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Mechanisms of neuropathic pain: the role of cytokines
Vol. 1, No. 4 2004
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Tamas Bartfai – Harold L. Dorris Neurological Research Center and The Scripps Research Institute, USA
DISEASE Pain MECHANISMS
Mechanisms of neuropathic pain: the role of cytokines Claudia Sommer*, Maria Scha¨fers Department of Neurology, University of Wu¨rzburg, Josef-Schneider-Strasse 11, 97080 Wu¨rzburg, Germany
Many experimental studies have provided evidence that pro-inflammatory cytokines induce or increase neuropathic pain. Direct receptor-mediated actions of cytokines have been demonstrated in addition to those involving further mediators. In spite of the redundancy and pleiotropy of the cytokine network, specific actions of individual cytokines have been identified. Preliminary clinical data point to a possible beneficial
Section Editor: Eija Kalso – Helsinki University Central Hospital, Finland Claudia Sommer and her coworkers at the University of Wu¨rzburg are pioneers in the research of cytokines and neuropathic pain. Proinflammatory cytokines increase the sensitivity of damaged neurones to nociceptive and non-nociceptive stimuli. Cytokines may be an important link between infections, inflammation and the development and maintenance of certain types of neuropathic pain. Blocking or inhibiting the effects of cytokines offers an exciting new approach to the prevention and treatment of some neuropathic pain states.
role of cytokine inhibition in patients with painful neuropathy or radiculopathy. Cytokines in neuropathic pain Introduction are extracellular signaling proteins phylogenetically related to opioid peptides [1] (see Glossary). They establish communication between the immune system and the nervous system, acting at hormonal concentrations through high-affinity receptors and producing endocrine, paracrine and autocrine effects. In contrast to circulating hormones, they exert their effects over short distances onto nearby cells. Cytokines are called ‘pleiotropic’, because of a broad range of redundant, frequently overlapping functions. Their activation or dysregulation is implied in a variety of disease states, for example, sepsis, rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease, multiple sclerosis and skin diseases. Some cytokines are labeled ‘pro-inflammatory’ or ‘Th1’, others ‘anti-inflammatory’ or ‘Th2’, depending on their effects on immune cells, in particular on lymphocytes. Recently, evidence has emerged that cytokines are involved in the generation of pain and hyperalgesia in inflammatory and neuropathic conditions.
CYTOKINES
*Corresponding author: (C. Sommer) [email protected] 1740-6765/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2004.11.018
Most of the evidence for a role of cytokines in neuropathic pain comes from animal studies using nerve injury models. In these models, cytokine levels are altered (mostly increased) in the peripheral and central nervous system (CNS). Modulation of the cytokine system either by application of agonists or antagonists or by transgenic approaches modulates pain behavior. Hypotheses on potential mechanisms involved in cytokine actions derived from in vitro studies. In addition, clinical studies measuring cytokine levels in humans with neuropathic pain or using drugs modulating cytokine function further support the idea of key role of cytokines in the development and maintenance of neuropathic pain states.
Evidence from animal models Cytokine regulation after nerve injury
In response to axonal lesions a series of inflammatory mediators, including cytokines are upregulated (for review see [2,3]). In a frequently used model of neuropathic pain, the chronic constriction injury (CCI) of the rat or mouse sciatic nerve, increased levels of tumor necrosis factor-alpha (TNF-A), interwww.drugdiscoverytoday.com
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Glossary Cytokines: extracellular signaling proteins within the immune system and between the immune system and nervous system. Etanercept: a TNF-receptor fusion protein that serves as a TNF-a inhibitor in human autoimmune disorders, mainly used in rheumatoid arthritis. Is beginning to be used for Crohn’s disease and sciatica. IL-1b: interleukin-1b, pro-inflammatory cytokine with algesic actions and with many properties similar to TNF-a. At higher endocrine concentrations it is associated with fever and formation of acute-phase plasma proteins in the liver. IL-4: interleukin-4, anti-inflammatory cytokine that has also been shown to have analgesic properties. IL-6: cytokine with mostly pro-inflammatory and algesic actions, member of the IL-6 cytokine family that includes leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). IL-10: interleukin-10, anti-inflammatory cytokine that has also been shown to have analgesic properties. Infliximab: monoclonal antibodies to TNF-a that serve as a TNF-a inhibitor in human autoimmune disorders. Nucleus pulposus: inner content of the spinal discs, surrounded by anulus fibrosus and released in the case of spinal disc herniation. TNF-a: tumor necrosis factor-alpha, pro-inflammatory cytokine, member of the ‘TNF-superfamily’, together with the structurally related peptide lymphotoxin (LT-a, formerly TNF-b) and several other structurally related proteins.
