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Cytokine involvement in dynorphin-induced allodynia
Pain 84 (2000) 159±167 www.elsevier.nl/locate/pain
Cytokine involvement in dynorphin-induced allodynia Tinna M. Laughlin a, John R. Bethea b, Robert P. Yezierski b, George L. Wilcox a,c,* a
b
Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA Department of Neurological Surgery and The Miami Project, University of Miami, Miami, FL 33136, USA c Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA Received 5 March 1999; received in revised form 25 May 1999; accepted 26 July 1999
Abstract Dynorphin A is an endogenous opioid peptide, which has previously been shown to produce a long-lasting allodynia and hyperalgesia in mice, behavioral states consistent with signs of clinically observed neuropathic pain. This dynorphin-induced allodynia was used as a pharmacological, central model of neuropathic pain. In this study, we examined the involvement of the cytokine IL-1b , the transcription factor nuclear factor kappa B (NF-k B), and de novo protein synthesis in the development of allodynia induced by intrathecal (i.t.) administration of dynorphin in male ICR mice. Pretreatment with the protein synthesis inhibitor cycloheximide (0.3±85 nmol), the NFk B inhibitor pyrrolidinedithiocarbamate (PDTC) (0.001±1000 pmol), the IL-1 receptor antagonist (IL-1ra) protein (0.01±100 ng), the caspase-1 inhibitor (YVAD) (0.1±300 pmol), and the anti-in¯ammatory cytokine IL-10 (0.1±300 ng) all dose-dependently reduced the induction of dynorphin-induced allodynia. Finally, IL-10 administered within the ®rst 24 h after the dynorphin insult prevented the development of chronic allodynia. These results demonstrate that the anti-in¯ammatory cytokines IL-10 and IL-1ra impede the development of dynorphin-induced allodynia. These results also suggest that production of new proteins through NF-k B activation is required for the induction of allodynia. We speculate that IL-1ra, IL-10, PDTC and cycloheximide interfere with the central pro-in¯ammatory cascade. Modulation of cytokine activity in the spinal cord may therefore prove to be an effective therapeutic strategy for the treatment of chronic pain. q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: IL-10; NF-k B; Interleukin-1beta; Cytokines; Protein synthesis; Neuropathic pain
1. Introduction Cytokines have long been associated with the peripheral in¯ammatory response, but recent data have revealed that cytokines also play a role in the pathology of brain injury and disease. Several studies have shown the involvement of cytokines in the pathology of several neurological diseases, including Alzheimer's disease (Grif®n et al., 1989), AIDS dementia complex (Merrill and Chen, 1991), multiple sclerosis (Hofman et al., 1986), and ischemic insult (Buttini et al., 1994; Loddick and Rothwell 1996). In addition to the neurodegenerative diseases, several groups have suggested that cytokines contribute directly to the pathology of neuropathic pain peripherally (Watkins et al., 1994; Wagner et al., 1998) and centrally (DeLeo et al., 1996; Watkins et al., 1997). In this study, we examined the involvement of the
* Corresponding author. Department of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church Street S.E., Minneapolis, MN 55455, USA. Tel.: 11-612-625-1474; fax: 11-612-625-3606. E-mail address: [email protected] (G.L. Wilcox)
interleukin-1 beta (IL-1b ) cytokine in the development of dynorphin-induced allodynia in mice. Pro-in¯ammatory cytokines, such as IL-1b , IL-6 and tumor necrosis factor-alpha (TNF-a ), are normally expressed in low concentrations in the brain; however, this expression increases after brain injury (Giulian and Lachman, 1985; Yan et al., 1992), ischemia (Buttini et al., 1994; Bhat et al., 1996), and in¯ammation (Wong et al., 1996; Woolf et al., 1997). These cytokines have also been shown to have a minimal constitutive expression in spinal cord, which increases after spinal cord injury (Wang et al., 1997; Bethea et al., 1999) and neuropathic pain (DeLeo et al., 1997). The enhanced expression of cytokines in these pathological states suggests that they have a role in the initiation or maintenance of the disease state. In this study, we investigated the involvement of the proin¯ammatory cytokine IL-1b in dynorphin-induced allodynia. IL-1b has been shown to mediate several responses in the CNS, including fever, anorexia, sleep and autonomic/ hormonal reactions (Dinarello, 1988). More importantly, several studies have indicated that IL-1b may have a role in nociception; for instance, central administration of IL-1b
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enhances the response of wide dynamic range neurons to noxious stimuli (Oka et al., 1994), dose-dependently enhances the amount of electrically evoked release of SP and CGRP from rat spinal cord slices (Malcangio et al., 1996), and induces mechanical hyperalgesia (Ferreira et al., 1988; Oka et al., 1993; Watkins et al., 1994; Yabuuchi et al., 1996). Blocking IL-1b activation of the IL-1 receptor with IL-1ra attenuated IL-1b -induced hyperalgesia (Oka et al., 1993), formalin-induced hyperalgesia (Watkins et al., 1997), and capsaicin-induced hyperalgesia (Davis and Perkins, 1996). In addition, the peripheral administration of formalin (Yabuuchi et al., 1996) and CFA (Woolf et al., 1997), which produce persistent nociceptive states, resulted in an increased expression of IL-1b . More recently, the levels of IL-1b in the synovial ¯uid have been shown to correlate with the level of pain in patients with chronic temporomandibular joint (TMJ) pain (Alstergren et al., 1998). Collectively, these studies lead us to test the hypothesis that central IL-1b is contributing to the development of dynorphin-induced allodynia in mice. 2. Materials and methods 2.1. Drugs and animals Cycloheximide and pyrrolidinedithiocarbamate (PDTC) were purchased from Sigma (St. Louis, MO), interleukin-1 receptor antagonist (IL-1ra) from R&D Systems (Minneapolis, MN), and caspase-1 inhibitor peptide Ac-Tyr-ValAla-Asp-aldehyde (YVAD) from Calbiochem (La Jolla, CA). Interleukin-10 (IL-10) was a gift from Schering Plough Pharmaceuticals, and dynorphin A (1-17) was provided by NIDA. All drugs were diluted in 0.9% saline and administered in a volume of 5 ml by intrathecal (i.t.) injections to awake male ICR mice (Harlan) using the method of Hylden and Wilcox (1980). All experiments were conducted so that the experimenter was blinded to the drug treatment. 2.2. Mechanical sensitivity assessment Mice were placed in individual 2 l bedding-lined beakers and allowed to adjust to the surroundings for 1 h prior to all behavioral testing. In order to study chronic nociception, we used the dynorphin-induced chronic allodynia model. In this model, a single intrathecal injection of dynorphin induces allodynia and hyperalgesia in mice (Laughlin et al., 1997) and rats (Vanderah et al., 1996) that last for at least 100 days; these behavioral states are consistent with signs of clinically observed neuropathic pain. Therefore, dynorphin-induced allodynia was used as a pharmacological model of neuropathic pain. Mechanical allodynia was determined with a set of von Frey ®laments modi®ed as previously described (Laughlin et al., 1997); these ®laments are used to stimulate the dorsal side of the hind paw. Individual von Frey ®laments were a gift from North Coast
