Neuroimmunologic approaches to the understanding and potential treatment of CRPS
By Donald C. Manning, MD, PhD

Although there has been a great deal of progress in defining CRPS, there is still controversy regarding the mechanisms involved. For example, if one looks at the high female predominance (75%) and the combination of inflammatory and neuropathic pain components, one may think of an autoimmune mechanism. The sympathetic nervous system involvement in at least a portion of CRPS subjects further complicates the understanding of this syndrome when one uses traditional approaches. It is time for a reevaluation of CRPS mechanisms to enable new therapeutic opportunities. To date there is no approved treatment for CRPS. Therapies used for neuropathic pain, directed at neuronal receptors and channels have given disappointing results in CRPS affording many patients, at best, only partial relief (1). Experimental and clinical evidence suggests that CRPS involves peripheral inflammation mechanisms and many aspects of CRPS outlined above are consistent with an immune mediated process resulting in a secondary activation of the nervous system. Neuroimmune approaches to CRPS and other neuropathic pain conditions are emerging as a viable way to think about and possibly treat these complex syndromes. Neuroimmune activation involves endothelial cells, microglia, and astrocytes which, when activated, produce proinflammatory mediators such as cytokines that induce and enhance the immune response to injury (2). Recently pro-inflammatory cytokines F and IL-6 levels were found to be elevated in blister fluid obtained from CRPS- involved limbs vs non-involved limbs (3).

Cytokine Mediators of Neuroimmune Activation
Cytokines are not typically stored in cells but are regulated by gene transcription following a stimulus. One cytokine can act on different cell types, limiting the utility of therapeutic administration of cytokines due to the development of many unwanted effects. Cytokines also are redundant in that multiple cytokines can exhibit the same functional effects. Antagonists to a single cytokine or alteration of a cytokine gene may have limited effects because other cytokines may be capable of compensating for the resulting loss of function. Cytokines can exert their actions in a local autocrine or paracrine manner or act systemically in an endocrine fashion. Cytokines typically act by binding to ultra-high-affinity receptors on the cell surface such that only a few molecules are needed to produce a response. The concentration of cytokines in a tissue or in the plasma is often low, and thus the inability to detect a particular cytokine does not mean that it is not acting in a given situation (4). TNF , IL-1 and IL-6 are the cytokines with the best-documented pathological roles in neuropathic pain.

IL-1 is found in dorsal root ganglion (DRG) neurons and in Schwann cells in the peripheral nervous system. IL-1 levels increase early in both inflammatory and nerve injury animal models of pain (5,6). Injection of exogenous IL-1 can induce thermal hyperalgesia and mechanical allodynia in the rat paw (7-9). However, direct nerve injury to the spinal root induces spinal glia activation and enhances expression of IL-1 bilaterally in the dorsal and ventral horns of the spinal cord. This finding suggests a central neuroimmune reaction (10) that is not confined to the innervation pattern of the nerve root. IL-1 can also attenuate the analgesic effects of morphine and may be related to morphine tolerance mechanisms. Shavit and colleagues (11) suggest that the analgesic effect of chronic morphine administration could be enhanced through inhibition of IL-1 function within the spinal cord.

TNF and its receptors can be up regulated following injury or inflammation/immune challenge. This increased TNF activity occurs in non-neuronal glial cells and macrophages and perhaps in neurons as well (12). Receptors for TNF are found on virtually all sensory neurons and most DRG neuronal cells (13) leading some to speculate that they may be "immunosensors" picking up signs of inflammation and activating neural reflex pathways (14). A linkage to immune-competent cells in the periphery may allow activation during inflammation and immune reactions and may drive illness behavior and fatigue syndromes (15). Exogenous administration of TNF can induce hyperalgesia and allodynia and administration of TNF inhibitors can reduce or prevent the increased nociceptor activation (16,17). TNF immunoreactivity is greater in Schwann cells of patients with painful neuropathy compared to those with nonpainful neuropathy (18). Soluble TNFR1 was also elevated in the serum of patients with mechanical allodynia as compared to patients without allodynia (18).

