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The interaction between IL-1β and morphine: possible mechanism of the deficiency of morphine-induced analgesia in diabetic mice
Pain 89 (2000) 39±45
www.elsevier.nl/locate/pain
The interaction between IL-1b and morphine: possible mechanism of the de®ciency of morphine-induced analgesia in diabetic mice Husamettin Gul, Oguzhan Yildiz*, Ahmet Dogrul, Ozgur Yesilyurt, Askin Isimer Department of Pharmacology, School of Medicine, Gulhane Military Medical Academy, 06018, Etlik, Ankara, Turkey Received 23 February 2000; received in revised form 1 May 2000; accepted 12 May 2000
Abstract It is known that diabetic mice are less sensitive to the analgesic effect of morphine. Some factor(s) derived from mononuclear cells, e.g. interleukin-1b (IL-1b), may be responsible for the diminished analgesic effect of morphine in diabetic mice. Therefore, we examined direct effects of IL-1b, intracerebroventricularly (i.c.v.), on morphine-induced analgesia, subcutaneously (s.c.), in diabetic and control mice by using the tail-¯ick test. Morphine at doses of 1, 2 and 5 mg/kg (s.c.) produced dose-dependent analgesia in diabetic and control mice but diabetic mice were less sensitive to the analgesic effect of morphine when compared to the controls. IL-1b at a dose of 0.1 ng/mouse produced analgesia in control mice but not in diabetics, whereas IL-1b at a dose of 10 ng/mouse produced a hyperalgesic effect both in diabetic and control mice. IL-1b at a dose of 1 ng/mouse has neither an analgesic nor a hyperalgesic effect in control and diabetic mice. Administration of a neutral (neither analgesic nor hyperalgesic) dose of IL-1b, 1 ng/mouse (i.c.v.), just prior to administration of morphine (s.c.) abolished the analgesic effect of morphine at doses of 1, 2 and 5 mg/kg in control mice and the analgesic effect of morphine became similar to that in diabetics. The diminished analgesic effect of morphine in diabetes was attenuated further with IL-1b at a dose of 1 ng/mouse (i.c.v.). These results suggest that the decreased analgesic effect of morphine in diabetes may be related to IL-1b. q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Interleukin-1 beta; Morphine; Streptozotocin; Diabetes; Mice
1. Introduction It is well established that animals with diabetes are significantly less sensitive than controls to the analgesic effect of morphine (Simon and Dewey, 1981; Raz et al., 1988; Kamei et al., 1992a). In humans, clinical studies show that morphine also has a limited analgesic effect in the treatment of painful diabetic neuropathy (James and Page, 1994; Galer, 1995). Morphine produces an analgesic effect mainly by m-opioid receptors; however, it has also a moderate af®nity for d- and k-opioid receptors (Emmerson et al., 1994). Kamei et al. (1992a) reported that mice with STZ-induced diabetes are selectively hyporesponsive to supraspinal mopioid receptor-mediated antinociception, but they respond normally to the activation of d- and k-opioid receptors. Although the exact mechanism is unknown, some studies suggest that certain factor(s) derived from spleen mononuclear cells may play an important role, directly or indirectly, * Corresponding author. Tel.: 190-312-3044766; fax: 190-3123234923. E-mail address: [email protected] (O. Yildiz).
in the selective reduction of m-agonist-mediated analgesia in experimental diabetes (Kamei et al., 1992b). There is a signi®cant increase in cytokine production in diabetes and certain cytokines, e.g. interleukin-1 (IL-1), may participate in the pathological events in diabetes (Ciampolillo et al., 1993; Espersen et al., 1993; Hussain et al., 1996). IL-1 plays important roles in the immune and in¯ammatory systems and it is involved in the local in¯ammatory and immune-mediated diseases which are associated with local pain (Dinarello, 1991). IL-1 when given intravenously produces hyperalgesia locally, as assessed by paw pressure test in rats (Ferreira et al., 1988; Follenfant et al., 1989), and enhances the acetylcholine-induced pain re¯ex in the isolated rabbit ear preparation (Schweizer et al., 1988). Blood-borne IL-1 may enter the brain comparably to other water-soluble compounds, such as morphine, known to cross the blood±brain barrier in suf®cient amounts to affect brain function (Banks et al., 1995). IL-1 is also produced in the brain and it plays a role as a neuromodulator in the brain (Katsuki et al., 1990; Minami et al., 1990, 1991; Yamaguchi et al., 1991). Brain-derived IL-1 produces a
0304-3959/00/$20.00 q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(00)00343-2
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H. Gul et al. / Pain 89 (2000) 39±45
variety of central nervous system-mediated responses, such as an increase in body temperature (Kluger, 1991), inducing slow-wave sleep (Krueger et al., 1984), anorexia (Mrosovsky et al., 1989), activating the hypothalamus-pituitary-adrenal axis (Berkenbosch et al., 1987) and attenuating neuronal activity in the hippocampus (Plata-Salaman and Ffrench-Mullen, 1992) and amygdala (Yu and ShinnickGallagher, 1994). The effects of centrally administered IL-1 on nociception have also been examined. Nakamura et al. (1988) demonstrated that intracisternal administration of IL-1a produces an analgesic effect, such as phenylquinolone-induced writhing (chemical stimulus) behavior in mice. In contrast, Oka et al. (1993) showed by using the hot plate method (thermal stimulus) that intracerebroventricular (i.c.v.) administration of IL-1b caused hyperalgesia in the rat. In a recent study, Yabuuchi et al. (1996) reported that IL-1 produced dose-dependent biphasic effects, i.e. hyperalgesia in low doses and analgesia in high doses, through the IL-1 receptor in the brain of rats with the paw pressure test (mechanical stimulus). Although these studies (using either thermal, chemical or mechanical stimuli) suggest that IL-1 has modulatory effects on nociceptive transmission, the direction of the effects, towards analgesia or hyperalgesia, is controversial. Previously, Kamei et al. (1992b) suggested that some factor(s) derived from spleen mononuclear cells may play a role in the reduction of the analgesic effect of morphine in diabetic mice. Raffa et al. (1993) postulated that cytokines may interact with opioid receptors and reduce the analgesic effect of morphine. However, to our knowledge, there is no study identifying the factor(s) which reduce the analgesic effect of morphine in diabetes. In this study, we characterized the analgesic and hyperalgesic effects of various doses of IL-1b and we aimed to analyze the effects of IL-1b on morphine-induced analgesia in STZ-diabetic and control mice by using the tail-¯ick test. 2. Materials and methods 2.1. Animals Adult male Balb/c mice (Breeding Colony of Research Center, Gulhane Military Medical School) weighing 25±30 g were used in the study. Animals were housed on a 12 h light/dark cycle (lights on from 08:00 h) at a constant ambient temperature (24 ^ 1)8C with normal mice chow and water available ad libitum during the study. Mice were handled in accordance with guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals (Zimmermann, 1983). 2.2. Induction of diabetes Diabetes was induced by a single intraperitoneal (i.p.)