leukin-1b (IL-1B) and interleukin-6 (IL-6) (see Glossary) have been shown at the mRNA, and partly also at the protein level [4–7] (Fig. 1). By contrast, protein levels of a prototypical antiinflammatory cytokine, interleukin-10 (IL-10) are decreased in the injured nerve [7]. Cytokines in peripheral nerves have been mostly localized to Schwann cells or macrophages, occasionally to fibroblasts (Fig. 2). In dorsal root ganglion (DRG) neurons, cytokine levels are not only increased after nerve injury [8], but there is also a phenotypic switch leading to TNFa expression in a population of medium size DRG neurons [9], indicating a possible alteration in the function of these neurons. Increases in cytokine expression have also been found in the lumbar spinal cord after CCI and in other partial nerve injury models [10–12] as well as in particular regions of the CNS [13,14]. In particular, spinal glia was found to be important in cytokine expression after nerve injury [15]. Studies using cytokine application or inhibition
Intra- or epineurial injection of IL-1b or TNF-a produces a concentration-dependent pain behavior in rats ([16,17], M. Zelenka et al., unpublished). Several investigators have shown an antihyperalgesic and antiallodynic effect of cytokine inhibition in models of neuropathic pain (for review and further references see [2]). Antagonism to IL-1 by using neutralizing antibodies to IL-1RI reduces thermal hyperalgesia and mechanical allodynia in mice with CCI and attenuates the endoneurial increase of TNF immunoreactivity. Intrathecal application of anti-IL-6 antibodies decreases tactile allodynia in the model of spinal nerve ligation (SNL). Epineurial injection of antibodies to the IL-6 receptor attenuates thermal hyperalgesia and 442
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mechanical allodynia in CCI. Pain-related behavior in CCI and other models is reduced by substances blocking TNF production, its release from the cell membrane or its function. The hyperalgesic action of TNF seems to be mediated by TNFR1, because neutralizing antibodies to TNFR1 but not to TNFR2 can reduce hyperalgesia. Anti-inflammatory cytokines like IL-4 and IL-10 seem to have antihyperalgesic actions in animal models of neuropathic pain [18]. IL-10 pre-treatment also reduces the hyperalgesic responses to intraplantar injections of carrageenin, IL-1b, IL-6 and TNF-a [19]. IL-4 delivered by a viral vector reduces behavioral signs of pain in an animal model of bone cancer [20], and intrathecally applied human IL-2 has a short-lived antinociceptive effect in the CCI-model [21]. Spinal administration of TNF-a antagonists prevents or reduces allodynia and hyperalgesia in animals with nerve injury and pain behavior. Whereas intrathecal IL-1b antagonists alone are seemingly without effect in nerve injury models, they synergize with TNF-a antagonists causing a further reduction of allodynia [22]. Spinal administration of IL-6, by contrast, appears to reduce electrically evoked C-fiber activity [23]. Furthermore, intrathecal application of inhibitors of glial metabolism also reduce neuropathic pain, indicating that indeed glial cells increase pain through the action of cytokines [24]. Nucleus pulposus, the material within the vertebral discs, is highly enriched with a variety of pro-inflammatory cytokines. Subsequent to disc herniation, this material probably comes into contact with the dorsal roots. Application of autologous nucleus pulposus to dorsal roots or experimental disc herniation in animals results in pain behavior as well as both ongoing and enhanced evoked activity in spinal nociceptive neurons [25]. Animals treated with either neutralizing antibodies to TNF-a directly on the nerve root or with systemic TNF-a antagonists show a marked reduction of both the neuronal activity and the pain behavior, implicating a role for TNF-a in the process [25]. Mechanisms of action
IL-1b, among other actions involving secondary production of nitric oxide, bradykinin or prostaglandins, has a direct excitatory action on nociceptive fibers, which are activated within 1 min by IL-1b application [26]. In a skin-nerve in vitro preparation, brief exposure of the skin to IL-1b facilitates heat-evoked calcitonin gene-related peptide release [27], which is a direct effect independent of changes in gene expression or receptor up-regulation. Brief applications of IL-1b to nociceptive neurons yielded a potentiation of heat-activated inward currents and a shift of activation thresholds towards lower temperatures without altering intracellular calcium levels. This IL-1b-induced heat sensitization is mediated by activation of protein kinases. IL-1 receptor is expressed in DRG neurons, such that IL-1b can act directly on sensory neurons to increase their susceptibility
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Figure 1. Relative gene expression of the pro-inflammatory cytokines IL-1b, TNF-a, the chemokine MCP-1 and the anti-inflammatory cytokine IL-10 in degenerating mouse nerve segments after chronic constriction injury (CCI; left panel) compared to sciatic nerve (SN) crush (right panel) as revealed by realtime RT-PCR and normalization against the housekeeping gene 18s RNA. Note the rapid increase in transcripts at day one after injury and the higher and more sustained expression levels after CCI. Abbreviations: Ctrl., Control nerves. Data from [6].