Medical, Inc. (San Jose, CA). The innocuous 0.4 mN (#2.44) ®lament was applied to the point of bending 3 times to the dorsal surface of the left and right hind paw for a total of 6 applications per mouse. We have found that this ®lament elicits paw withdrawal responses only in mice exposed to allodynia-inducing manipulations such as intrathecally applied dynorphin (Laughlin et al., 1997). 2.3. Statistical analysis The response frequency of foot withdrawals for each time point was expressed as the number of positive responses divided by 6; this number was then multiplied by 100 to represent percent response. A mean and standard error of the mean (SEM) were calculated for each group from the percent response frequency. The seven day time course data for each animal were converted to a single point by adding the individual percent response frequency scores and dividing by the number of time points summed; then a mean ^ SEM was generated from these values. This mean was referred to as the area under the curve (AUC). The data were tested for signi®cance using analysis of variance (ANOVA), and statistical differences between groups were further analyzed with Dunnett's test for multiple compansons to a control (saline) group. P values of less than 0.05 were considered statistically signi®cant. 3. Results 3.1. Role of protein synthesis in dynorphin-induced allodynia Through the use of a protein synthesis inhibitor, we ®rst examined whether a genetic program is contributing to the mechanism of dynorphin-induced allodynia. A single systemic injection of the protein synthesis inhibitor cycloheximide has previously been shown to transiently suppress the synthesis of proteins in the rat brain (Pavlik and Teisinger, 1980). A single intrathecal pretreatment (30 mm) with cycloheximide (0.3±85 nmol) dose-dependently inhibited the induction of dynorphin-induced chronic allodynia (Fig. 1). Pretreatment with 3 and 85 nmol but not 0.3 nmol signi®cantly inhibited dynorphin-induced allodynia (Fig. 1B). Cycloheximide preceding a saline injection (control group) induced a small allodynia at the higher doses, 3 and 85 nmol (Fig. 1B). These results imply that events requiring protein synthesis are required during the establishment of the chronic allodynic state. 3.2. Role of interleukin-1b in dynorphin-induced allodynia Because IL-1b has been shown to have a role in nociception, we sought to determine the role of IL-b in dynorphininduced chronic allodynia. A 30 min pretreatment with the interleukin-1 receptor antagonist (0.01±100 ng) dose-
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Fig. 1. Involvement of protein synthesis in dynorphin-induced allodynia. (A) Time-course of the effect of a 30 min pretreatment with the protein synthesis inhibitor cycloheximide. Treatment with 85 nmol cycloheximide (open circles) fully inhibited allodynia, 3 nmol cycloheximide (open crosses) partially inhibited allodynia, and 0.3 nmol cycloheximide (open triangles) had no effect on development of dynorphin-induced chronic allodynia (squares). (B) Dose±response curve for cycloheximide treatment. Cycloheximide administered 30 min before 3 nmol dynorphin dose-dependently prevented the induction of allodynia (circles), whereas cycloheximide preceded by a saline injection produced no signi®cant allodynia (triangles). Mechanical allodynia was determined by application of the 0.4 mN von Frey ®lament to the dorsal hind paw. Time course data from part (A) were converted to area under the curve (AUC) for part (B). * P , 0:05 from the saline group (diamond), # P , 0:05 from the dynorphin 1 saline positive control group (square); n 8±10 mice per group.
Fig. 2. Effect of pretreatment with the interleukin-1 receptor antagonist (IL-1ra) in dynorphin-induced allodynia. (A) Time-course of the effect of a 30 min pretreatment with IL-1ra. Treatment with 1 ng IL-10 (open circles) but not 0.01 ng IL-1ra (open triangles) inhibited the development of dynorphin-induced chronic allodynia (squares). (B) Dose±response curve for IL1ra treatment. IL-1ra administered 30 min before 3 nmol dynorphin dosedependently prevented the induction of allodynia (circles). IL-1ra preceding a saline injection (triangles) produced no signi®cant allodynia at the 0.01±1 ng dose, whereas 100±300 ng IL-1ra induced allodynia. Mechanical allodynia was determined by application of the 0.4 mN von Frey ®lament to the dorsal hind paw. Time course data from part (A) were converted to area under the curve (AUC) for part (B). * P , 0:05 from the saline group (diamond), # P , 0:05 from the dynorphin 1 saline positive control group (square). n 8±10 mice per group, except n 3 mice for the 0.01, 0.1 and 100 ng IL-1ra plus saline groups.
dependently inhibited the induction of dynorphin-induced allodynia (Fig. 2). IL-1ra pretreatment signi®cantly decreased the response frequency from the dynorphin control in a dose-dependent manner: 0.1 ng and 1 ng were effective whereas 0.01 ng and 100 ng were not (Fig. 2B). These results suggest that dynorphin-induced allodynia may involve the activation of the IL-1 receptor. IL-1ra administered preceding a saline injection (control group) induced allodynia at the higher doses, 100 and 300 ng (Fig. 2B); this effect may account for the failure of 100 ng IL-1ra to inhibit dynorphin-induced allodynia and suggests that IL-1ra is behaviorally toxic at higher doses.
Caspase-1 (also known as interleukin-1b converting enzyme or ICE) is a protease that processes pro-interleukin-1b to the active form of IL-1b (Cerref® et al., 1992; Thornberry et al., 1992). Thus, caspase-1 inhibitors block the formation of active IL-1b . A 30 min pretreatment with the caspase-1 inhibitor, YVAD (0.2±200 pmol) dose-dependently blocked induction of allodynia (Fig. 3). All doses (0.2 pmol, 20 pmol and 200 pmol) were effective in reducing allodynia and none produced allodynia alone (Fig. 3B). The 0.2 pmol dose of YVAD initially inhibited allodynia, but the allodynia gradually returned to dynorphin 1 saline control group levels by day 5 (Fig. 3A). These results, together with the IL-1ra data, indicate that activation of
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Fig. 3. Involvement of the caspase-1 enzyme in dynorphin-induced allodynia. (A) Time-course of the effect of a 30 min pretreatment with the caspase-1 inhibitor, YVAD. Treatment with 20 pmol YVAD (open circles) prevented the development of dynorphin-induced chronic allodynia (squares). On the other hand, 0.2 pmol YVAD (open triangles) initially inhibited allodynia, and the allodynia gradually returned to dynorphin 1 saline control group (squares) levels (B) Dose±response curve for YVAD. YVAD administered 30 min before 3 nmol dynorphin dose-dependently prevented the induction of allodynia (circles); 200 pmol of YVAD preceding a saline injection did not produce signi®cant allodynia (triangle). Mechanical allodynia was determined by application of the 0.4 mN von Frey ®lament to the dorsal hind paw. Time course data from part (A) were converted to area under the curve (AUC) for part (B). * P , 0:05 from the saline group (diamond), # P , 0:05 from the dynorphin 1 saline positive control group (square); n 7±10 mice per group.