injection of streptozotocin (STZ) (170 mg/kg body weight) after an overnight fast. STZ was dissolved in 3 mM citric acid buffer (pH 4.5) immediately before injection. Agematched non-diabetic mice received similar volumes of vehicle alone by the same route. Mice with serum glucose levels above 400 mg/dl were considered diabetic. 2.3. Drugs Mouse IL-1b and streptozotocin were purchased from Sigma (Deisenhofen, Germany). Morphine hydrochloride was purchased from Sandoz Chemical Co. (Sandoz, Switzerland). 2.4. Drug administration IL-1b was dissolved in phosphate-buffered saline (PBS, pH 7.4) containing 0.1% human serum albumin and it was administered i.c.v. as 5 ml/mouse (according to the method of Haley and McCormick, 1957). Morphine hydrochloride was dissolved in saline and administered subcutaneously (s.c.). 2.5. Measuring the tail-¯ick latency and MPE The analgesic response was evaluated by the tail-¯ick test. All animals were restrained for 30 min for adaptation and they were in restrainers during the tail-¯ick measurements. The intensity of a heat lamp was set to provide a predrug (baseline) latency time in the tail-¯ick response of 3±5 s. The intensity of the heat lamp was the same for all measurements in control and diabetic mice throughout the study. A cut-off time of 12 s was used. The percentage of the maximum possible effect (%MPE) was calculated for each animal using the following formula (Dewey et al., 1970): %MPE 100 £
test latency 2 pre-drug latency 12 2 pre-drug latency
The analgesic responses were measured 2 weeks after induction of diabetes in both diabetic and non-diabetic control mice. Mice were treated with either morphine hydrochloride (1, 2, and 5 mg/kg body weight, s.c.) and IL-1b (0.1, 1 and 10 ng/mouse, i.c.v.) alone or with a combination of morphine hydrochloride (1, 2, and 5 mg/kg body weight, s.c.) and IL-1b (1 ng/mouse, i.c.v., as a non-analgesic and non-hyperalgesic dose). In a group of mice we measured the effect of vehicle of IL-1b (i.c.v.) (PBS, pH 7.4, containing 0.1% human serum albumin) alone and in combination with morphine (5 mg/kg, s.c.) in the tail-¯ick test. Each mouse was used once for different doses of IL-1b, morphine or for the combination of both. Tail-¯ick latencies were measured just before and 30, 60 and 90 min after the administration of the drugs. To test the possible pyrogenic effect of IL-1b, rectal temperature was measured in a separate group of mice by using a pyrogen test instrument (Ellab, Copenhagen, Denmark) before and 30, 60, 120 and 180 min
H. Gul et al. / Pain 89 (2000) 39±45
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after the administration of IL-1b. All of the studies were done between 09:00 h and 13:00 h. 2.6. Statistical analysis Results were presented as the mean ^ SEM of the %MPE. For statistical comparisons among the groups, one-way analysis of variance (ANOVA) was performed followed by Duncan's multiple range test. A paired or unpaired Student's t-test was used to compare the values from two groups when necessary. P , 0:05 was considered signi®cant. 3. Results 3.1. Effects of morphine on the tail-¯ick test in control and diabetic mice Subcutaneous administration of morphine (1, 2 and 5 mg/ kg) induced a dose-dependent analgesic effect both in control and diabetic mice (Fig. 1). While baseline tail¯ick latencies were not different between control (4.22 ^ 0.24 s, n 76) and diabetic (4.02 ^ 0.2 s, n 65) mice, morphine-produced analgesia in control mice was higher than in diabetic mice at 30 min at a dose of 1 mg/kg (P , 0:001), at 30 and 60 min at a dose of 2 mg/ kg (P , 0:01 and P , 0:05, respectively), and at 30, 60 and 90 min at a dose of 5 mg/kg (P , 0:001) (Fig. 1).
Fig. 1. Effect of morphine (1, 2 and 5 mg/kg, s.c.) on the tail-¯ick test both in control and diabetic mice. Results are presented as the mean ^ SEM of the %MPE. *P , 0:001, versus diabetic 1 mg/kg morphine (at 30 min; Student's t-test); **P , 0:01, versus diabetic 2 mg/kg (at 30 min; Student's t-test); ***P , 0:05, versus diabetic 2 mg/kg (at 60 min; Student's t-test); # P , 0:001, versus diabetic 5 mg/kg (at 30, 60 and 90 min; Student's t-test).
Fig. 2. Effect of i.c.v. injection of IL-1b (0.1, 1 and 10 ng) on the tail-¯ick test both in control and diabetic mice. Results are presented as the mean ^ SEM of the %MPE. *P , 0:05, versus control 1 IL-1b (10 ng/mouse, i.c.v.; at 30 min; ANOVA, post hoc Duncan's multiple range test); **P , 0:05, versus diabetic 1 IL-1b (0.1 ng/mouse, i.c.v.; at 60 and 90 min; Student's t-test); ***P , 0:01, versus diabetic 1 IL-1b (10 ng/mouse, i.c.v.; at 60 min; ANOVA, post hoc Duncan's multiple range test); # P , 0:05, versus control 1 IL-1b (1, 10 ng/mouse, i.c.v.; at 60 and 90 min; ANOVA, post hoc Duncan's multiple range test).
3.2. Effects of IL-1b on the tail-¯ick test in control and diabetic mice We examined the effects of i.c.v. injection of IL-1b alone with the tail-¯ick test. IL-1b produced an analgesic effect at a dose of 0.1 ng/mouse in control mice but not in diabetics (Fig. 2). The analgesic effect of IL-1b at a dose of 0.1 ng/ mouse was markedly higher than the effect of IL-1b at a dose of 1 ng/mouse in control mice at 60 and 90 min (P , 0:05). IL-1b at a dose of 1 ng/mouse produced neither an analgesic nor a hyperalgesic effect in control and diabetic mice (Fig. 2). IL-1b produced a hyperalgesic effect at a dose of 10 ng/mouse in control and diabetic mice (Fig. 2). In control mice, the hyperalgesic effect of high dose of IL1b (10 ng/mouse) was signi®cantly different when compared to its effect at a dose of 0.1 ng/mouse at 30, 60 and 90 min and it was also different than the effect of 1 ng/ mouse at 30 min (P , 0:05, respectively). In diabetic mice, the hyperalgesic effect of a high dose of IL-1b (10 ng/ mouse) was signi®cantly different at 30 min when compared to the effect of 0.1 and 1 ng/mouse IL-1b. Although the analgesic effect of a low dose (0.1 ng/mouse) of IL-1b continued at 120 and 180 min (%MPE 14.8 ^ 6 and 11.7 ^ 4, respectively, not shown in Fig. 2), the hyperalgesic effect of a high dose (10 ng/mouse) of IL-1b was reduced at 120 and 180 min (%MPE 24.1 ^ 2.8 and 25.3 ^ 2.5, respectively, not shown in Fig. 2) in control
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H. Gul et al. / Pain 89 (2000) 39±45
mice. In contrast to control mice, the hyperalgesic effect of a high dose of IL-1b (10 ng) was reversed to analgesia at 120 and 180 min in diabetic mice (%MPE 2.3 ^ 3.4 and 5.7 ^ 4.9, respectively, not shown in Fig. 2). 3.3. Effects of IL-1b in combination with morphine on the tail-¯ick test in control and diabetic mice The vehicle of IL-1b (PBS 1 0.1% human serum albumin) did not produce any analgesic or hyperalgesic effects alone and it also had no effect on morphine (5 mg/kg)induced analgesia both in control and diabetic mice (Table 1). The neutral dose of IL-1b (1 ng/mouse, i.c.v.) blocked the analgesic effect of 1 mg/kg morphine and it induced hyperalgesia (P , 0:001 at 30 and 60 min, P , 0:01 at 90 min). IL-1b (1 ng/mouse, i.c.v.) reduced the analgesic effect of morphine at a dose of 2 mg/kg at 30 and 60 min (P , 0:001 and P , 0:01, respectively) and it also reduced the analgesic effect of morphine at a dose of 5 mg/kg at 30, 60 and 90 min (P , 0:001) in control mice (Fig. 3). In diabetic mice, IL-1b (1 ng/mouse, i.c.v.) blocked the analgesic effect of morphine at a dose of 1 mg/kg, and it induced signi®cant hyperalgesia at 60 min only (P , 0:001). IL-1b blocked the analgesic effect of morphine at a dose of 2 mg/kg and it induced hyperalgesia at 30, 60 (P , 0:001) and 90 min (P , 0:01). In addition, IL-1b (1 ng/mouse) signi®cantly reduced the analgesic effect of morphine at a dose of 5 mg/kg at 30 min (P , 0:05) (Fig. 3). 3.4. Effect of IL-1b on rectal temperature IL-1b at doses of 0.1, 1 and 10 ng/mouse (i.c.v.) and its vehicle did not signi®cantly change the rectal temperature at 30, 60, 120 and 180 min after the injections (Table 2). 4. Discussion In the present study, the diabetic mice were signi®cantly less sensitive to the analgesic effect of morphine than nondiabetic mice and this ®nding is consistent with previous studies (Simon and Dewey, 1981; Raz et al., 1988; Kamei et al., 1992a). The major ®nding of our study is that a
Fig. 3. Effect of IL-1b (0.1, 1 and 10 ng/mouse, i.c.v.) on morphine-induced analgesia in control (upper) and diabetic (lower) mice. Results are presented as the mean ^ SEM of the %MPE. Student's t-test was used for statistical comparisons. (Upper) *P , 0:001, versus control 1 morphine (1 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 30 and 60 min); **P , 0:001, control 1 morphine (1 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 90 min); ***P , 0:001, versus control 1 morphine (2 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 30 min); #P , 0:01, versus control 1 morphine (2 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 60 min); ##P , 0:001, control 1 morphine (5 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 30, 60 and 90 min). (Lower) *P , 0:001, versus diabetic 1 morphine (1 mg/kg) 1 IL-1b (1 ng/ mouse, i.c.v.; at 60 min); **P , 0:001, versus diabetic 1 morphine (2 mg/ kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 30 and 60 min); ***P , 0:01, versus diabetic 1 morphine (2 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 90 min); #P , 0:05, versus diabetic 1 morphine (5 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 60 min).