to noxious heat [28]. Similar effects have been shown for IL-6 in conjunction with the soluble IL-6 receptor. TNF-a lowers mechanical activation thresholds in C nociceptors of the rat sural nerve when injected subcutaneously [29], a result that might be due to an acute TNF-induced decrease in K+ conductance [30] or to a protein kinase Adependent mechanism [31]. In vitro perfusion of TNF-a to dorsal root ganglia (DRG) elicits neuronal discharges in both A and C-fibers (Fig. 3). The firing frequency is markedly higher and the discharge longer-lasting after nerve injury, indicating an increased sensitivity of injured afferent neurons to TNF-a [32]. Injection or perfusion of TNF-a into or onto rat DRGs in vivo induces allodynia. Subthreshold quantities of TNF-a injected into a DRG when its spinal nerve is ligated results
in faster onset of allodynia and increased spontaneous pain behavior. Thus, there is strong in vivo and in vitro evidence that nerve injury results in increased endogenous TNF-a and that injured nerve fibers are sensitized to the excitatory effects of TNF-a. Receptor-mediated activation of protein kinases or calcium mobilization in sensory neurons may confer the sensitizing effects that TNF-a has on nociceptors in vitro [33]. Downstream of TNF-a receptor activation, hyperalgesia induced by nerve injury is mediated via p38 MAPK [34]. The TNF-a inhibitor etanercept reduces both allodynia and p38 MAPK phosphorylation in the SNL-model of neuropathic pain, indicating that the TNF-a-p38 signal transduction cascade in the DRG is a significant participant in the generation of mechanical allodynia following nerve injury (Fig. 4). www.drugdiscoverytoday.com
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Figure 2. TNF immunolocalization in rat lumbar dorsal root ganglion (DRG) neurons and sciatic nerve four days after CCI: single labeling on serial sections demonstrating a heavy co-localization (yellow) of TNF (red) and the neuronal marker PGP 9.5 (green). Double staining demonstrating almost no co-localization of TNF-a (red) and ED1 (green). In the sciatic nerve, TNF-a (red) co-localizes (yellow) with the Schwann cell marker S-100 (green). There is almost no co-localization of TNF-a and ED1-positive macrophages (green).
In addition to the direct actions on nerve fibers, indirect actions of the cytokines mediated by other algogenic compounds, are likely in many paradigms (Fig. 5). Nerve growth factor (NGF) is one candidate mediator of cytokine-induced hyperalgesia. For example, IL-1b induces transcription of NGF in Schwann cells [35], and IL-1b-induced hyperalgesia can be prevented by anti-NGF antibodies [36], indicating that IL-1b hyperalgesia is mediated via the increase in NGF in inflamed nerves. Furthermore, prostaglandins, bradykinin and neuropeptides might mediate cytokine–hyperalgesia. TNF-a enhances the sensitivity to capsaicin of rat sensory neurons [37], probably via the neuronal production of prostaglandins. Long-time exposure of primary afferent neurons to IL-1b induces substance P release via the cyclooxygenase (COX)-2 system [38]. IL-6 induces the production of galanin in sensory neurons [39]. These changes in neurotransmitter expression might contribute to alter pain processing after nerve injury. An interesting finding is the possible relevance of spinal cytokines to opioid tolerance. In rats, chronic administration of morphine activates spinal glia to upregulated pro-inflammatory cytokines, a process which is further enhanced in nerve injured rats. Inhibition of pro-inflammatory cytokines at the spinal level restores acute morphine antinociception in these rats and reverses the development of morphine tolerance [40]. Thus, the long-term response to opioids in neuropathic pain might be improved by concomitant modulation of the cytokine system.
Evidence from human diseases Information about the role of cytokines in neuropathic pain in humans is derived from studies correlating cytokine levels 444
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Figure 3. In vitro extracellular dorsal root single fiber recording. (a) Schematic of the preparation. The ganglion is suspended in the middle chamber; the dorsal root and spinal nerve are in the adjacent mineral oilfilled chambers for recording (R) and stimulation (S), respectively. (b) Activity was recorded from one A-delta fiber while the DRG was perfused with TNF-a. The fiber responds with firing in response to perfusion of the DRG with TNF-a (here 100 pg/ml).
in body fluids or biopsied tissues and from a few, mostly uncontrolled, treatment trials. A striking correlation between cytokine levels and neuropathic pain was observed in patients with leprosy, where a subgroup of patients with elevated serum levels of TNF-a and IL-1b suffer from excruciating pain [41]. Treatment with thalidomide reduces TNF-a secretion in peripheral blood mononuclear cells by >90% and greatly reduces pain in these patients [42]. In other human neuropathies, preliminary data also point to a correlation between cytokine expression and pain [43,44]. In a series of sural nerve biopsies, cytokine levels were increased more often in patients with painful neuropa-
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Figure 4. Pain-behavior induced by spinal nerve ligation (SNL) is attenuated by systemic inhibition of TNF and intrathecal inhibition of the MAPK p38. (a) Treatment with the TNF antagonist etanercept attenuates mechanical allodynia induced by SNL. (b) Treatment with the p38 inhibitor SB203580 attenuates mechanical allodynia if started two days before SNL, but not if starting seven days after SNL. Data from [34].