the IL-1b signaling cascade is required for the development of dynorphin-induced allodynia. 3.3. Effect of interleukin-10 treatment Having demonstrated that the pro-in¯ammatory cytokine IL-1b is involved in dynorphin-induced allodynia, we next determined the potential of the anti-in¯ammatory cytokine IL-10 to prevent the induction of allodynia. IL-10 is an antiin¯ammatory cytokine that, among other actions, inhibits the synthesis and secretion of pro-in¯ammatory cytokines, such as IL-1b , IL-6, and TNF-a (Fiorentino et al., 1991; Balasingam and Yong, 1996; Rongione et al., 1997; Wagner
Fig. 4. Effect of pretreatment with the interleukin-10 anti-in¯ammatory cytokine in dynorphin-induced allodynia. (A) Time-course of the effect of a 30 min pretreatment with IL-10. Pretreatment with 1 ng IL-10 (open circles) but not 0.1 ng IL-10 (open squares) prevented the development of dynorphin-induced chronic allodynia (triangles). (B) Dose±response curve of IL-10 treatment. IL-10 administered 30 min before 3 nmol dynorphin dose-dependently prevented the induction of allodynia (circles). IL-10 preceding a saline injection (triangles) did not produce any signi®cant allodynia at the 0.1 and 1 ng dose. Mechanical allodynia was determined by application of the 0.4 mN von Frey ®lament to the dorsal hind paw. Time course data from part (A) were converted to area under the curve (AUC) for part (B). * P , 0:05 from the saline group (diamond), # P , 0:05 from the dynorphin 1 saline postive control group (square); n 7±10 mice per group.
et al., 1998). A 30 min pretreatment with IL-10 (0.1±10 ng) dose-dependently inhibited the induction of dynorphininduced allodynia (Fig. 4). Pretreatment with 0.6 ng, 1 ng and 10 ng IL-10 signi®cantly decreased the response frequency from the dynorphin 1 saline control group, whereas 0.1 ng and 0.3 ng IL-10 were ineffective (Fig. 4B). IL-10 has been shown with repeated doses to cause an increase in tissue damage following injury (Bethea et al., 1999; Brewer et al., 1999); in addition, we have shown that intrathecal administration of 300 ng IL-10 by itself induces signi®cant allodynia (data not shown). This effect may explain why the IL-10 dose±response curve (Fig. 4B) starts to turn upward at high doses, limiting its maximum ef®cacy. These results, along with the cycloheximide results, suggest that the synthesis and secretion of pro-
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in¯ammatory cytokines are involved in the development of dynorphin-induced allodynia. To determine the therapeutic potential of IL-10 in preexisting conditions, we next determined the ability of IL-10 post-treatment to diminish dynorphin-induced allodynia. IL-10 (1 ng, i.t.), administered sequentially at three different time points, 30 min, 6 h, and 24 h after dynorphin, prevented the development of dynorphin-induced allodynia (Fig. 5). Mice administered 1 ng of IL-10 once at 30 min after dynorphin were allodynic 1 day after dynorphin; thereafter, the allodynia was signi®cantly reduced to saline control levels (Fig. 5). Finally, a single administration of 1 ng IL-10 at 24 h after dynorphin did not reduce allodynia (Fig. 5). Thus, there appears to be a critical period and duration of less then 24 h after insult in which IL-10 may be therapeutic. 3.4. Effect of NF-k B inhibition Having shown that protein synthesis (Fig. 1) and, more speci®cally, the synthesis of pro-in¯ammatory cytokines (Fig. 4) may be involved in the development of dynorphin-induced allodynia, we next examined the possible role of the transcriptional factor, NF-k B, in this model. Pyrrolidinedithiocarbamate (PDTC) is an antioxidant that has been shown to be a potent inhibitor of the activation of NF-k B, (Schreck et al., 1992; Cooper et al., 1998; Heese et al., 1998; Iuvone et al., 1998). A 30 min pretreatment with PDTC (1 fmol±100 nmol) dose-dependently inhibited the induction of dynorphin-induced allodynia (Fig. 6). Pretreatment with 1, 10, 100, and 1000 pmol PDTC signi®-
Fig. 5. Effect of post-treatment with IL-10 in dynorphin-induced allodynia. Both repetitive administration of 1 ng IL-10 (30 min, 6 h, 24 h after 3 nmol dynorphin; open triangles), and a single treatment of 1 ng IL-10 at 30 min after dynorphin (open circles) signi®cantly reduced chronic allodynia. Administration of 1 ng IL-10 1 day after dynorphin (open crosses) injection did not alleviate dynorphin-induced chronic allodynia (squares). The mean ^ SEM represents the percent response frequency of foot withdrawals to 6 applications of the 0.4 mN ®lament to the dorsal hind paw. n 8±10 mice per group.
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cantly reduced mechanical allodynia, 0.5 pmol PDTC produced a small reduction in the amount of allodynia, and 1 fmol PDTC had no effect (Fig. 6). Saline or PDTC administered preceding a saline injection produced no signi®cant allodynia (Fig. 6). These results suggest that transcriptional regulation through NF-k B may be required for the development of a chronic allodynic state. 4. Discussion The present study demonstrates that the IL-1b cytokine system is required for the development of dynorphininduced allodynia. First, pretreatment with IL-1ra (0.01± 100 ng) and YVAD (0.1±300 pmol) dose-dependently inhibited the induction of dynorphin-induced allodynia, results which support the involvement of the pro-in¯ammatory cytokine IL-1b in the development of dynorphininduced allodynia. Second, pretreatment with the protein synthesis inhibitor cycloheximide (0.3±85 nmol), the NFk B inhibitor PDTC (0.001±1000 pmol) and the anti-in¯ammatory cytokine IL-10 (0.1±300 ng) dose-dependently inhibited the induction of dynorphin-induced allodynia, results that suggest the involvement of the transcription and synthesis of cytokine/cytokine-induced proteins in dynorphin-induced allodynia. Finally, IL-10, administered within the ®rst 24 h after the dynorphin insult, signi®cantly reduced the development of dynorphin-induced chronic allodynia, suggesting a therapeutic application for this anti-in¯ammatory cytokine. 4.1. Involvement of IL-1b in dynorphin-induced allodynia In this study, we have shown that the pro-in¯ammatory cytokine IL-1b contributes to the development of dynorphin-induced allodynia. IL-1b may be contributing to the development of this allodynia through several mechanisms. Cytokines are thought to be neurotoxic indirectly through the release of nitric oxide, reactive oxygen species, eicosanoids and excitatory amino acids (Toulmond et al., 1996). Alternatively, IL-1b may sensitize spinal cord neurons through an up-regulation of nociceptive peptide expression, such as substance P (Shadiack et al., 1993; Jeanjean et al., 1995) and nerve growth factor (NGF) (Lindholm et al., 1987; Sa®eh-Garabedian et al., 1995). In addition to changes in protein expression, IL-1b may contribute to the synaptic plasticity and hyperexcitable state of spinal cord neurons, because it has been shown that pro-in¯ammatory cytokines enhance miniature endplate potentials at the neuromuscular junction (Caratsch et al., 1994), enhance the response of wide dynamic range (WDR) neurons to noxious stimuli (Oka et al., 1994), and produce long term potentiation (Patterson and Nawa, 1993). Finally, IL-1b may be inducing damage by inhibiting glial cells' ability to remove glutamate from the synaptic cleft, thus contributing to an excitotoxic state (Chao et al., 1995b).