neutral (neither analgesic nor hyperalgesic) dose of IL-1b (1 ng/mouse, i.c.v.) abolished the analgesic effect of morphine in control mice and the analgesic effect of
Table 1 Effect of vehicle of IL-1b (PBS 1 0.1% human serum albumin) alone (intracerebroventricularly) and on morphine-induced (subcutaneously) analgesia a Groups (n)
Control vehicle (6) Diabetic vehicle (9) Control morphine 5 mg/kg (10) Control morphine 5 mg/kg 1 vehicle i.c.v. (6) Diabetic morphine 5 mg/kg (7) Diabetic morphine 5 mg/kg 1 vehicle (6) a
Results are presented as the mean ^ SEM %MPE.
%MPE (min) 30
60
90
2.9 ^ 1.83 1.26 ^ 3.63 82.8 ^ 9.5 90.36 ^ 6.16 26.4 ^ 8.9 34.42 ^ 2.47
20.4 ^ 1.87 24.78 ^ 3.74 90.4 ^ 5.1 96.77 ^ 3.23 17.4 ^ 2.8 17.68 ^ 1.37
0.3 ^ 1.83 22.96 ^ 3.1 84.7 ^ 11.2 95.25 ^ 3.04 15.3 ^ 2.5 11.94 ^ 2.72
H. Gul et al. / Pain 89 (2000) 39±45
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Table 2 Effect of IL-1b and its vehicle (PBS 1 0.1% human serum albumin) on rectal temperature of mice (n 5 for all groups) a IL-1b (i.c.v.)
0.1 ng 1 ng 10 ng Vehicle a
Before injection
38.78 ^ 0.16 37.86 ^ 0.19 37.92 ^ 0.17 37.66 ^ 0.14
After injection (min) 30
60
120
180
38.12 ^ 0.19 38.32 ^ 0.32 37.80 ^ 0.29 37.72 ^ 0.19
37.94 ^ 0.15 37.96 ^ 0.25 37.52 ^ 0.34 37.76 ^ 0.13
37.80 ^ 0.23 38.24 ^ 0.29 37.66 ^ 0.35 37.92 ^ 0.26
37.96 ^ 0.05 37.64 ^ 0.19 37.30 ^ 0.16 37.86 ^ 0.24
Results are presented as the mean ^ SEM temperature (8C).
morphine became similar to that in diabetics. We also found that the diminished analgesic effect of morphine in diabetes was attenuated further with IL-1b at a dose of 1 ng/mouse (i.c.v.). Previously, Mathiasen et al. (1987) reported that beige-J mice, a type of mutant mice with immunological de®ciencies, had more resistance to the analgesic effect of morphine and other m-opioid receptor agonists than control mice. Kimball and Raffa (1989) showed that the transfer of mononuclear spleen cells obtained from beige-J mice to normal littermates resulted in a signi®cant reduction of the analgesic effect of morphine similar to that in beige-J mice. Monocytes from beige-J mice were found to have a higher basal activity and were able to secrete considerably higher levels of IL-1 than were their littermates (Raffa et al., 1993). Interestingly, Kamei et al. (1992b) demonstrated that the defect of the analgesic effect to m-opioid receptor agonist in diabetic mice was recovered by splenectomy. They speculated that this recovery of analgesic effect was due to depression of some endogen agents derived from spleen rather than the genetic de®ciency of m-opioid receptor synthesis. Kamei et al. (1992b) suggested that some factor(s) derived from spleen mononuclear cells might play an important direct or indirect role in the selective reduction in m-agonist-mediated analgesia in diabetic mice. Interestingly, Ciampolillo et al. (1993) and Espersen et al. (1993) reported that IL-1b was increased at the onset of insulin-dependent diabetes mellitus (IDDM) in humans. On the contrary, Luger et al. (1988), Mooradian et al. (1991), and Ohno et al. (1993) reported that there was a decrease in the secretion of IL-1b from stimulated monocytes in human subjects with IDDM. In addition, Netea et al. (1997) reported that ex vivo lipopolisaccharide-stimulated IL-1b production in newly diagnosed IDDM patients was signi®cantly increased compared to long-term IDDM patients and healthy controls. They also demonstrated that the ratio of IL-1 receptor antagonist/interleukin-1 (IL-1ra/ IL-1) was signi®cantly decreased in newly diagnosed IDDM patients and that the circulating level of IL-1ra was increased in long-term IDDM patients. IL-1b and IL-1ra may cross the blood±brain barrier (Banks et al., 1995) and may affect the antinociceptive action of morphine. Our study is the ®rst to demonstrate that IL-1b may be the factor which may reduce morphine-induced analgesia in diabetes.