thies. Recently, an increase in serum IL-8 was identified as a predictor for the development of postherpetic neuralgia (PHN) after acute herpes zoster [45]. If this finding holds true in further case series, IL-8 might represent not only a predictor but also a possible target for the prevention of PHN. Several studies have reported increased levels of cytokines in the vicinity of herniated discs and a correlation of their presence to sciatica [46–48]. Inhibitors of TNF-a have been used successfully for patients with chronic nerve root pain, but up to now only data from case reports and uncontrolled studies are available [49]. Occasionally, TNF-a inhibitors have been used to treat inflammatory neuropathies, but data on pain are lacking in these reports [50]. One remarkable case report describes remission of long standing complex regional pain syndrome (CRPS) after treatment with thalidomide for Behc¸et’s disease [51]. Anti-TNF-a strategies have been used in various nonneuropathic painful conditions like AIDS-associated proctitis, rheumatoid arthritis and HIV-associated aphthous ulcers (for review on earlier studies see [52]). Many other reports have followed since the advent of etanercept and infliximab, for example, a controlled trial showing a beneficial effect of etanercept in ankylosing spondylitis [53], and a prospective study with historical controls using infliximab in low back pain [54] (Table 1). Controlled trials are warranted to establish the role of cytokine inhibition in these painful conditions. Obviously, caution is necessary because of the pleiotropy of the cytokines. Treatment with antagonists might not only reduce the target symptom (pain) but also have undesired side effects in other systems. Furthermore, given the redundancy of the cytokine system, blockade of one cytokine may be compensated by upregulation of others with similar effects. Therefore, combined antagonistic strategies might be necessary.
Summary and conclusions Numerous experimental studies provide evidence that proinflammatory cytokines induce or facilitate neuropathic Table 1. Key therapies – pre-clinical to market Target
Therapy against target
Stage of development
Advantages/Disadvantages
References
TNF-a
Etanercept (dimeric TNFR2 fusion protein) Infliximab (chimeric monocloncal TNF antibody) Adalimumab (human IgG1 anti-tumor necrosis factor alpha monoclonal antibody)
On market for rheumatoid arthritis, Crohn’s disease Case reports for low back pain, sciatica
Mostly well-tolerated Rare side effects are: injections site reactions, rash, respiratory tract infections, for example, tuberculosis, demyelinating disorders, aplastic anemia, worsening of congestive heart failure, back pain
[54–59]
IL-1
Anakinra (IL-1 inhibitor)
On market for rheumatoid arthritis
Unknown
[60]
IL-2, IL-4, IL-10
Gene therapy
Experimental use for cancer pain, neuropathic pain, inflammatory pain
Unknown
[21,61]
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Figure 5. Possible currently discussed mechanisms by which TNF as the prototypical proinflammatory cytokine induces neuropathic pain: TNF exerts pleitropic effects on Schwann cells, endothelial cells, axons, dorsal root ganglia neurons and the CNS. Abbreviations: EC, endothelial cells; NGF, nerve growth factor; CGRP: calcitonin gene-related peptide; DRG, dorsal root ganglion; CNS: central nervous system; MAPK: mitogen-activated protein kinases; PKA, proteinkinase A; SPA, spontaneous activity.
pain. Direct receptor-mediated actions of cytokines on afferent nerve fibers as well as cytokine effects involving further mediators have been reported. Furthermore, endogenous cytokine levels may modulate the response to opioids. Cytokine levels are rapidly and markedly upregulated in peripheral nerves, DRGs and in the spinal cord after peripheral nerve injuries. Whereas direct application of exogenous pro-inflammatory cytokines induces pain, blockade of these cytokines or application of anti-inflammatory cytokines reduces pain behavior in most experimental paradigms. A finding of particular clinical importance may be the involvement of TNF-a released from spinal discs in the generation and maintenance of sciatica. Cytokine measurements might identify patients at risk to develop chronic pain associated to their neuropathic conditions, as in the examples of peripheral neuropathies and PHN. Case reports and uncontrolled trials point to a positive effect of TNF-a inhibition in sciatica and possibly other neuropathic conditions. Because of the pleiotropy and redun-
Outstanding questions What are the molecular mechanisms through which cytokines induce or maintain pain? Could p38 mitogen-activated protein kinase be an alternative target for pain control?
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How can we identify those patients in whom cytokines play a major role in the generation of pain? Is blockade of one cytokine (as exemplified in the TNF inhibitors) a valid strategy for pain control in selected diseases, or would a more successful approach involve shifting the balance between pro- and anti-inflammatory cytokines?
dancy of the cytokine system, the successful approach might not be the inhibition of one particular cytokine but the strategies shifting the balance between pro- and anti-inflammatory cytokines in properly selected patients.