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Fig. 6. Involvement of NF-k B in dynorphin-induced allodynia. (A) Timecourse of the effect of a 30 min pretreatment with the NF-k B inhibitor PDTC. Treatment with 10 pmol PDTC (open circles) but not 0.5 pmol (open crosses) inhibited the development of dynorphin-induced chronic allodynia (squares). (B) Dose±response curve of PDTC treatment. PDTC (0.001±10 000 pmol) administered 30 min before 3 nmol dynorphin dosedependently prevented the induction of allodynia (circles). PDTC (100± 10 000 pmol) preceding by a saline injection did not produce signi®cant allodynia (triangles). Mechanical allodynia was determined by application of the 0.4 mN von Frey ®lament to the dorsal hind paw. Time course data from part (A) were converted to area under the curve (AUC) for part (B). * P , 0:05 from the saline group (diamond), # p , 0:05 from the dynorphin 1 saline positive control group (square). n 8±10 mice per group, except n 12 for the dynorphin 1 saline group, n 15 for the saline 1 saline group, and n 17 for the 0.1 nmol PDTC 1 dynorphin group.
4.2. Involvement of transcription and translation in dynorphin-induced allodynia Cytokine production is regulated primarily at the transcriptional level (Baeuerle and Henkel, 1994; O'Neill and Kaltschmidt, 1997). Transcription factors provide the link between receptor-driven cytoplasmic signaling events and changes in gene expression. The ®rst part of this study demonstrated the involvement of protein synthesis in the development of chronic allodynia, because pretreatment
with cycloheximide dose-dependently reduced dynorphininduced allodynia (Fig. 1). The last part of this study demonstrated the involvement of the transcription factor NF-k B in the development of dynorphin-induced allodynia, because pretreatment with PDTC dose-dependently inhibited allodynia (Fig. 6). These results suggest that dynorphin may be initiating a genetic program through the activation of NFk B that results in the induction of a chronic pain state. The transcription factor NF-k B is one of several transcription factors that links early signaling with changes in gene expression (Baeuerle and Henkel, 1994). Many different stimuli, such as pro-in¯ammatory cytokines, nerve growth factor, and protein kinase C can activate NF-k B, whereas NF-k B regulates the expression of several proteins with nociceptive sequelae, such as pro-in¯ammatory cytokines, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2) and pre-pro-dynorphin (Barnes and Adcock, 1997; O'Neill and Kaltschmidt, 1997). NF-k B is constitutively expressed in low concentrations in the brain; however, expression increases after challenges such as seizure (Rong and Baudry, 1996), ischemia (Kaltschmidt et al., 1994; Salminen et al., 1995; Clemens et al., 1997), trauma (Yang et al., 1995; Perez-Otano et al., 1996), Alzheimer's disease (Terai et al., 1996), spinal cord injury (Bethea et al., 1998), and capsaicin-induced hyperalgesia (Chen et al., 1998). This pro®le suggests that activation of NF-k B may trigger the synthesis of a series of proteins that participate in some of the negative sequelae associated with these syndromes (Bethea et al., 1998; Brewer et al., 1999). This interpretation is supported by the observation that aspirin-induced inhibition of NF-k B activation is associated with neuroprotection against glutamate-induced cell death (Grilli et al., 1996). The relevance of NF-k B to nociception is supported by the observation that administration of hyperalgesic doses of NGF in vivo activates NF-k B in dorsal root gangla (Wood, 1995), and capsaicin-induced hyperalgesia is associated with enhanced expression of NF-k B (Chen et al., 1998). 4.3. Inhibition of the cytokine cascade PDTC, cycloheximide, and IL-10 were all used to disrupt a possible positive feed-forward loop occurring between pro-in¯ammatory cytokines and NF-k B. PDTC likely prevented NF-k B-initiated transcription, and cycloheximide probably prevented the translation of proteins possibly transcribed under the control of NF-k B. Inhibition of protein synthesis with cycloheximide has been shown to reduce the area of tissue damage following spinal cord injury (Yakovlev and Faden, 1994; Liu et al., 1997), ischemia (Goto et al., 1990; Aronowski et al., 1997; Kharlamov et al., 1997), and seizures (Jones et al., 1992; Williams and Jope, 1994), suggesting that a genetic program could contribute to the pathogenesis of these injuries. IL-10 may also inhibit a positive feed-forward loop through inhibition of the initial induction and subsequent ampli®cation of pro-in¯am-
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matory cytokines (Fiorentino et al., 1991; Balasingam and Yong, 1996; Rongione et al., 1997; Wagner et al., 1998), possibly through the inhibition of NF-k B activation (Wang et al., 1995; Lentsch et al., 1997; Ehrlich et al., 1998). In agreement with our results, a single peripheral administration of IL-10 has been shown to decrease nerve injuryinduced thermal hyperalgesia (Wagner et al., 1998) and intradermal cytokine-induced mechanical hyperalgesia (Poole et al., 1995). Administration of IL-10 has been shown to be neuroprotective in the quisqulate (Brewer et al., 1999) and weight drop (Bethea et al., 1999) models of spinal cord injury. In addition, the fact that levels of IL-10 increase in the autoimmune encephalomyelitis (EAE) spinal cord at clinical recovery suggests that IL-10 participates in the recovery from EAE (Issazadeh et al., 1995). Future treatment with IL-10 may provide a new therapeutic strategy for chronic pain patients by reducing the production of cytokines.
Furthermore, modulation of cytokine activity in the spinal cord may prove to be an effective therapeutic strategy for the treatment of chronic pain.
4.4. Conclusions
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The fact that treatment with the anti-in¯ammatory cytokine IL-10 reverses dynorphin-induced allodynia suggests that dynorphin is inducing the pro-in¯ammory cytokine system in the mouse spinal cord. Dynorphin has been shown to both inhibit (Kong et al., 1997) and enhance (Chao et al., 1995a) production of cytokines; thus, intrathecal administration of dynorphin could modulate the immune function of microglia and astrocytes in brain to induce chronic allodynia. This conclusion leads us to speculate that administration of dynorphin, or the enhanced expression of dynorphin during many chronic pain states (Cox et al., 1985; Iadarola et al., 1988; Kajander et al., 1990), may be initiating a cascade beginning with the NMDA receptor and resulting in the activation of NF-k B, consequently increasing the expression of cytokines, dynorphin itself, and other excitatory peptides. In support of this conclusion, dynorphin has been shown to interact with NMDA in addition to opioid receptors, and many of dynorphin's adverse effects, such as paralysis, chronic allodynia, and expanded receptor ®eld sizes, are dependent on activation of NMDA rather than of opioid receptors (Dubner and Ruda, 1992). Also, activation of NMDA receptors has been shown to cause an increase in the NF-k B activity (Guerrini et al., 1995; Kaltschmidt et al., 1995; Grilli et al., 1996). Subsequently, NF-KB could perhaps up-regulate the expression of nociceptive agents such as pro-in¯ammatory cytokines, nitric oxide synthase, cyclooxygenase and dynorphin (O'Neill and Kaltschmidt, 1997), any of which might damage spinal neurons, generate nociceptive signals and activate NF-k B (Bethea et al., 1998). In conclusion, we have shown that the pro-in¯ammatory cytokine IL-1b is involved in the mechanism of action of dynorphin-induced allodynia. In the future, determining how and where cytokines are interacting will provide greater insight into the mechanism of neuropathic pain.
Acknowledgements We thank Dr. L.S. Stone and Ms. Kristin L. Schreiber for their comments on this manuscript. Dynorphin A (1-17) was provided by NIDA, and interleukin-10 (IL-10) was a generous gift from Schering Plough Pharmaceuticals. Individual von Frey ®laments were a gift from North Coast Medical, Inc. (San Jose, CA). Supported by NIDA/R01-DA-04274 and R01-DA-01933 to GLW; NIDA training grant T32 DA07097 supported TML.