The mechanism of diminishing the analgesic effect of morphine by IL-1b is not clear. It was reported that there were no differences in m-opioid receptor binding sites between spontaneously- or streptozotocin-diabetic and corresponding control mice (Brase et al., 1987). Morphine produces an analgesic effect mainly by m-opioid receptors and therefore the differences in the analgesic effect of morphine between diabetic and control mice are not attributable to density, size and distribution of m-opioid receptors. However, it is possible that a m-opioid receptor± effector coupling mechanism may be changed in diabetes and this change would result in the reduction of the analgesic effect of morphine in diabetic mice. We investigated the acute effects of IL-1b on morphine-induced analgesia; therefore, it was not possible that IL-1b could change the opioid receptor density. We think that IL-1b may reduce the analgesic effect of morphine by changing its receptor±effector coupling mechanism. However, further investigation is needed to clarify this point. IL-1b may produce fever (Busbridge et al., 1989; Dascombe et al., 1989) and decreased vigilance (Opp et al., 1991) which may in¯uence the results of behavioral testing for nociception. In the present study, IL-1b had no effect on rectal temperature in mice at the doses used. Recently, Fox et al. (1999) reported a critical evaluation of a STZ model of painful diabetic neuropathy in the rat. They found that STZ-induced diabetes in rats produced long-term resting mechanical, but not thermal hyperalgesia. They suggested that a discrepancy between the presence of mechanical and thermal hyperalgesia was important and speci®c for diabetic rats. They hypothesized that discomfort and also handling of animals with general ill health might contribute to false positive readings in the mechanical hyperalgesia paw pressure test. In the present study, we observed no difference in the thermal nociception threshold between diabetic and control mice. While this observation is in agreement with some previous studies (Raz et al., 1988; Fox et al., 1999), some authors reported a thermal hyperalgesia or hypoalgesia (Forman et al., 1986; Lee and McCarty, 1992). We used the tail-¯ick test to evaluate thermal nociception. We employed 30 min of restraining time and thus performed the experiments with minimal handling. We demonstrated that IL-1b has biphasic effects in mice with the tail-¯ick test. IL-1b produced analgesia at the low
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H. Gul et al. / Pain 89 (2000) 39±45
dose (0.1 ng/mouse, i.c.v.) but it produced hyperalgesia at the high dose (10 ng/mouse, i.c.v.). IL-1b did not produce any analgesic or hyperalgesic effect at 1 ng/mouse (i.c.v.). There are a number of con¯icting data about the antinociceptive effect of IL-1b in the literature. While Nakamura et al. (1988) demonstrated that i.c.v. administration of IL-1b had an analgesic effect in the phenylquinolone-induced writhing test, Oka et al. (1993) discovered that i.c.v. administration of IL-1b had a hyperalgesic effect in the hot plate test. In contrast to our ®ndings, Yabuuchi et al. (1996) reported that IL-1b produced biphasic dose-dependent effects (hyperalgesia with low doses and analgesia with high doses) in the paw pressure test. These con¯icting results may be due to differences in experimental conditions, such as methods of testing nociception, animal species or the doses of IL-1. None of these studies used the tail-¯ick test in mice to test the antinociceptive effect of IL-1b (i.c.v.). Previously, Yabuuchi et al. (1996) demonstrated that the hyperalgesic effect of lower doses of IL-1b on mechanical nociception was mediated by a cyclooxygenase pathway. Na-salicylate, a cyclooxygenase inhibitor, caused the lower doses of IL-1b to induce an analgesic rather than a hyperalgesic effect. The reason for this inversion is still unclear. In addition, they also found that the analgesic effect of higher doses of IL-1b was blocked by a-helical CRF, the antagonist of CRF. In view of these ®ndings and the present data, further investigations are needed to analyze the mechanisms of the biphasic effect of IL-1b. In conclusion, IL-1b has biphasic effects, analgesia at the low dose and hyperalgesia at the high dose, in thermal nociception in control mice. Streptozotocin-diabetic mice are signi®cantly less sensitive than controls to the analgesic effect of morphine and IL-1b may be related to this diminished analgesic effect. However, further investigations are needed to clarify the mechanism of interaction between IL1b and morphine. Acknowledgements We thank Novartis for ®nancial support and Ali Gursoy Erturk of Novartis for his help throughout the study. We also thank Dr Zafer Goren for his helpful comments on methods. References Banks WA, Kastin AJ, Broadwell RD. Passage of cytokines across the blood-brain barrier. Neuroimmunomodulation 1995;2:241±248. Berkenbosch F, van Oers J, del Rey A, Tilders F, Besedovsky H. Corticotropin-releasing factor-producing neurons in the rat activated by interleukin-1. Science 1987;238:524±526. Brase DA, Han YH, Dewey WL. Effects of glucose and diabetes on binding of naloxone and dihydromorphine to opiate receptors in mouse brain. Diabetes 1987;36:1173±1177. Busbridge NJ, Dascombe MJ, Tilders FJ, van Oers JW, Linton EA,
Rothwell NJ. Central activation of thermogenesis and fever by interleukin-1 beta and interleukin-1 alpha involves different mechanisms. Biochem Biophys Res Commun 1989;162:591±596. Ciampolillo A, Guastamacchia E, Caragiulo L, Lollino G, De Robertis O, Lattanzi V, Giorgino R. In vitro secretion of interleukin-1 beta and interferon-gamma by peripheral blood lymphomononuclear cells in diabetic patients. Diabetes Res Clin Pract 1993;21:87±93. Dascombe MJ, Rothwell NJ, Sagay BO, Stock MJ. Pyrogenic and thermogenic effects of interleukin-1 beta in the rat. Am J Physiol 1989;256:E7±E11. Dewey WL, Harris LS, Howes JF, Nuite JA. The effect of various neurohumoral modulators on the activity of morphine and the narcotic antagonists in the tail-¯ick and phenylquinone tests. J Pharmacol Exp Ther 1970;175:435±442. Dinarello CA. Interleukin-1 and interleukin-1 antagonism. Blood 1991;77:1627±1652. Emmerson PJ, Liu MR, Woods JH, Medzihradssky F. Binding af®nity and selectivity of opioids at mu, delta, and kappa receptors in monkey brain membranes. J Pharmacol Exp Ther 1994;271:1630±1637. Espersen GT, Mathiesen O, Grunnet N, Jensen S, Ditzel J. Cytokine plasma levels and lymphocyte subsets in patients with newly diagnosed insulindependent (type 1) diabetes mellitus before and following initial insulin treatment. APMIS 1993;101:703±706. Ferreira SH, Lorenzetti BB, Bristow AF, Poole S. Interleukin-1 beta as a potent hyperalgesic agent antagonized by a tripeptide analogue. Nature 1988;334:698±700. Follenfant RL, Nakamura-Craig M, Henderson B, Higgs GA. Inhibition by neuropeptides of interleukin-1 beta-induced, prostaglandin-independent hyperalgesia. Br J Pharmacol 1989;98:41±43. Forman LJ, Estilow S, Lewis M, Vasilenko P. Streptozotocin diabetes alters immunoreactive beta-endorphin levels and pain perception after 8 weeks in female rats. Diabetes 1986;35:1309±1313. Fox A, Eastwood C, Gentry C, Manning D, Urban L. Critical evaluation of the streptozotocin model of painful diabetic neuropathy in the rat. Pain 1999;81:307±316. Galer BS. Neuropathic pain of peripheral origin: advances in pharmacologic treatment. Neurology 1995;45:S17±S25. Haley TJ, McCormick WG. Pharmacological effects produced by intracerebral injections of drugs in the conscious mouse. Br J Pharmacol 1957;12:12±15. Hussain MJ, Peakman M, Gallati H, Lo SS, Hawa M, Viberti GC, Watkins PJ, Leslie RD, Vergani D. Elevated serum levels of macrophagederived cytokines precede and accompany the onset of IDDM. Diabetologia 1996;39:60±69. James JS, Page JC. Painful diabetic peripheral neuropathy. A stepwise approach to treatment. J Am Pediatr Med Assoc 1994;84:439±447. Kamei J, Ohhashi Y, Aoki T, Kawasima N, Kasuya Y. Streptozotocininduced diabetes selectively alters the potency of analgesia produced by mu-opioid agonists, but not by delta- and kappa-opioid agonists. Brain Res 1992a;571:199±203. Kamei J, Kawashima N, Kasuya Y. Role of spleen or spleen products in the de®ciency in morphine-induced analgesia in diabetic mice. Brain Res 1992b;576:139±142. Katsuki H, Nakai S, Hirai Y, Akaji K, Kiso Y, Satoh M. Interleukin-1 beta inhibits long-term potentiation in the CA3 region of mouse hippocampal slices. Eur J Pharmacol 1990;181:323±326. Kimball ES, Raffa RB. Obligatory role of B cells and adherent accessory cells in the transfer of a defect in morphine-mediated antinociception in C57BL/6J-bg/bg (beige-J) mice. J Neuroimmunol 1989;22:185± 192. Kluger MJ. Fever: role of pyrogens and cryogens. Physiol Rev 1991;71:93± 127. Krueger JM, Walter J, Dinarello CA, Wolff SM, Chedid L. Sleep-promoting effects of endogenous pyrogen (interleukin-1). Am J Physiol 1984;246:R994±R999. Lee JH, McCarty R. Pain threshold in diabetic rats: effects of good versus poor diabetic control. Pain 1992;50:231±236.