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55 Moreland, L.W. et al. (1997) Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein. N. Engl. J. Med. 337, 141–147 56 Maini, R. et al. (1999) Infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial. ATTRACT Study Group. Lancet 354, 1932–1939 57 Lipsky, P.E. et al. (2000) nfliximab and methotrexate in the treatment of rheumatoid arthritis. Anti-tumor necrosis factor trial in Rheumatoid ATTRACT Study Group. N. Engl. J. Med. 22, 1594–1602
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Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Tamas Bartfai – Harold L. Dorris Neurological Research Center and The Scripps Research Institute, USA
DISEASE Pain MECHANISMS
Mechanisms of neuropathic pain: the role of cytokines Claudia Sommer*, Maria Scha¨fers Department of Neurology, University of Wu¨rzburg, Josef-Schneider-Strasse 11, 97080 Wu¨rzburg, Germany
Many experimental studies have provided evidence that pro-inflammatory cytokines induce or increase neuropathic pain. Direct receptor-mediated actions of cytokines have been demonstrated in addition to those involving further mediators. In spite of the redundancy and pleiotropy of the cytokine network, specific actions of individual cytokines have been identified. Preliminary clinical data point to a possible beneficial
Section Editor: Eija Kalso – Helsinki University Central Hospital, Finland Claudia Sommer and her coworkers at the University of Wu¨rzburg are pioneers in the research of cytokines and neuropathic pain. Proinflammatory cytokines increase the sensitivity of damaged neurones to nociceptive and non-nociceptive stimuli. Cytokines may be an important link between infections, inflammation and the development and maintenance of certain types of neuropathic pain. Blocking or inhibiting the effects of cytokines offers an exciting new approach to the prevention and treatment of some neuropathic pain states.
role of cytokine inhibition in patients with painful neuropathy or radiculopathy. Cytokines in neuropathic pain Introduction are extracellular signaling proteins phylogenetically related to opioid peptides [1] (see Glossary). They establish communication between the immune system and the nervous system, acting at hormonal concentrations through high-affinity receptors and producing endocrine, paracrine and autocrine effects. In contrast to circulating hormones, they exert their effects over short distances onto nearby cells. Cytokines are called ‘pleiotropic’, because of a broad range of redundant, frequently overlapping functions. Their activation or dysregulation is implied in a variety of disease states, for example, sepsis, rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease, multiple sclerosis and skin diseases. Some cytokines are labeled ‘pro-inflammatory’ or ‘Th1’, others ‘anti-inflammatory’ or ‘Th2’, depending on their effects on immune cells, in particular on lymphocytes. Recently, evidence has emerged that cytokines are involved in the generation of pain and hyperalgesia in inflammatory and neuropathic conditions.
CYTOKINES
*Corresponding author: (C. Sommer) [email protected] 1740-6765/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2004.11.018
Most of the evidence for a role of cytokines in neuropathic pain comes from animal studies using nerve injury models. In these models, cytokine levels are altered (mostly increased) in the peripheral and central nervous system (CNS). Modulation of the cytokine system either by application of agonists or antagonists or by transgenic approaches modulates pain behavior. Hypotheses on potential mechanisms involved in cytokine actions derived from in vitro studies. In addition, clinical studies measuring cytokine levels in humans with neuropathic pain or using drugs modulating cytokine function further support the idea of key role of cytokines in the development and maintenance of neuropathic pain states.
Evidence from animal models Cytokine regulation after nerve injury
In response to axonal lesions a series of inflammatory mediators, including cytokines are upregulated (for review see [2,3]). In a frequently used model of neuropathic pain, the chronic constriction injury (CCI) of the rat or mouse sciatic nerve, increased levels of tumor necrosis factor-alpha (TNF-A), interwww.drugdiscoverytoday.com
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Glossary Cytokines: extracellular signaling proteins within the immune system and between the immune system and nervous system. Etanercept: a TNF-receptor fusion protein that serves as a TNF-a inhibitor in human autoimmune disorders, mainly used in rheumatoid arthritis. Is beginning to be used for Crohn’s disease and sciatica. IL-1b: interleukin-1b, pro-inflammatory cytokine with algesic actions and with many properties similar to TNF-a. At higher endocrine concentrations it is associated with fever and formation of acute-phase plasma proteins in the liver. IL-4: interleukin-4, anti-inflammatory cytokine that has also been shown to have analgesic properties. IL-6: cytokine with mostly pro-inflammatory and algesic actions, member of the IL-6 cytokine family that includes leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). IL-10: interleukin-10, anti-inflammatory cytokine that has also been shown to have analgesic properties. Infliximab: monoclonal antibodies to TNF-a that serve as a TNF-a inhibitor in human autoimmune disorders. Nucleus pulposus: inner content of the spinal discs, surrounded by anulus fibrosus and released in the case of spinal disc herniation. TNF-a: tumor necrosis factor-alpha, pro-inflammatory cytokine, member of the ‘TNF-superfamily’, together with the structurally related peptide lymphotoxin (LT-a, formerly TNF-b) and several other structurally related proteins.