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Cytokine involvement in dynorphin-induced allodynia Tinna M. Laughlin a, John R. Bethea b, Robert P. Yezierski b, George L. Wilcox a,c,* a
b
Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA Department of Neurological Surgery and The Miami Project, University of Miami, Miami, FL 33136, USA c Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA Received 5 March 1999; received in revised form 25 May 1999; accepted 26 July 1999
Abstract Dynorphin A is an endogenous opioid peptide, which has previously been shown to produce a long-lasting allodynia and hyperalgesia in mice, behavioral states consistent with signs of clinically observed neuropathic pain. This dynorphin-induced allodynia was used as a pharmacological, central model of neuropathic pain. In this study, we examined the involvement of the cytokine IL-1b , the transcription factor nuclear factor kappa B (NF-k B), and de novo protein synthesis in the development of allodynia induced by intrathecal (i.t.) administration of dynorphin in male ICR mice. Pretreatment with the protein synthesis inhibitor cycloheximide (0.3±85 nmol), the NFk B inhibitor pyrrolidinedithiocarbamate (PDTC) (0.001±1000 pmol), the IL-1 receptor antagonist (IL-1ra) protein (0.01±100 ng), the caspase-1 inhibitor (YVAD) (0.1±300 pmol), and the anti-in¯ammatory cytokine IL-10 (0.1±300 ng) all dose-dependently reduced the induction of dynorphin-induced allodynia. Finally, IL-10 administered within the ®rst 24 h after the dynorphin insult prevented the development of chronic allodynia. These results demonstrate that the anti-in¯ammatory cytokines IL-10 and IL-1ra impede the development of dynorphin-induced allodynia. These results also suggest that production of new proteins through NF-k B activation is required for the induction of allodynia. We speculate that IL-1ra, IL-10, PDTC and cycloheximide interfere with the central pro-in¯ammatory cascade. Modulation of cytokine activity in the spinal cord may therefore prove to be an effective therapeutic strategy for the treatment of chronic pain. q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: IL-10; NF-k B; Interleukin-1beta; Cytokines; Protein synthesis; Neuropathic pain
1. Introduction Cytokines have long been associated with the peripheral in¯ammatory response, but recent data have revealed that cytokines also play a role in the pathology of brain injury and disease. Several studies have shown the involvement of cytokines in the pathology of several neurological diseases, including Alzheimer's disease (Grif®n et al., 1989), AIDS dementia complex (Merrill and Chen, 1991), multiple sclerosis (Hofman et al., 1986), and ischemic insult (Buttini et al., 1994; Loddick and Rothwell 1996). In addition to the neurodegenerative diseases, several groups have suggested that cytokines contribute directly to the pathology of neuropathic pain peripherally (Watkins et al., 1994; Wagner et al., 1998) and centrally (DeLeo et al., 1996; Watkins et al., 1997). In this study, we examined the involvement of the
* Corresponding author. Department of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church Street S.E., Minneapolis, MN 55455, USA. Tel.: 11-612-625-1474; fax: 11-612-625-3606. E-mail address: [email protected] (G.L. Wilcox)
interleukin-1 beta (IL-1b ) cytokine in the development of dynorphin-induced allodynia in mice. Pro-in¯ammatory cytokines, such as IL-1b , IL-6 and tumor necrosis factor-alpha (TNF-a ), are normally expressed in low concentrations in the brain; however, this expression increases after brain injury (Giulian and Lachman, 1985; Yan et al., 1992), ischemia (Buttini et al., 1994; Bhat et al., 1996), and in¯ammation (Wong et al., 1996; Woolf et al., 1997). These cytokines have also been shown to have a minimal constitutive expression in spinal cord, which increases after spinal cord injury (Wang et al., 1997; Bethea et al., 1999) and neuropathic pain (DeLeo et al., 1997). The enhanced expression of cytokines in these pathological states suggests that they have a role in the initiation or maintenance of the disease state. In this study, we investigated the involvement of the proin¯ammatory cytokine IL-1b in dynorphin-induced allodynia. IL-1b has been shown to mediate several responses in the CNS, including fever, anorexia, sleep and autonomic/ hormonal reactions (Dinarello, 1988). More importantly, several studies have indicated that IL-1b may have a role in nociception; for instance, central administration of IL-1b
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enhances the response of wide dynamic range neurons to noxious stimuli (Oka et al., 1994), dose-dependently enhances the amount of electrically evoked release of SP and CGRP from rat spinal cord slices (Malcangio et al., 1996), and induces mechanical hyperalgesia (Ferreira et al., 1988; Oka et al., 1993; Watkins et al., 1994; Yabuuchi et al., 1996). Blocking IL-1b activation of the IL-1 receptor with IL-1ra attenuated IL-1b -induced hyperalgesia (Oka et al., 1993), formalin-induced hyperalgesia (Watkins et al., 1997), and capsaicin-induced hyperalgesia (Davis and Perkins, 1996). In addition, the peripheral administration of formalin (Yabuuchi et al., 1996) and CFA (Woolf et al., 1997), which produce persistent nociceptive states, resulted in an increased expression of IL-1b . More recently, the levels of IL-1b in the synovial ¯uid have been shown to correlate with the level of pain in patients with chronic temporomandibular joint (TMJ) pain (Alstergren et al., 1998). Collectively, these studies lead us to test the hypothesis that central IL-1b is contributing to the development of dynorphin-induced allodynia in mice. 2. Materials and methods 2.1. Drugs and animals Cycloheximide and pyrrolidinedithiocarbamate (PDTC) were purchased from Sigma (St. Louis, MO), interleukin-1 receptor antagonist (IL-1ra) from R&D Systems (Minneapolis, MN), and caspase-1 inhibitor peptide Ac-Tyr-ValAla-Asp-aldehyde (YVAD) from Calbiochem (La Jolla, CA). Interleukin-10 (IL-10) was a gift from Schering Plough Pharmaceuticals, and dynorphin A (1-17) was provided by NIDA. All drugs were diluted in 0.9% saline and administered in a volume of 5 ml by intrathecal (i.t.) injections to awake male ICR mice (Harlan) using the method of Hylden and Wilcox (1980). All experiments were conducted so that the experimenter was blinded to the drug treatment. 2.2. Mechanical sensitivity assessment Mice were placed in individual 2 l bedding-lined beakers and allowed to adjust to the surroundings for 1 h prior to all behavioral testing. In order to study chronic nociception, we used the dynorphin-induced chronic allodynia model. In this model, a single intrathecal injection of dynorphin induces allodynia and hyperalgesia in mice (Laughlin et al., 1997) and rats (Vanderah et al., 1996) that last for at least 100 days; these behavioral states are consistent with signs of clinically observed neuropathic pain. Therefore, dynorphin-induced allodynia was used as a pharmacological model of neuropathic pain. Mechanical allodynia was determined with a set of von Frey ®laments modi®ed as previously described (Laughlin et al., 1997); these ®laments are used to stimulate the dorsal side of the hind paw. Individual von Frey ®laments were a gift from North Coast
