement of nociceptive thresholds only after the administration of the dose of 20 mg/kg. The immune actions of tramadol were unexpected since the activation of central m-opiate receptors has classically been associated with immunosuppression (Taub et al., 1991;
Table 1 Effects of chronic treatment (2 weeks) with tramadol (20 mg/kg) on different immune parameters
Splenocyte proliferation (cpm [3H]thymidine) ConA 2.5 mg/ml ConA 5.0 mg/ml ConA 10 mg /ml NK activity (% specific lysis) E:T 200:1 E:T 100:1 IL-2 production (units/ml) Values are mean ± SD.
Saline
Tramadol (last injection 24 h earlier)
Tramadol (last injection 1 h earlier)
55600 ± 16230 83050 ± 28100 61000 ± 21500
56890 ± 12563 87000 ± 30500 66450 ± 25750
53874 ± 19856 82500 ± 25200 59400 ± 18000
16.2 ± 7.2 15.4 ± 7.1 202.5 ± 57.7
17.8 ± 9 16.8 ± 5 227 ± 108
21.2 ± 9.6 19.4 ± 4.8 188.5 ± 59.68
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Fig. 6. Effects on immune parameters of tramadol (10 and 20 mg/kg s.c.) after 2 weeks of treatment with saline. Panel A: ConA-induced splenocyte proliferation; panel B: NK activity; panel C: IL-2 production. Values are means ± SD. *P , 0.01 vs. saline group (0).
Band et al., 1992). A wide literature has described the suppressive effects of morphine and endogenous opioids on many immune functions, including lymphocyte proliferation, IL-2 production and NK activity (Band et al., 1992; Manfredi et al., 1993; Peterson et al., 1993; Panerai et al., 1995). However, in very recent work, the opiate derived compound OHM3295 has been shown to potentiate splenic NK activity in vivo throughout the activation of m- and kopioid receptors (Baker et al., 1995). Therefore, we cannot rule out the possibility that the enhancement of immune function induced by tramadol could be mediated via opiate receptors. Further experiments in order to elucidate this point are particularly difficult since, in our hands, classical opioid antagonists such as naloxone and naltrexone exert immunostimulatory activity by themselves (Manfredi et al., 1993). It is well known that the antinociceptive effects of tramadol are mediated not only via an opioid mechanism, but also via a separate, non-opioid, mechanism, due to the inhibition of neuronal uptake of noradrenaline and serotonin (Kayser et al., 1992; Raffa et al., 1992). It is therefore also possible that the immune effects of this drug on the immune system could be mediated by the activation of the monoaminergic pathways. The involvement of catecholamine and serotonin systems in neural-immune interactions has been studied using different experimental models. Both enhancement and reduction of immune response have been related to the activation of noradrenergic system, providing evidence for the heterogeneity of sympathetic nervous system regulation of lymphocyte function (Livnat et al., 1985; Madden et al., 1994). On the other hand, the increase of serotoninergic tone has been frequently associated with stimulation of NK activity and lymphocyte proliferation (Steplewsky and Vogel, 1985; Gabrilovac et al., 1992). Similarly, drugs which increase serotoninergic tone such as d-fenfluoramine (Petrovic et al., 1990) and fluoxetine (Sacerdote, unpublished results), stimulate immune function in rodents.
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Therefore, the activation of the serotoninergic system might be involved in the immune effects induced by the acute administration of tramadol. Consistent with the results obtained by other authors (Dayer et al., 1994), we did not observe any tolerance to the antinociceptive activity of tramadol. On the contrary, after 14 days of treatment, the immune effects of the drug were lost. This observation suggests that a dissociation exists between the mechanism of action of tramadol on nociceptive thresholds and on immune responses. At present, we do not have an explanation for the loss of the immunological effects induced by tramadol after chronic administration. However, it is important to note that an adaptation of the immune system has been reported to the effects of pharmacological (Bryant et al., 1988) as well as physiological, e.g., stress (Khansari et al., 1990; Sacerdote et al., 1994) stimuli. Tolerance also develops to the immunosuppressive effects of morphine (Bryant et al., 1988). Some types of mild stressors, such as handling and novelty, have been shown to enhance some immune responses (Wood et al., 1993; Dhabhar and McEwen, 1996).However, since we observed that the immune responses were also increased by acute injections of tramadol after 2 weeks of treatment with saline, during which potential tolerance to stress should have developed, we can exclude that we were actually observing the development of tolerance to these stressors rather than to the drug administration. The clinical value of our observations might be relevant. Indeed, the use of classical narcotic drugs with immunosuppressive effects is contraindicated in many situations where the immune system is compromised, such as in cancer patients (Provinciali et al., 1991) or in the postoperative period (Hammer et al., 1992; Saito et al., 1992). In this respect, the availability of a drug, like tramadol, which lacks immunosuppressive side-effects may represent a favorable therapeutic alternative to opiates for the treatment of acute pain.
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