In healthy animals just the administration of IL-1 or TNF can produce symptoms of neuropathic pain. Spinal neuron activation by these cytokines can produce long-term alterations in neuronal excitability. Infusion of IL-1 or TNF for adjuvant cancer chemotherapy has been associated with an incidence of nearly 50% of pain syndromes or complaints of pain and tenderness at the injection site (19 - 21). Whereas TNF and IL-1 play important roles in the initiation of persistent neuropathic pain, delayed IL-6 production is a factor in the maintenance of such pain (5).

Il-6, on the other hand, has many properties of a circulating hormone as well as a local mediator and serves to communicate between the CNS and the periphery in a bidirectional manner (22). Induction of IL-6 is a general arousal signal to the entire body (22). IL-6 acts through a specific receptor that is expressed on lymphocytes, macrophages, and other immune cells. Within the nervous system, mRNA for both IL-6 and its receptor are expressed in the hippocampus, neocortex, cerebellum, neurons, and astrocytes (23). IL-6 mRNA is expressed at low levels in many cell types including monocytes, macrophages, endothelial cells, fibroblasts, mast cells, adipocytes, microglia, muscle cells and spinal cord neurons in the dorsal horn and is up regulated following peripheral nerve injury, where it may play a role in nociceptive processing at the local and spinal level (24). Membrane depolarization and neuronal activity itself can induce IL-6 in neurons (25).

IL-6 can increase cold allodynia (26). Evidence is accumulating for IL-6 contributions to human pain states. An IL-6 gene variation associated with increased expression and plasma levels of IL-6 has been identified in patients with herniated disks characterized by sciatica (27). Patients with persistent pain 8 weeks after diskectomy had a significantly elevated IL-6 level compared to pain-free volunteers (28).

Cytokine involvement in chronic pain derives from a much broader view of injury-related behavior termed the "sickness response." This response is composed of a wide range of changes initiated by a peripheral immune or inflammatory challenge (29). The sickness response includes fever, increased white blood count, activation of the hypothalamic-pituitary-adrenal axis, sympathetic nervous system arousal, decreased social interaction, decreased food and water intake, and increased sensitivity to pain (30). Chronic pain is often associated with behavioral and cognitive alterations. Increased levels of IL-6 following diskectomy have been associated with depressed mood, increased self-reported stress, and altered morning cortisol secretion (28). Immune activation also has been associated with a decrease in mood and cognitive function, a common adverse effect of cytokine administration for cancer (31). IL-6 has also been associated with inhibition of certain types of learning and memory (32). These findings suggest that some of the behavioral consequences of chronic pain may have an origin in increased IL-6 levels.

Several anti-inflammatory molecules including the cytokines IL-4 and IL-10 serve to dampen or inhibit the activity of pro-algesic compounds. In normal tissue states, an intricate balance is present between pro-inflammatory and anti-inflammatory mediators. Malfunction or absence of these endogenous anti-inflammatory molecules could produce chronic pain and inflammation. IL-10 is the prototypical anti-inflammatory cytokine. It can act in an antagonistic manner to reverse or oppose many of the actions of pro-inflammatory cytokines. IL-10 can inhibit the production, release, and activity of TNF , IL-1 , and IL-6; and can down regulate the receptors for pro-inflammatory cytokines (reviewed in 33). IL-10 can be secreted from infiltrating macrophages and lymphocytes to suppress ongoing inflammation. Augmentation of IL-10 appears to be attractive for managing neuropathic pain associated with glial activation because IL-10 inhibits only the pathological functions and increased cytokine activity and does not alter basal activity. Gene therapy methods have been developed to augment IL-10 release, but the delivery system is awkward. IL-10 therapy may be complicated because this cytokine can down regulate the expression of its own receptor in an autocrine or paracrine negative feedback system (34). Studies with IL-10 knockout animals or administration of anti-IL-10 antibodies have demonstrated decreased thermal hyperalgesia, suggesting that endogenous IL-10 contributes to nociception (35). Clearly, more work is needed to elucidate this area.