leukin-1b (IL-1B) and interleukin-6 (IL-6) (see Glossary) have been shown at the mRNA, and partly also at the protein level [4–7] (Fig. 1). By contrast, protein levels of a prototypical antiinflammatory cytokine, interleukin-10 (IL-10) are decreased in the injured nerve [7]. Cytokines in peripheral nerves have been mostly localized to Schwann cells or macrophages, occasionally to fibroblasts (Fig. 2). In dorsal root ganglion (DRG) neurons, cytokine levels are not only increased after nerve injury [8], but there is also a phenotypic switch leading to TNFa expression in a population of medium size DRG neurons [9], indicating a possible alteration in the function of these neurons. Increases in cytokine expression have also been found in the lumbar spinal cord after CCI and in other partial nerve injury models [10–12] as well as in particular regions of the CNS [13,14]. In particular, spinal glia was found to be important in cytokine expression after nerve injury [15]. Studies using cytokine application or inhibition
Intra- or epineurial injection of IL-1b or TNF-a produces a concentration-dependent pain behavior in rats ([16,17], M. Zelenka et al., unpublished). Several investigators have shown an antihyperalgesic and antiallodynic effect of cytokine inhibition in models of neuropathic pain (for review and further references see [2]). Antagonism to IL-1 by using neutralizing antibodies to IL-1RI reduces thermal hyperalgesia and mechanical allodynia in mice with CCI and attenuates the endoneurial increase of TNF immunoreactivity. Intrathecal application of anti-IL-6 antibodies decreases tactile allodynia in the model of spinal nerve ligation (SNL). Epineurial injection of antibodies to the IL-6 receptor attenuates thermal hyperalgesia and 442
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mechanical allodynia in CCI. Pain-related behavior in CCI and other models is reduced by substances blocking TNF production, its release from the cell membrane or its function. The hyperalgesic action of TNF seems to be mediated by TNFR1, because neutralizing antibodies to TNFR1 but not to TNFR2 can reduce hyperalgesia. Anti-inflammatory cytokines like IL-4 and IL-10 seem to have antihyperalgesic actions in animal models of neuropathic pain [18]. IL-10 pre-treatment also reduces the hyperalgesic responses to intraplantar injections of carrageenin, IL-1b, IL-6 and TNF-a [19]. IL-4 delivered by a viral vector reduces behavioral signs of pain in an animal model of bone cancer [20], and intrathecally applied human IL-2 has a short-lived antinociceptive effect in the CCI-model [21]. Spinal administration of TNF-a antagonists prevents or reduces allodynia and hyperalgesia in animals with nerve injury and pain behavior. Whereas intrathecal IL-1b antagonists alone are seemingly without effect in nerve injury models, they synergize with TNF-a antagonists causing a further reduction of allodynia [22]. Spinal administration of IL-6, by contrast, appears to reduce electrically evoked C-fiber activity [23]. Furthermore, intrathecal application of inhibitors of glial metabolism also reduce neuropathic pain, indicating that indeed glial cells increase pain through the action of cytokines [24]. Nucleus pulposus, the material within the vertebral discs, is highly enriched with a variety of pro-inflammatory cytokines. Subsequent to disc herniation, this material probably comes into contact with the dorsal roots. Application of autologous nucleus pulposus to dorsal roots or experimental disc herniation in animals results in pain behavior as well as both ongoing and enhanced evoked activity in spinal nociceptive neurons [25]. Animals treated with either neutralizing antibodies to TNF-a directly on the nerve root or with systemic TNF-a antagonists show a marked reduction of both the neuronal activity and the pain behavior, implicating a role for TNF-a in the process [25]. Mechanisms of action
IL-1b, among other actions involving secondary production of nitric oxide, bradykinin or prostaglandins, has a direct excitatory action on nociceptive fibers, which are activated within 1 min by IL-1b application [26]. In a skin-nerve in vitro preparation, brief exposure of the skin to IL-1b facilitates heat-evoked calcitonin gene-related peptide release [27], which is a direct effect independent of changes in gene expression or receptor up-regulation. Brief applications of IL-1b to nociceptive neurons yielded a potentiation of heat-activated inward currents and a shift of activation thresholds towards lower temperatures without altering intracellular calcium levels. This IL-1b-induced heat sensitization is mediated by activation of protein kinases. IL-1 receptor is expressed in DRG neurons, such that IL-1b can act directly on sensory neurons to increase their susceptibility
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Figure 1. Relative gene expression of the pro-inflammatory cytokines IL-1b, TNF-a, the chemokine MCP-1 and the anti-inflammatory cytokine IL-10 in degenerating mouse nerve segments after chronic constriction injury (CCI; left panel) compared to sciatic nerve (SN) crush (right panel) as revealed by realtime RT-PCR and normalization against the housekeeping gene 18s RNA. Note the rapid increase in transcripts at day one after injury and the higher and more sustained expression levels after CCI. Abbreviations: Ctrl., Control nerves. Data from [6].