H. Gul et al. / Pain 89 (2000) 39±45 Luger A, Schernthaner G, Urbanski A, Luger TA. Cytokine production in patients with newly diagnosed insulin-dependent (type I) diabetes mellitus. Eur J Clin Invest 1988;18:233±236. Mathiasen JR, Raffa RB, Vaught JL. C57BL/6J-bgj (beige) mice: differential sensitivity in the tail ¯ick test to centrally administered mu- and delta-opioid receptor agonists. Life Sci 1987;40:1989±1994. Minami M, Kuraishi Y, Yamaguchi T, Nakai S, Hirai Y, Satoh M. Convulsants induce interleukin-1 beta messenger RNA in rat brain. Biochem Biophys Res Commun 1990;171:832±837. Minami M, Kuraishi Y, Yamaguchi T, Nakai S, Hirai Y, Satoh M. Immobilization stress induces interleukin-1 beta mRNA in the rat hypothalamus. Neurosci Lett 1991;123:254±256. Mooradian AD, Reed RL, Meredith KE, Scuderi P. Serum levels of tumor necrosis factor and IL-1 alpha and IL-1 beta in diabetic patients. Diabetes Care 1991;14:63±65. Mrosovsky N, Molony LA, Conn CA, Kluger MJ. Anorexic effects of interleukin-1 in the rat. Am J Physiol 1989;257:R1315±R1321. Nakamura H, Nakanishi K, Kita A, Kadokawa T. Interleukin-1 induces analgesia in mice by a central action. Eur J Pharmacol 1988;149:49±54. Netea MG, Hancu N, Blok WL, Grigorescu-Sido P, Popa L, Popa V, van der Meer JW. Interleukin 1 beta, tumour necrosis factor-alpha and interleukin 1 receptor antagonist in newly diagnosed insulin-dependent diabetes mellitus: comparison to long-standing diabetes and healthy individuals. Cytokine 1997;9:284±287. Ohno Y, Aoki N, Nishimura A. In vitro production of interleukin-1, interleukin-6, and tumor necrosis factor-alpha in insulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1993;77:1072±1077. Oka T, Aou S, Hori T. Intracerebroventricular injection of interleukin-1 beta induces hyperalgesia in rats. Brain Res 1993;624:61±68.
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Opp MR, Obal Jr F, Krueger JM. Interleukin 1 alters rat sleep: temporal and dose related effects. Am J Physiol 1991;260:R52±R58. Plata-Salaman CR, Ffrench-Mullen JM. Interleukin-1 beta depresses calcium currents in CA1 hippocampal neurons at pathophysiological concentrations. Brain Res Bull 1992;29:221±223. Raffa RB, Mathiasen JR, Kimball ES, Vaught JL. The combined immunological and antinociceptive defects of beige-j mice: the possible existence of a `m-repression'. Life Sci 1993;52:1±8. Raz I, Hasdai D, Seltzer Z, Melmed RN. Effect of hyperglycemia on pain perception and on ef®cacy of morphine analgesia in rats. Diabetes 1988;37:1253±1259. Schweizer A, Feige U, Fontana A, Muller K, Dinarello CA. Interleukin-1 enhances pain re¯exes. Mediation through increased prostaglandin E2 levels. Agents Actions 1988;25:246±251. Simon GS, Dewey WL. Narcotics and diabetes. I. The effects of streptozotocin-induced diabetes on the antinociceptive potency of morphine. J Pharmacol Exp Ther 1981;218:318±323. Yabuuchi K, Nishiyori A, Minami M, Satoh M. Biphasic effects of intracerebroventricular interleukin-1 beta on mechanical nociception in the rat. Eur J Pharmacol 1996;300:59±65. Yamaguchi T, Kuraishi Y, Minami M, Yabuuchi K, Satoh M. Involvement of central beta-adrenoceptors in the induction of hypothalamic interleukin-1 beta mRNA by methamphetamine. Neurosci Res 1991;12:432±439. Yu B, Shinnick-Gallagher P. Interleukin-1 beta inhibits synaptic transmission and induces membrane hyperpolarization in amygdala neurons. J Pharmacol Exp Ther 1994;271:590±600. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109±110.
www.elsevier.nl/locate/pain
The interaction between IL-1b and morphine: possible mechanism of the de®ciency of morphine-induced analgesia in diabetic mice Husamettin Gul, Oguzhan Yildiz*, Ahmet Dogrul, Ozgur Yesilyurt, Askin Isimer Department of Pharmacology, School of Medicine, Gulhane Military Medical Academy, 06018, Etlik, Ankara, Turkey Received 23 February 2000; received in revised form 1 May 2000; accepted 12 May 2000
Abstract It is known that diabetic mice are less sensitive to the analgesic effect of morphine. Some factor(s) derived from mononuclear cells, e.g. interleukin-1b (IL-1b), may be responsible for the diminished analgesic effect of morphine in diabetic mice. Therefore, we examined direct effects of IL-1b, intracerebroventricularly (i.c.v.), on morphine-induced analgesia, subcutaneously (s.c.), in diabetic and control mice by using the tail-¯ick test. Morphine at doses of 1, 2 and 5 mg/kg (s.c.) produced dose-dependent analgesia in diabetic and control mice but diabetic mice were less sensitive to the analgesic effect of morphine when compared to the controls. IL-1b at a dose of 0.1 ng/mouse produced analgesia in control mice but not in diabetics, whereas IL-1b at a dose of 10 ng/mouse produced a hyperalgesic effect both in diabetic and control mice. IL-1b at a dose of 1 ng/mouse has neither an analgesic nor a hyperalgesic effect in control and diabetic mice. Administration of a neutral (neither analgesic nor hyperalgesic) dose of IL-1b, 1 ng/mouse (i.c.v.), just prior to administration of morphine (s.c.) abolished the analgesic effect of morphine at doses of 1, 2 and 5 mg/kg in control mice and the analgesic effect of morphine became similar to that in diabetics. The diminished analgesic effect of morphine in diabetes was attenuated further with IL-1b at a dose of 1 ng/mouse (i.c.v.). These results suggest that the decreased analgesic effect of morphine in diabetes may be related to IL-1b. q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Interleukin-1 beta; Morphine; Streptozotocin; Diabetes; Mice
1. Introduction It is well established that animals with diabetes are significantly less sensitive than controls to the analgesic effect of morphine (Simon and Dewey, 1981; Raz et al., 1988; Kamei et al., 1992a). In humans, clinical studies show that morphine also has a limited analgesic effect in the treatment of painful diabetic neuropathy (James and Page, 1994; Galer, 1995). Morphine produces an analgesic effect mainly by m-opioid receptors; however, it has also a moderate af®nity for d- and k-opioid receptors (Emmerson et al., 1994). Kamei et al. (1992a) reported that mice with STZ-induced diabetes are selectively hyporesponsive to supraspinal mopioid receptor-mediated antinociception, but they respond normally to the activation of d- and k-opioid receptors. Although the exact mechanism is unknown, some studies suggest that certain factor(s) derived from spleen mononuclear cells may play an important role, directly or indirectly, * Corresponding author. Tel.: 190-312-3044766; fax: 190-3123234923. E-mail address: [email protected] (O. Yildiz).