Medical, Inc. (San Jose, CA). The innocuous 0.4 mN (#2.44) ®lament was applied to the point of bending 3 times to the dorsal surface of the left and right hind paw for a total of 6 applications per mouse. We have found that this ®lament elicits paw withdrawal responses only in mice exposed to allodynia-inducing manipulations such as intrathecally applied dynorphin (Laughlin et al., 1997). 2.3. Statistical analysis The response frequency of foot withdrawals for each time point was expressed as the number of positive responses divided by 6; this number was then multiplied by 100 to represent percent response. A mean and standard error of the mean (SEM) were calculated for each group from the percent response frequency. The seven day time course data for each animal were converted to a single point by adding the individual percent response frequency scores and dividing by the number of time points summed; then a mean ^ SEM was generated from these values. This mean was referred to as the area under the curve (AUC). The data were tested for signi®cance using analysis of variance (ANOVA), and statistical differences between groups were further analyzed with Dunnett's test for multiple compansons to a control (saline) group. P values of less than 0.05 were considered statistically signi®cant. 3. Results 3.1. Role of protein synthesis in dynorphin-induced allodynia Through the use of a protein synthesis inhibitor, we ®rst examined whether a genetic program is contributing to the mechanism of dynorphin-induced allodynia. A single systemic injection of the protein synthesis inhibitor cycloheximide has previously been shown to transiently suppress the synthesis of proteins in the rat brain (Pavlik and Teisinger, 1980). A single intrathecal pretreatment (30 mm) with cycloheximide (0.3±85 nmol) dose-dependently inhibited the induction of dynorphin-induced chronic allodynia (Fig. 1). Pretreatment with 3 and 85 nmol but not 0.3 nmol signi®cantly inhibited dynorphin-induced allodynia (Fig. 1B). Cycloheximide preceding a saline injection (control group) induced a small allodynia at the higher doses, 3 and 85 nmol (Fig. 1B). These results imply that events requiring protein synthesis are required during the establishment of the chronic allodynic state. 3.2. Role of interleukin-1b in dynorphin-induced allodynia Because IL-1b has been shown to have a role in nociception, we sought to determine the role of IL-b in dynorphininduced chronic allodynia. A 30 min pretreatment with the interleukin-1 receptor antagonist (0.01±100 ng) dose-
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Fig. 1. Involvement of protein synthesis in dynorphin-induced allodynia. (A) Time-course of the effect of a 30 min pretreatment with the protein synthesis inhibitor cycloheximide. Treatment with 85 nmol cycloheximide (open circles) fully inhibited allodynia, 3 nmol cycloheximide (open crosses) partially inhibited allodynia, and 0.3 nmol cycloheximide (open triangles) had no effect on development of dynorphin-induced chronic allodynia (squares). (B) Dose±response curve for cycloheximide treatment. Cycloheximide administered 30 min before 3 nmol dynorphin dose-dependently prevented the induction of allodynia (circles), whereas cycloheximide preceded by a saline injection produced no signi®cant allodynia (triangles). Mechanical allodynia was determined by application of the 0.4 mN von Frey ®lament to the dorsal hind paw. Time course data from part (A) were converted to area under the curve (AUC) for part (B). * P , 0:05 from the saline group (diamond), # P , 0:05 from the dynorphin 1 saline positive control group (square); n 8±10 mice per group.
Fig. 2. Effect of pretreatment with the interleukin-1 receptor antagonist (IL-1ra) in dynorphin-induced allodynia. (A) Time-course of the effect of a 30 min pretreatment with IL-1ra. Treatment with 1 ng IL-10 (open circles) but not 0.01 ng IL-1ra (open triangles) inhibited the development of dynorphin-induced chronic allodynia (squares). (B) Dose±response curve for IL1ra treatment. IL-1ra administered 30 min before 3 nmol dynorphin dosedependently prevented the induction of allodynia (circles). IL-1ra preceding a saline injection (triangles) produced no signi®cant allodynia at the 0.01±1 ng dose, whereas 100±300 ng IL-1ra induced allodynia. Mechanical allodynia was determined by application of the 0.4 mN von Frey ®lament to the dorsal hind paw. Time course data from part (A) were converted to area under the curve (AUC) for part (B). * P , 0:05 from the saline group (diamond), # P , 0:05 from the dynorphin 1 saline positive control group (square). n 8±10 mice per group, except n 3 mice for the 0.01, 0.1 and 100 ng IL-1ra plus saline groups.
dependently inhibited the induction of dynorphin-induced allodynia (Fig. 2). IL-1ra pretreatment signi®cantly decreased the response frequency from the dynorphin control in a dose-dependent manner: 0.1 ng and 1 ng were effective whereas 0.01 ng and 100 ng were not (Fig. 2B). These results suggest that dynorphin-induced allodynia may involve the activation of the IL-1 receptor. IL-1ra administered preceding a saline injection (control group) induced allodynia at the higher doses, 100 and 300 ng (Fig. 2B); this effect may account for the failure of 100 ng IL-1ra to inhibit dynorphin-induced allodynia and suggests that IL-1ra is behaviorally toxic at higher doses.
Caspase-1 (also known as interleukin-1b converting enzyme or ICE) is a protease that processes pro-interleukin-1b to the active form of IL-1b (Cerref® et al., 1992; Thornberry et al., 1992). Thus, caspase-1 inhibitors block the formation of active IL-1b . A 30 min pretreatment with the caspase-1 inhibitor, YVAD (0.2±200 pmol) dose-dependently blocked induction of allodynia (Fig. 3). All doses (0.2 pmol, 20 pmol and 200 pmol) were effective in reducing allodynia and none produced allodynia alone (Fig. 3B). The 0.2 pmol dose of YVAD initially inhibited allodynia, but the allodynia gradually returned to dynorphin 1 saline control group levels by day 5 (Fig. 3A). These results, together with the IL-1ra data, indicate that activation of
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Fig. 3. Involvement of the caspase-1 enzyme in dynorphin-induced allodynia. (A) Time-course of the effect of a 30 min pretreatment with the caspase-1 inhibitor, YVAD. Treatment with 20 pmol YVAD (open circles) prevented the development of dynorphin-induced chronic allodynia (squares). On the other hand, 0.2 pmol YVAD (open triangles) initially inhibited allodynia, and the allodynia gradually returned to dynorphin 1 saline control group (squares) levels (B) Dose±response curve for YVAD. YVAD administered 30 min before 3 nmol dynorphin dose-dependently prevented the induction of allodynia (circles); 200 pmol of YVAD preceding a saline injection did not produce signi®cant allodynia (triangle). Mechanical allodynia was determined by application of the 0.4 mN von Frey ®lament to the dorsal hind paw. Time course data from part (A) were converted to area under the curve (AUC) for part (B). * P , 0:05 from the saline group (diamond), # P , 0:05 from the dynorphin 1 saline positive control group (square); n 7±10 mice per group.