to noxious heat [28]. Similar effects have been shown for IL-6 in conjunction with the soluble IL-6 receptor. TNF-a lowers mechanical activation thresholds in C nociceptors of the rat sural nerve when injected subcutaneously [29], a result that might be due to an acute TNF-induced decrease in K+ conductance [30] or to a protein kinase Adependent mechanism [31]. In vitro perfusion of TNF-a to dorsal root ganglia (DRG) elicits neuronal discharges in both A and C-fibers (Fig. 3). The firing frequency is markedly higher and the discharge longer-lasting after nerve injury, indicating an increased sensitivity of injured afferent neurons to TNF-a [32]. Injection or perfusion of TNF-a into or onto rat DRGs in vivo induces allodynia. Subthreshold quantities of TNF-a injected into a DRG when its spinal nerve is ligated results
in faster onset of allodynia and increased spontaneous pain behavior. Thus, there is strong in vivo and in vitro evidence that nerve injury results in increased endogenous TNF-a and that injured nerve fibers are sensitized to the excitatory effects of TNF-a. Receptor-mediated activation of protein kinases or calcium mobilization in sensory neurons may confer the sensitizing effects that TNF-a has on nociceptors in vitro [33]. Downstream of TNF-a receptor activation, hyperalgesia induced by nerve injury is mediated via p38 MAPK [34]. The TNF-a inhibitor etanercept reduces both allodynia and p38 MAPK phosphorylation in the SNL-model of neuropathic pain, indicating that the TNF-a-p38 signal transduction cascade in the DRG is a significant participant in the generation of mechanical allodynia following nerve injury (Fig. 4). www.drugdiscoverytoday.com
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Figure 2. TNF immunolocalization in rat lumbar dorsal root ganglion (DRG) neurons and sciatic nerve four days after CCI: single labeling on serial sections demonstrating a heavy co-localization (yellow) of TNF (red) and the neuronal marker PGP 9.5 (green). Double staining demonstrating almost no co-localization of TNF-a (red) and ED1 (green). In the sciatic nerve, TNF-a (red) co-localizes (yellow) with the Schwann cell marker S-100 (green). There is almost no co-localization of TNF-a and ED1-positive macrophages (green).
In addition to the direct actions on nerve fibers, indirect actions of the cytokines mediated by other algogenic compounds, are likely in many paradigms (Fig. 5). Nerve growth factor (NGF) is one candidate mediator of cytokine-induced hyperalgesia. For example, IL-1b induces transcription of NGF in Schwann cells [35], and IL-1b-induced hyperalgesia can be prevented by anti-NGF antibodies [36], indicating that IL-1b hyperalgesia is mediated via the increase in NGF in inflamed nerves. Furthermore, prostaglandins, bradykinin and neuropeptides might mediate cytokine–hyperalgesia. TNF-a enhances the sensitivity to capsaicin of rat sensory neurons [37], probably via the neuronal production of prostaglandins. Long-time exposure of primary afferent neurons to IL-1b induces substance P release via the cyclooxygenase (COX)-2 system [38]. IL-6 induces the production of galanin in sensory neurons [39]. These changes in neurotransmitter expression might contribute to alter pain processing after nerve injury. An interesting finding is the possible relevance of spinal cytokines to opioid tolerance. In rats, chronic administration of morphine activates spinal glia to upregulated pro-inflammatory cytokines, a process which is further enhanced in nerve injured rats. Inhibition of pro-inflammatory cytokines at the spinal level restores acute morphine antinociception in these rats and reverses the development of morphine tolerance [40]. Thus, the long-term response to opioids in neuropathic pain might be improved by concomitant modulation of the cytokine system.
Evidence from human diseases Information about the role of cytokines in neuropathic pain in humans is derived from studies correlating cytokine levels 444
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Figure 3. In vitro extracellular dorsal root single fiber recording. (a) Schematic of the preparation. The ganglion is suspended in the middle chamber; the dorsal root and spinal nerve are in the adjacent mineral oilfilled chambers for recording (R) and stimulation (S), respectively. (b) Activity was recorded from one A-delta fiber while the DRG was perfused with TNF-a. The fiber responds with firing in response to perfusion of the DRG with TNF-a (here 100 pg/ml).
in body fluids or biopsied tissues and from a few, mostly uncontrolled, treatment trials. A striking correlation between cytokine levels and neuropathic pain was observed in patients with leprosy, where a subgroup of patients with elevated serum levels of TNF-a and IL-1b suffer from excruciating pain [41]. Treatment with thalidomide reduces TNF-a secretion in peripheral blood mononuclear cells by >90% and greatly reduces pain in these patients [42]. In other human neuropathies, preliminary data also point to a correlation between cytokine expression and pain [43,44]. In a series of sural nerve biopsies, cytokine levels were increased more often in patients with painful neuropa-
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Figure 4. Pain-behavior induced by spinal nerve ligation (SNL) is attenuated by systemic inhibition of TNF and intrathecal inhibition of the MAPK p38. (a) Treatment with the TNF antagonist etanercept attenuates mechanical allodynia induced by SNL. (b) Treatment with the p38 inhibitor SB203580 attenuates mechanical allodynia if started two days before SNL, but not if starting seven days after SNL. Data from [34].