in the selective reduction of m-agonist-mediated analgesia in experimental diabetes (Kamei et al., 1992b). There is a signi®cant increase in cytokine production in diabetes and certain cytokines, e.g. interleukin-1 (IL-1), may participate in the pathological events in diabetes (Ciampolillo et al., 1993; Espersen et al., 1993; Hussain et al., 1996). IL-1 plays important roles in the immune and in¯ammatory systems and it is involved in the local in¯ammatory and immune-mediated diseases which are associated with local pain (Dinarello, 1991). IL-1 when given intravenously produces hyperalgesia locally, as assessed by paw pressure test in rats (Ferreira et al., 1988; Follenfant et al., 1989), and enhances the acetylcholine-induced pain re¯ex in the isolated rabbit ear preparation (Schweizer et al., 1988). Blood-borne IL-1 may enter the brain comparably to other water-soluble compounds, such as morphine, known to cross the blood±brain barrier in suf®cient amounts to affect brain function (Banks et al., 1995). IL-1 is also produced in the brain and it plays a role as a neuromodulator in the brain (Katsuki et al., 1990; Minami et al., 1990, 1991; Yamaguchi et al., 1991). Brain-derived IL-1 produces a
0304-3959/00/$20.00 q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(00)00343-2
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H. Gul et al. / Pain 89 (2000) 39±45
variety of central nervous system-mediated responses, such as an increase in body temperature (Kluger, 1991), inducing slow-wave sleep (Krueger et al., 1984), anorexia (Mrosovsky et al., 1989), activating the hypothalamus-pituitary-adrenal axis (Berkenbosch et al., 1987) and attenuating neuronal activity in the hippocampus (Plata-Salaman and Ffrench-Mullen, 1992) and amygdala (Yu and ShinnickGallagher, 1994). The effects of centrally administered IL-1 on nociception have also been examined. Nakamura et al. (1988) demonstrated that intracisternal administration of IL-1a produces an analgesic effect, such as phenylquinolone-induced writhing (chemical stimulus) behavior in mice. In contrast, Oka et al. (1993) showed by using the hot plate method (thermal stimulus) that intracerebroventricular (i.c.v.) administration of IL-1b caused hyperalgesia in the rat. In a recent study, Yabuuchi et al. (1996) reported that IL-1 produced dose-dependent biphasic effects, i.e. hyperalgesia in low doses and analgesia in high doses, through the IL-1 receptor in the brain of rats with the paw pressure test (mechanical stimulus). Although these studies (using either thermal, chemical or mechanical stimuli) suggest that IL-1 has modulatory effects on nociceptive transmission, the direction of the effects, towards analgesia or hyperalgesia, is controversial. Previously, Kamei et al. (1992b) suggested that some factor(s) derived from spleen mononuclear cells may play a role in the reduction of the analgesic effect of morphine in diabetic mice. Raffa et al. (1993) postulated that cytokines may interact with opioid receptors and reduce the analgesic effect of morphine. However, to our knowledge, there is no study identifying the factor(s) which reduce the analgesic effect of morphine in diabetes. In this study, we characterized the analgesic and hyperalgesic effects of various doses of IL-1b and we aimed to analyze the effects of IL-1b on morphine-induced analgesia in STZ-diabetic and control mice by using the tail-¯ick test. 2. Materials and methods 2.1. Animals Adult male Balb/c mice (Breeding Colony of Research Center, Gulhane Military Medical School) weighing 25±30 g were used in the study. Animals were housed on a 12 h light/dark cycle (lights on from 08:00 h) at a constant ambient temperature (24 ^ 1)8C with normal mice chow and water available ad libitum during the study. Mice were handled in accordance with guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals (Zimmermann, 1983). 2.2. Induction of diabetes Diabetes was induced by a single intraperitoneal (i.p.)
injection of streptozotocin (STZ) (170 mg/kg body weight) after an overnight fast. STZ was dissolved in 3 mM citric acid buffer (pH 4.5) immediately before injection. Agematched non-diabetic mice received similar volumes of vehicle alone by the same route. Mice with serum glucose levels above 400 mg/dl were considered diabetic. 2.3. Drugs Mouse IL-1b and streptozotocin were purchased from Sigma (Deisenhofen, Germany). Morphine hydrochloride was purchased from Sandoz Chemical Co. (Sandoz, Switzerland). 2.4. Drug administration IL-1b was dissolved in phosphate-buffered saline (PBS, pH 7.4) containing 0.1% human serum albumin and it was administered i.c.v. as 5 ml/mouse (according to the method of Haley and McCormick, 1957). Morphine hydrochloride was dissolved in saline and administered subcutaneously (s.c.). 2.5. Measuring the tail-¯ick latency and MPE The analgesic response was evaluated by the tail-¯ick test. All animals were restrained for 30 min for adaptation and they were in restrainers during the tail-¯ick measurements. The intensity of a heat lamp was set to provide a predrug (baseline) latency time in the tail-¯ick response of 3±5 s. The intensity of the heat lamp was the same for all measurements in control and diabetic mice throughout the study. A cut-off time of 12 s was used. The percentage of the maximum possible effect (%MPE) was calculated for each animal using the following formula (Dewey et al., 1970): %MPE 100 £
test latency 2 pre-drug latency 12 2 pre-drug latency
The analgesic responses were measured 2 weeks after induction of diabetes in both diabetic and non-diabetic control mice. Mice were treated with either morphine hydrochloride (1, 2, and 5 mg/kg body weight, s.c.) and IL-1b (0.1, 1 and 10 ng/mouse, i.c.v.) alone or with a combination of morphine hydrochloride (1, 2, and 5 mg/kg body weight, s.c.) and IL-1b (1 ng/mouse, i.c.v., as a non-analgesic and non-hyperalgesic dose). In a group of mice we measured the effect of vehicle of IL-1b (i.c.v.) (PBS, pH 7.4, containing 0.1% human serum albumin) alone and in combination with morphine (5 mg/kg, s.c.) in the tail-¯ick test. Each mouse was used once for different doses of IL-1b, morphine or for the combination of both. Tail-¯ick latencies were measured just before and 30, 60 and 90 min after the administration of the drugs. To test the possible pyrogenic effect of IL-1b, rectal temperature was measured in a separate group of mice by using a pyrogen test instrument (Ellab, Copenhagen, Denmark) before and 30, 60, 120 and 180 min
H. Gul et al. / Pain 89 (2000) 39±45
41
after the administration of IL-1b. All of the studies were done between 09:00 h and 13:00 h. 2.6. Statistical analysis Results were presented as the mean ^ SEM of the %MPE. For statistical comparisons among the groups, one-way analysis of variance (ANOVA) was performed followed by Duncan's multiple range test. A paired or unpaired Student's t-test was used to compare the values from two groups when necessary. P , 0:05 was considered signi®cant. 3. Results 3.1. Effects of morphine on the tail-¯ick test in control and diabetic mice Subcutaneous administration of morphine (1, 2 and 5 mg/ kg) induced a dose-dependent analgesic effect both in control and diabetic mice (Fig. 1). While baseline tail¯ick latencies were not different between control (4.22 ^ 0.24 s, n 76) and diabetic (4.02 ^ 0.2 s, n 65) mice, morphine-produced analgesia in control mice was higher than in diabetic mice at 30 min at a dose of 1 mg/kg (P , 0:001), at 30 and 60 min at a dose of 2 mg/ kg (P , 0:01 and P , 0:05, respectively), and at 30, 60 and 90 min at a dose of 5 mg/kg (P , 0:001) (Fig. 1).
Fig. 1. Effect of morphine (1, 2 and 5 mg/kg, s.c.) on the tail-¯ick test both in control and diabetic mice. Results are presented as the mean ^ SEM of the %MPE. *P , 0:001, versus diabetic 1 mg/kg morphine (at 30 min; Student's t-test); **P , 0:01, versus diabetic 2 mg/kg (at 30 min; Student's t-test); ***P , 0:05, versus diabetic 2 mg/kg (at 60 min; Student's t-test); # P , 0:001, versus diabetic 5 mg/kg (at 30, 60 and 90 min; Student's t-test).