the IL-1b signaling cascade is required for the development of dynorphin-induced allodynia. 3.3. Effect of interleukin-10 treatment Having demonstrated that the pro-in¯ammatory cytokine IL-1b is involved in dynorphin-induced allodynia, we next determined the potential of the anti-in¯ammatory cytokine IL-10 to prevent the induction of allodynia. IL-10 is an antiin¯ammatory cytokine that, among other actions, inhibits the synthesis and secretion of pro-in¯ammatory cytokines, such as IL-1b , IL-6, and TNF-a (Fiorentino et al., 1991; Balasingam and Yong, 1996; Rongione et al., 1997; Wagner
Fig. 4. Effect of pretreatment with the interleukin-10 anti-in¯ammatory cytokine in dynorphin-induced allodynia. (A) Time-course of the effect of a 30 min pretreatment with IL-10. Pretreatment with 1 ng IL-10 (open circles) but not 0.1 ng IL-10 (open squares) prevented the development of dynorphin-induced chronic allodynia (triangles). (B) Dose±response curve of IL-10 treatment. IL-10 administered 30 min before 3 nmol dynorphin dose-dependently prevented the induction of allodynia (circles). IL-10 preceding a saline injection (triangles) did not produce any signi®cant allodynia at the 0.1 and 1 ng dose. Mechanical allodynia was determined by application of the 0.4 mN von Frey ®lament to the dorsal hind paw. Time course data from part (A) were converted to area under the curve (AUC) for part (B). * P , 0:05 from the saline group (diamond), # P , 0:05 from the dynorphin 1 saline postive control group (square); n 7±10 mice per group.
et al., 1998). A 30 min pretreatment with IL-10 (0.1±10 ng) dose-dependently inhibited the induction of dynorphininduced allodynia (Fig. 4). Pretreatment with 0.6 ng, 1 ng and 10 ng IL-10 signi®cantly decreased the response frequency from the dynorphin 1 saline control group, whereas 0.1 ng and 0.3 ng IL-10 were ineffective (Fig. 4B). IL-10 has been shown with repeated doses to cause an increase in tissue damage following injury (Bethea et al., 1999; Brewer et al., 1999); in addition, we have shown that intrathecal administration of 300 ng IL-10 by itself induces signi®cant allodynia (data not shown). This effect may explain why the IL-10 dose±response curve (Fig. 4B) starts to turn upward at high doses, limiting its maximum ef®cacy. These results, along with the cycloheximide results, suggest that the synthesis and secretion of pro-
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in¯ammatory cytokines are involved in the development of dynorphin-induced allodynia. To determine the therapeutic potential of IL-10 in preexisting conditions, we next determined the ability of IL-10 post-treatment to diminish dynorphin-induced allodynia. IL-10 (1 ng, i.t.), administered sequentially at three different time points, 30 min, 6 h, and 24 h after dynorphin, prevented the development of dynorphin-induced allodynia (Fig. 5). Mice administered 1 ng of IL-10 once at 30 min after dynorphin were allodynic 1 day after dynorphin; thereafter, the allodynia was signi®cantly reduced to saline control levels (Fig. 5). Finally, a single administration of 1 ng IL-10 at 24 h after dynorphin did not reduce allodynia (Fig. 5). Thus, there appears to be a critical period and duration of less then 24 h after insult in which IL-10 may be therapeutic. 3.4. Effect of NF-k B inhibition Having shown that protein synthesis (Fig. 1) and, more speci®cally, the synthesis of pro-in¯ammatory cytokines (Fig. 4) may be involved in the development of dynorphin-induced allodynia, we next examined the possible role of the transcriptional factor, NF-k B, in this model. Pyrrolidinedithiocarbamate (PDTC) is an antioxidant that has been shown to be a potent inhibitor of the activation of NF-k B, (Schreck et al., 1992; Cooper et al., 1998; Heese et al., 1998; Iuvone et al., 1998). A 30 min pretreatment with PDTC (1 fmol±100 nmol) dose-dependently inhibited the induction of dynorphin-induced allodynia (Fig. 6). Pretreatment with 1, 10, 100, and 1000 pmol PDTC signi®-
Fig. 5. Effect of post-treatment with IL-10 in dynorphin-induced allodynia. Both repetitive administration of 1 ng IL-10 (30 min, 6 h, 24 h after 3 nmol dynorphin; open triangles), and a single treatment of 1 ng IL-10 at 30 min after dynorphin (open circles) signi®cantly reduced chronic allodynia. Administration of 1 ng IL-10 1 day after dynorphin (open crosses) injection did not alleviate dynorphin-induced chronic allodynia (squares). The mean ^ SEM represents the percent response frequency of foot withdrawals to 6 applications of the 0.4 mN ®lament to the dorsal hind paw. n 8±10 mice per group.
163
cantly reduced mechanical allodynia, 0.5 pmol PDTC produced a small reduction in the amount of allodynia, and 1 fmol PDTC had no effect (Fig. 6). Saline or PDTC administered preceding a saline injection produced no signi®cant allodynia (Fig. 6). These results suggest that transcriptional regulation through NF-k B may be required for the development of a chronic allodynic state. 4. Discussion The present study demonstrates that the IL-1b cytokine system is required for the development of dynorphininduced allodynia. First, pretreatment with IL-1ra (0.01± 100 ng) and YVAD (0.1±300 pmol) dose-dependently inhibited the induction of dynorphin-induced allodynia, results which support the involvement of the pro-in¯ammatory cytokine IL-1b in the development of dynorphininduced allodynia. Second, pretreatment with the protein synthesis inhibitor cycloheximide (0.3±85 nmol), the NFk B inhibitor PDTC (0.001±1000 pmol) and the anti-in¯ammatory cytokine IL-10 (0.1±300 ng) dose-dependently inhibited the induction of dynorphin-induced allodynia, results that suggest the involvement of the transcription and synthesis of cytokine/cytokine-induced proteins in dynorphin-induced allodynia. Finally, IL-10, administered within the ®rst 24 h after the dynorphin insult, signi®cantly reduced the development of dynorphin-induced chronic allodynia, suggesting a therapeutic application for this anti-in¯ammatory cytokine. 4.1. Involvement of IL-1b in dynorphin-induced allodynia In this study, we have shown that the pro-in¯ammatory cytokine IL-1b contributes to the development of dynorphin-induced allodynia. IL-1b may be contributing to the development of this allodynia through several mechanisms. Cytokines are thought to be neurotoxic indirectly through the release of nitric oxide, reactive oxygen species, eicosanoids and excitatory amino acids (Toulmond et al., 1996). Alternatively, IL-1b may sensitize spinal cord neurons through an up-regulation of nociceptive peptide expression, such as substance P (Shadiack et al., 1993; Jeanjean et al., 1995) and nerve growth factor (NGF) (Lindholm et al., 1987; Sa®eh-Garabedian et al., 1995). In addition to changes in protein expression, IL-1b may contribute to the synaptic plasticity and hyperexcitable state of spinal cord neurons, because it has been shown that pro-in¯ammatory cytokines enhance miniature endplate potentials at the neuromuscular junction (Caratsch et al., 1994), enhance the response of wide dynamic range (WDR) neurons to noxious stimuli (Oka et al., 1994), and produce long term potentiation (Patterson and Nawa, 1993). Finally, IL-1b may be inducing damage by inhibiting glial cells' ability to remove glutamate from the synaptic cleft, thus contributing to an excitotoxic state (Chao et al., 1995b).
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Fig. 6. Involvement of NF-k B in dynorphin-induced allodynia. (A) Timecourse of the effect of a 30 min pretreatment with the NF-k B inhibitor PDTC. Treatment with 10 pmol PDTC (open circles) but not 0.5 pmol (open crosses) inhibited the development of dynorphin-induced chronic allodynia (squares). (B) Dose±response curve of PDTC treatment. PDTC (0.001±10 000 pmol) administered 30 min before 3 nmol dynorphin dosedependently prevented the induction of allodynia (circles). PDTC (100± 10 000 pmol) preceding by a saline injection did not produce signi®cant allodynia (triangles). Mechanical allodynia was determined by application of the 0.4 mN von Frey ®lament to the dorsal hind paw. Time course data from part (A) were converted to area under the curve (AUC) for part (B). * P , 0:05 from the saline group (diamond), # p , 0:05 from the dynorphin 1 saline positive control group (square). n 8±10 mice per group, except n 12 for the dynorphin 1 saline group, n 15 for the saline 1 saline group, and n 17 for the 0.1 nmol PDTC 1 dynorphin group.