thies. Recently, an increase in serum IL-8 was identified as a predictor for the development of postherpetic neuralgia (PHN) after acute herpes zoster [45]. If this finding holds true in further case series, IL-8 might represent not only a predictor but also a possible target for the prevention of PHN. Several studies have reported increased levels of cytokines in the vicinity of herniated discs and a correlation of their presence to sciatica [46–48]. Inhibitors of TNF-a have been used successfully for patients with chronic nerve root pain, but up to now only data from case reports and uncontrolled studies are available [49]. Occasionally, TNF-a inhibitors have been used to treat inflammatory neuropathies, but data on pain are lacking in these reports [50]. One remarkable case report describes remission of long standing complex regional pain syndrome (CRPS) after treatment with thalidomide for Behc¸et’s disease [51]. Anti-TNF-a strategies have been used in various nonneuropathic painful conditions like AIDS-associated proctitis, rheumatoid arthritis and HIV-associated aphthous ulcers (for review on earlier studies see [52]). Many other reports have followed since the advent of etanercept and infliximab, for example, a controlled trial showing a beneficial effect of etanercept in ankylosing spondylitis [53], and a prospective study with historical controls using infliximab in low back pain [54] (Table 1). Controlled trials are warranted to establish the role of cytokine inhibition in these painful conditions. Obviously, caution is necessary because of the pleiotropy of the cytokines. Treatment with antagonists might not only reduce the target symptom (pain) but also have undesired side effects in other systems. Furthermore, given the redundancy of the cytokine system, blockade of one cytokine may be compensated by upregulation of others with similar effects. Therefore, combined antagonistic strategies might be necessary.
Summary and conclusions Numerous experimental studies provide evidence that proinflammatory cytokines induce or facilitate neuropathic Table 1. Key therapies – pre-clinical to market Target
Therapy against target
Stage of development
Advantages/Disadvantages
References
TNF-a
Etanercept (dimeric TNFR2 fusion protein) Infliximab (chimeric monocloncal TNF antibody) Adalimumab (human IgG1 anti-tumor necrosis factor alpha monoclonal antibody)
On market for rheumatoid arthritis, Crohn’s disease Case reports for low back pain, sciatica
Mostly well-tolerated Rare side effects are: injections site reactions, rash, respiratory tract infections, for example, tuberculosis, demyelinating disorders, aplastic anemia, worsening of congestive heart failure, back pain
[54–59]
IL-1
Anakinra (IL-1 inhibitor)
On market for rheumatoid arthritis
Unknown
[60]
IL-2, IL-4, IL-10
Gene therapy
Experimental use for cancer pain, neuropathic pain, inflammatory pain
Unknown
[21,61]
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Figure 5. Possible currently discussed mechanisms by which TNF as the prototypical proinflammatory cytokine induces neuropathic pain: TNF exerts pleitropic effects on Schwann cells, endothelial cells, axons, dorsal root ganglia neurons and the CNS. Abbreviations: EC, endothelial cells; NGF, nerve growth factor; CGRP: calcitonin gene-related peptide; DRG, dorsal root ganglion; CNS: central nervous system; MAPK: mitogen-activated protein kinases; PKA, proteinkinase A; SPA, spontaneous activity.
pain. Direct receptor-mediated actions of cytokines on afferent nerve fibers as well as cytokine effects involving further mediators have been reported. Furthermore, endogenous cytokine levels may modulate the response to opioids. Cytokine levels are rapidly and markedly upregulated in peripheral nerves, DRGs and in the spinal cord after peripheral nerve injuries. Whereas direct application of exogenous pro-inflammatory cytokines induces pain, blockade of these cytokines or application of anti-inflammatory cytokines reduces pain behavior in most experimental paradigms. A finding of particular clinical importance may be the involvement of TNF-a released from spinal discs in the generation and maintenance of sciatica. Cytokine measurements might identify patients at risk to develop chronic pain associated to their neuropathic conditions, as in the examples of peripheral neuropathies and PHN. Case reports and uncontrolled trials point to a positive effect of TNF-a inhibition in sciatica and possibly other neuropathic conditions. Because of the pleiotropy and redun-
Outstanding questions What are the molecular mechanisms through which cytokines induce or maintain pain? Could p38 mitogen-activated protein kinase be an alternative target for pain control?
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How can we identify those patients in whom cytokines play a major role in the generation of pain? Is blockade of one cytokine (as exemplified in the TNF inhibitors) a valid strategy for pain control in selected diseases, or would a more successful approach involve shifting the balance between pro- and anti-inflammatory cytokines?
dancy of the cytokine system, the successful approach might not be the inhibition of one particular cytokine but the strategies shifting the balance between pro- and anti-inflammatory cytokines in properly selected patients.
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