Fig. 2. Effect of i.c.v. injection of IL-1b (0.1, 1 and 10 ng) on the tail-¯ick test both in control and diabetic mice. Results are presented as the mean ^ SEM of the %MPE. *P , 0:05, versus control 1 IL-1b (10 ng/mouse, i.c.v.; at 30 min; ANOVA, post hoc Duncan's multiple range test); **P , 0:05, versus diabetic 1 IL-1b (0.1 ng/mouse, i.c.v.; at 60 and 90 min; Student's t-test); ***P , 0:01, versus diabetic 1 IL-1b (10 ng/mouse, i.c.v.; at 60 min; ANOVA, post hoc Duncan's multiple range test); # P , 0:05, versus control 1 IL-1b (1, 10 ng/mouse, i.c.v.; at 60 and 90 min; ANOVA, post hoc Duncan's multiple range test).
3.2. Effects of IL-1b on the tail-¯ick test in control and diabetic mice We examined the effects of i.c.v. injection of IL-1b alone with the tail-¯ick test. IL-1b produced an analgesic effect at a dose of 0.1 ng/mouse in control mice but not in diabetics (Fig. 2). The analgesic effect of IL-1b at a dose of 0.1 ng/ mouse was markedly higher than the effect of IL-1b at a dose of 1 ng/mouse in control mice at 60 and 90 min (P , 0:05). IL-1b at a dose of 1 ng/mouse produced neither an analgesic nor a hyperalgesic effect in control and diabetic mice (Fig. 2). IL-1b produced a hyperalgesic effect at a dose of 10 ng/mouse in control and diabetic mice (Fig. 2). In control mice, the hyperalgesic effect of high dose of IL1b (10 ng/mouse) was signi®cantly different when compared to its effect at a dose of 0.1 ng/mouse at 30, 60 and 90 min and it was also different than the effect of 1 ng/ mouse at 30 min (P , 0:05, respectively). In diabetic mice, the hyperalgesic effect of a high dose of IL-1b (10 ng/ mouse) was signi®cantly different at 30 min when compared to the effect of 0.1 and 1 ng/mouse IL-1b. Although the analgesic effect of a low dose (0.1 ng/mouse) of IL-1b continued at 120 and 180 min (%MPE 14.8 ^ 6 and 11.7 ^ 4, respectively, not shown in Fig. 2), the hyperalgesic effect of a high dose (10 ng/mouse) of IL-1b was reduced at 120 and 180 min (%MPE 24.1 ^ 2.8 and 25.3 ^ 2.5, respectively, not shown in Fig. 2) in control
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H. Gul et al. / Pain 89 (2000) 39±45
mice. In contrast to control mice, the hyperalgesic effect of a high dose of IL-1b (10 ng) was reversed to analgesia at 120 and 180 min in diabetic mice (%MPE 2.3 ^ 3.4 and 5.7 ^ 4.9, respectively, not shown in Fig. 2). 3.3. Effects of IL-1b in combination with morphine on the tail-¯ick test in control and diabetic mice The vehicle of IL-1b (PBS 1 0.1% human serum albumin) did not produce any analgesic or hyperalgesic effects alone and it also had no effect on morphine (5 mg/kg)induced analgesia both in control and diabetic mice (Table 1). The neutral dose of IL-1b (1 ng/mouse, i.c.v.) blocked the analgesic effect of 1 mg/kg morphine and it induced hyperalgesia (P , 0:001 at 30 and 60 min, P , 0:01 at 90 min). IL-1b (1 ng/mouse, i.c.v.) reduced the analgesic effect of morphine at a dose of 2 mg/kg at 30 and 60 min (P , 0:001 and P , 0:01, respectively) and it also reduced the analgesic effect of morphine at a dose of 5 mg/kg at 30, 60 and 90 min (P , 0:001) in control mice (Fig. 3). In diabetic mice, IL-1b (1 ng/mouse, i.c.v.) blocked the analgesic effect of morphine at a dose of 1 mg/kg, and it induced signi®cant hyperalgesia at 60 min only (P , 0:001). IL-1b blocked the analgesic effect of morphine at a dose of 2 mg/kg and it induced hyperalgesia at 30, 60 (P , 0:001) and 90 min (P , 0:01). In addition, IL-1b (1 ng/mouse) signi®cantly reduced the analgesic effect of morphine at a dose of 5 mg/kg at 30 min (P , 0:05) (Fig. 3). 3.4. Effect of IL-1b on rectal temperature IL-1b at doses of 0.1, 1 and 10 ng/mouse (i.c.v.) and its vehicle did not signi®cantly change the rectal temperature at 30, 60, 120 and 180 min after the injections (Table 2). 4. Discussion In the present study, the diabetic mice were signi®cantly less sensitive to the analgesic effect of morphine than nondiabetic mice and this ®nding is consistent with previous studies (Simon and Dewey, 1981; Raz et al., 1988; Kamei et al., 1992a). The major ®nding of our study is that a
Fig. 3. Effect of IL-1b (0.1, 1 and 10 ng/mouse, i.c.v.) on morphine-induced analgesia in control (upper) and diabetic (lower) mice. Results are presented as the mean ^ SEM of the %MPE. Student's t-test was used for statistical comparisons. (Upper) *P , 0:001, versus control 1 morphine (1 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 30 and 60 min); **P , 0:001, control 1 morphine (1 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 90 min); ***P , 0:001, versus control 1 morphine (2 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 30 min); #P , 0:01, versus control 1 morphine (2 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 60 min); ##P , 0:001, control 1 morphine (5 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 30, 60 and 90 min). (Lower) *P , 0:001, versus diabetic 1 morphine (1 mg/kg) 1 IL-1b (1 ng/ mouse, i.c.v.; at 60 min); **P , 0:001, versus diabetic 1 morphine (2 mg/ kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 30 and 60 min); ***P , 0:01, versus diabetic 1 morphine (2 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 90 min); #P , 0:05, versus diabetic 1 morphine (5 mg/kg) 1 IL-1b (1 ng/mouse, i.c.v.; at 60 min).
neutral (neither analgesic nor hyperalgesic) dose of IL-1b (1 ng/mouse, i.c.v.) abolished the analgesic effect of morphine in control mice and the analgesic effect of
Table 1 Effect of vehicle of IL-1b (PBS 1 0.1% human serum albumin) alone (intracerebroventricularly) and on morphine-induced (subcutaneously) analgesia a Groups (n)
Control vehicle (6) Diabetic vehicle (9) Control morphine 5 mg/kg (10) Control morphine 5 mg/kg 1 vehicle i.c.v. (6) Diabetic morphine 5 mg/kg (7) Diabetic morphine 5 mg/kg 1 vehicle (6) a
Results are presented as the mean ^ SEM %MPE.
%MPE (min) 30
60
90
2.9 ^ 1.83 1.26 ^ 3.63 82.8 ^ 9.5 90.36 ^ 6.16 26.4 ^ 8.9 34.42 ^ 2.47
20.4 ^ 1.87 24.78 ^ 3.74 90.4 ^ 5.1 96.77 ^ 3.23 17.4 ^ 2.8 17.68 ^ 1.37
0.3 ^ 1.83 22.96 ^ 3.1 84.7 ^ 11.2 95.25 ^ 3.04 15.3 ^ 2.5 11.94 ^ 2.72
H. Gul et al. / Pain 89 (2000) 39±45
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Table 2 Effect of IL-1b and its vehicle (PBS 1 0.1% human serum albumin) on rectal temperature of mice (n 5 for all groups) a IL-1b (i.c.v.)