4.2. Involvement of transcription and translation in dynorphin-induced allodynia Cytokine production is regulated primarily at the transcriptional level (Baeuerle and Henkel, 1994; O'Neill and Kaltschmidt, 1997). Transcription factors provide the link between receptor-driven cytoplasmic signaling events and changes in gene expression. The ®rst part of this study demonstrated the involvement of protein synthesis in the development of chronic allodynia, because pretreatment
with cycloheximide dose-dependently reduced dynorphininduced allodynia (Fig. 1). The last part of this study demonstrated the involvement of the transcription factor NF-k B in the development of dynorphin-induced allodynia, because pretreatment with PDTC dose-dependently inhibited allodynia (Fig. 6). These results suggest that dynorphin may be initiating a genetic program through the activation of NFk B that results in the induction of a chronic pain state. The transcription factor NF-k B is one of several transcription factors that links early signaling with changes in gene expression (Baeuerle and Henkel, 1994). Many different stimuli, such as pro-in¯ammatory cytokines, nerve growth factor, and protein kinase C can activate NF-k B, whereas NF-k B regulates the expression of several proteins with nociceptive sequelae, such as pro-in¯ammatory cytokines, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2) and pre-pro-dynorphin (Barnes and Adcock, 1997; O'Neill and Kaltschmidt, 1997). NF-k B is constitutively expressed in low concentrations in the brain; however, expression increases after challenges such as seizure (Rong and Baudry, 1996), ischemia (Kaltschmidt et al., 1994; Salminen et al., 1995; Clemens et al., 1997), trauma (Yang et al., 1995; Perez-Otano et al., 1996), Alzheimer's disease (Terai et al., 1996), spinal cord injury (Bethea et al., 1998), and capsaicin-induced hyperalgesia (Chen et al., 1998). This pro®le suggests that activation of NF-k B may trigger the synthesis of a series of proteins that participate in some of the negative sequelae associated with these syndromes (Bethea et al., 1998; Brewer et al., 1999). This interpretation is supported by the observation that aspirin-induced inhibition of NF-k B activation is associated with neuroprotection against glutamate-induced cell death (Grilli et al., 1996). The relevance of NF-k B to nociception is supported by the observation that administration of hyperalgesic doses of NGF in vivo activates NF-k B in dorsal root gangla (Wood, 1995), and capsaicin-induced hyperalgesia is associated with enhanced expression of NF-k B (Chen et al., 1998). 4.3. Inhibition of the cytokine cascade PDTC, cycloheximide, and IL-10 were all used to disrupt a possible positive feed-forward loop occurring between pro-in¯ammatory cytokines and NF-k B. PDTC likely prevented NF-k B-initiated transcription, and cycloheximide probably prevented the translation of proteins possibly transcribed under the control of NF-k B. Inhibition of protein synthesis with cycloheximide has been shown to reduce the area of tissue damage following spinal cord injury (Yakovlev and Faden, 1994; Liu et al., 1997), ischemia (Goto et al., 1990; Aronowski et al., 1997; Kharlamov et al., 1997), and seizures (Jones et al., 1992; Williams and Jope, 1994), suggesting that a genetic program could contribute to the pathogenesis of these injuries. IL-10 may also inhibit a positive feed-forward loop through inhibition of the initial induction and subsequent ampli®cation of pro-in¯am-
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matory cytokines (Fiorentino et al., 1991; Balasingam and Yong, 1996; Rongione et al., 1997; Wagner et al., 1998), possibly through the inhibition of NF-k B activation (Wang et al., 1995; Lentsch et al., 1997; Ehrlich et al., 1998). In agreement with our results, a single peripheral administration of IL-10 has been shown to decrease nerve injuryinduced thermal hyperalgesia (Wagner et al., 1998) and intradermal cytokine-induced mechanical hyperalgesia (Poole et al., 1995). Administration of IL-10 has been shown to be neuroprotective in the quisqulate (Brewer et al., 1999) and weight drop (Bethea et al., 1999) models of spinal cord injury. In addition, the fact that levels of IL-10 increase in the autoimmune encephalomyelitis (EAE) spinal cord at clinical recovery suggests that IL-10 participates in the recovery from EAE (Issazadeh et al., 1995). Future treatment with IL-10 may provide a new therapeutic strategy for chronic pain patients by reducing the production of cytokines.
Furthermore, modulation of cytokine activity in the spinal cord may prove to be an effective therapeutic strategy for the treatment of chronic pain.
4.4. Conclusions
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The fact that treatment with the anti-in¯ammatory cytokine IL-10 reverses dynorphin-induced allodynia suggests that dynorphin is inducing the pro-in¯ammory cytokine system in the mouse spinal cord. Dynorphin has been shown to both inhibit (Kong et al., 1997) and enhance (Chao et al., 1995a) production of cytokines; thus, intrathecal administration of dynorphin could modulate the immune function of microglia and astrocytes in brain to induce chronic allodynia. This conclusion leads us to speculate that administration of dynorphin, or the enhanced expression of dynorphin during many chronic pain states (Cox et al., 1985; Iadarola et al., 1988; Kajander et al., 1990), may be initiating a cascade beginning with the NMDA receptor and resulting in the activation of NF-k B, consequently increasing the expression of cytokines, dynorphin itself, and other excitatory peptides. In support of this conclusion, dynorphin has been shown to interact with NMDA in addition to opioid receptors, and many of dynorphin's adverse effects, such as paralysis, chronic allodynia, and expanded receptor ®eld sizes, are dependent on activation of NMDA rather than of opioid receptors (Dubner and Ruda, 1992). Also, activation of NMDA receptors has been shown to cause an increase in the NF-k B activity (Guerrini et al., 1995; Kaltschmidt et al., 1995; Grilli et al., 1996). Subsequently, NF-KB could perhaps up-regulate the expression of nociceptive agents such as pro-in¯ammatory cytokines, nitric oxide synthase, cyclooxygenase and dynorphin (O'Neill and Kaltschmidt, 1997), any of which might damage spinal neurons, generate nociceptive signals and activate NF-k B (Bethea et al., 1998). In conclusion, we have shown that the pro-in¯ammatory cytokine IL-1b is involved in the mechanism of action of dynorphin-induced allodynia. In the future, determining how and where cytokines are interacting will provide greater insight into the mechanism of neuropathic pain.
Acknowledgements We thank Dr. L.S. Stone and Ms. Kristin L. Schreiber for their comments on this manuscript. Dynorphin A (1-17) was provided by NIDA, and interleukin-10 (IL-10) was a generous gift from Schering Plough Pharmaceuticals. Individual von Frey ®laments were a gift from North Coast Medical, Inc. (San Jose, CA). Supported by NIDA/R01-DA-04274 and R01-DA-01933 to GLW; NIDA training grant T32 DA07097 supported TML.
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