0.1 ng 1 ng 10 ng Vehicle a
Before injection
38.78 ^ 0.16 37.86 ^ 0.19 37.92 ^ 0.17 37.66 ^ 0.14
After injection (min) 30
60
120
180
38.12 ^ 0.19 38.32 ^ 0.32 37.80 ^ 0.29 37.72 ^ 0.19
37.94 ^ 0.15 37.96 ^ 0.25 37.52 ^ 0.34 37.76 ^ 0.13
37.80 ^ 0.23 38.24 ^ 0.29 37.66 ^ 0.35 37.92 ^ 0.26
37.96 ^ 0.05 37.64 ^ 0.19 37.30 ^ 0.16 37.86 ^ 0.24
Results are presented as the mean ^ SEM temperature (8C).
morphine became similar to that in diabetics. We also found that the diminished analgesic effect of morphine in diabetes was attenuated further with IL-1b at a dose of 1 ng/mouse (i.c.v.). Previously, Mathiasen et al. (1987) reported that beige-J mice, a type of mutant mice with immunological de®ciencies, had more resistance to the analgesic effect of morphine and other m-opioid receptor agonists than control mice. Kimball and Raffa (1989) showed that the transfer of mononuclear spleen cells obtained from beige-J mice to normal littermates resulted in a signi®cant reduction of the analgesic effect of morphine similar to that in beige-J mice. Monocytes from beige-J mice were found to have a higher basal activity and were able to secrete considerably higher levels of IL-1 than were their littermates (Raffa et al., 1993). Interestingly, Kamei et al. (1992b) demonstrated that the defect of the analgesic effect to m-opioid receptor agonist in diabetic mice was recovered by splenectomy. They speculated that this recovery of analgesic effect was due to depression of some endogen agents derived from spleen rather than the genetic de®ciency of m-opioid receptor synthesis. Kamei et al. (1992b) suggested that some factor(s) derived from spleen mononuclear cells might play an important direct or indirect role in the selective reduction in m-agonist-mediated analgesia in diabetic mice. Interestingly, Ciampolillo et al. (1993) and Espersen et al. (1993) reported that IL-1b was increased at the onset of insulin-dependent diabetes mellitus (IDDM) in humans. On the contrary, Luger et al. (1988), Mooradian et al. (1991), and Ohno et al. (1993) reported that there was a decrease in the secretion of IL-1b from stimulated monocytes in human subjects with IDDM. In addition, Netea et al. (1997) reported that ex vivo lipopolisaccharide-stimulated IL-1b production in newly diagnosed IDDM patients was signi®cantly increased compared to long-term IDDM patients and healthy controls. They also demonstrated that the ratio of IL-1 receptor antagonist/interleukin-1 (IL-1ra/ IL-1) was signi®cantly decreased in newly diagnosed IDDM patients and that the circulating level of IL-1ra was increased in long-term IDDM patients. IL-1b and IL-1ra may cross the blood±brain barrier (Banks et al., 1995) and may affect the antinociceptive action of morphine. Our study is the ®rst to demonstrate that IL-1b may be the factor which may reduce morphine-induced analgesia in diabetes.
The mechanism of diminishing the analgesic effect of morphine by IL-1b is not clear. It was reported that there were no differences in m-opioid receptor binding sites between spontaneously- or streptozotocin-diabetic and corresponding control mice (Brase et al., 1987). Morphine produces an analgesic effect mainly by m-opioid receptors and therefore the differences in the analgesic effect of morphine between diabetic and control mice are not attributable to density, size and distribution of m-opioid receptors. However, it is possible that a m-opioid receptor± effector coupling mechanism may be changed in diabetes and this change would result in the reduction of the analgesic effect of morphine in diabetic mice. We investigated the acute effects of IL-1b on morphine-induced analgesia; therefore, it was not possible that IL-1b could change the opioid receptor density. We think that IL-1b may reduce the analgesic effect of morphine by changing its receptor±effector coupling mechanism. However, further investigation is needed to clarify this point. IL-1b may produce fever (Busbridge et al., 1989; Dascombe et al., 1989) and decreased vigilance (Opp et al., 1991) which may in¯uence the results of behavioral testing for nociception. In the present study, IL-1b had no effect on rectal temperature in mice at the doses used. Recently, Fox et al. (1999) reported a critical evaluation of a STZ model of painful diabetic neuropathy in the rat. They found that STZ-induced diabetes in rats produced long-term resting mechanical, but not thermal hyperalgesia. They suggested that a discrepancy between the presence of mechanical and thermal hyperalgesia was important and speci®c for diabetic rats. They hypothesized that discomfort and also handling of animals with general ill health might contribute to false positive readings in the mechanical hyperalgesia paw pressure test. In the present study, we observed no difference in the thermal nociception threshold between diabetic and control mice. While this observation is in agreement with some previous studies (Raz et al., 1988; Fox et al., 1999), some authors reported a thermal hyperalgesia or hypoalgesia (Forman et al., 1986; Lee and McCarty, 1992). We used the tail-¯ick test to evaluate thermal nociception. We employed 30 min of restraining time and thus performed the experiments with minimal handling. We demonstrated that IL-1b has biphasic effects in mice with the tail-¯ick test. IL-1b produced analgesia at the low
44
H. Gul et al. / Pain 89 (2000) 39±45
dose (0.1 ng/mouse, i.c.v.) but it produced hyperalgesia at the high dose (10 ng/mouse, i.c.v.). IL-1b did not produce any analgesic or hyperalgesic effect at 1 ng/mouse (i.c.v.). There are a number of con¯icting data about the antinociceptive effect of IL-1b in the literature. While Nakamura et al. (1988) demonstrated that i.c.v. administration of IL-1b had an analgesic effect in the phenylquinolone-induced writhing test, Oka et al. (1993) discovered that i.c.v. administration of IL-1b had a hyperalgesic effect in the hot plate test. In contrast to our ®ndings, Yabuuchi et al. (1996) reported that IL-1b produced biphasic dose-dependent effects (hyperalgesia with low doses and analgesia with high doses) in the paw pressure test. These con¯icting results may be due to differences in experimental conditions, such as methods of testing nociception, animal species or the doses of IL-1. None of these studies used the tail-¯ick test in mice to test the antinociceptive effect of IL-1b (i.c.v.). Previously, Yabuuchi et al. (1996) demonstrated that the hyperalgesic effect of lower doses of IL-1b on mechanical nociception was mediated by a cyclooxygenase pathway. Na-salicylate, a cyclooxygenase inhibitor, caused the lower doses of IL-1b to induce an analgesic rather than a hyperalgesic effect. The reason for this inversion is still unclear. In addition, they also found that the analgesic effect of higher doses of IL-1b was blocked by a-helical CRF, the antagonist of CRF. In view of these ®ndings and the present data, further investigations are needed to analyze the mechanisms of the biphasic effect of IL-1b. In conclusion, IL-1b has biphasic effects, analgesia at the low dose and hyperalgesia at the high dose, in thermal nociception in control mice. Streptozotocin-diabetic mice are signi®cantly less sensitive than controls to the analgesic effect of morphine and IL-1b may be related to this diminished analgesic effect. However, further investigations are needed to clarify the mechanism of interaction between IL1b and morphine. Acknowledgements We thank Novartis for ®nancial support and Ali Gursoy Erturk of Novartis for his help throughout the study. We also thank Dr Zafer Goren for his helpful comments on methods. References Banks WA, Kastin AJ, Broadwell RD. Passage of cytokines across the blood-brain barrier. Neuroimmunomodulation 1995;2:241±248. Berkenbosch F, van Oers J, del Rey A, Tilders F, Besedovsky H. Corticotropin-releasing factor-producing neurons in the rat activated by interleukin-1. Science 1987;238:524±526. Brase DA, Han YH, Dewey WL. Effects of glucose and diabetes on binding of naloxone and dihydromorphine to opiate receptors in mouse brain. Diabetes 1987;36:1173±1177. Busbridge NJ, Dascombe MJ, Tilders FJ, van Oers JW, Linton EA,
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