Cytokine Interaction with the Sympathetic Nervous System
Sympathetic influence over macrophage and immune cell cytokine release provides a mechanism for the sympathetic nervous system to influence the somatosensory system through a neuroimmune mechanism. This may partially account for sympathetic involvement in the maintenance of CRPS. In the periphery, norepinephrine acting through the 2-adrenergic receptor increased the production and release of TNF in macrophages (36) but inhibits IL-6, whereas -adrenergic receptor stimulation decreased TNF production in macrophages and microglia (37, 38) and in concert with cortisol stimulates IL-6 production (39). It is clear therefore that the sympathetic regulation of TNF is different than the regulation of IL-6. Thus the sympathetic nervous system can fine-tune cytokine release but this is dependent upon the relative alpha or beta-adrenergic receptor tone of the tissues (22). This complexity suggests that merely blocking regional sympathetic input may have detrimental or at best unpredictable effects on CRPS signs and symptoms.
Centrally TNF is localized in neurons in several norepinephrine-rich areas of the brain including the locus ceruleus and the hippocampus (40) and can, along with 2-adrenergic receptors inhibit norepinephrine release. During persistent neuropathic pain TNF levels increase in these brain regions, and there is greater 2-adrenergic receptor/TNF -induced inhibition of norepinephrine release, resulting in decreased norepinephrine function (41). Infusion of anti-TNF antibodies reverses the thermal hyperalgesia and hyperalgesia. In naive rats, intracerebroventricular infusions of recombinant TNF ?can induce thermal hyperalgesia and mechanical allodynia (42). Administration of a tricyclic antidepressant reduces neuron-localized TNF . And the usual TNF inhibition of norepinephrine release reverses to facilitation. These findings suggest that TNF is involved in therapeutic actions of tricyclic antidepressants in pain and depression that are often associated in CRPS.

Glial Cell Involvement in Chronic Pain
Cerebrospinal fluid levels of the cytokines IL-6 and IL-1 (but not TNF ) are elevated in patients with CRPS relative to patients without pain or with radiculopathy pain at equivalent intensity not related to CRPS (44). This suggests a specific association between cytokines and the symptoms of CRPS. There were no differences in plasma or systemic levels of these cytokines. The source of these cytokines is not fully established at this time. The initial cytokine release in response to injury derives from Schwann cells enveloping peripheral nerves, endothelial cells, tissue resident macrophages and mast cells (45). Later release is due to blood derived macrophages and immune cells. Non-neuronal cells such as microglia in the central nervous system are correlates of resident macrophages in the periphery and are the principle sources of the pro-inflammatory cytokines IL-1 , IL-6, and TNF as well as the anti-inflammatory cytokine IL-10 (46). Microglia can be activated by intense sensory afferent stimulation as well as by peripherally derived cytokines that are actively transported across the blood-brain barrier. It has become increasingly clear that microglial activation is critical to the development of neuropathic pain, but may be just the first step in a cascade of immune responses to injury expressed in the CNS.

Microglia and astrocytes (another type of non-neural glial cell) can communicate with other glia over long distances with in the CNS. These inter-glial connections do not follow neuroanatomical patterns and can lead to activation bilaterally and at great distances within the spinal cord. The net result is that areas of pain can spread well beyond the region of original injury and can easily spread bilaterally and to other regions of the body. Invoking glial cells may account for the spreading symptoms of CRPS more readily than invoking non-anatomical connections between distant neurons. Activated microglia through the release proinflammatory cytokines activate astrocytes
(another type of non-neural glial cell). Once activated, astrocytes can maintain hyperalgesia and allodynia independent of microglia. The point of conversion to astrocyte-driven sensitization appears to occur within the first 24 hours following injury. Astrocytes have a "cellular memory" in that intracellular calcium responses are greatly amplified when astrocytes have previously been repetitively stimulated or exposed to strong synaptic activity (47, 48). This finding could account for the reports of pain reactivation with new injury, especially in cases of complex regional pain syndrome.

Emerging and Potential Therapies
The systems and mechanisms above represent a departure from the traditional thinking about CRPS and neuropathic pain. Targeting therapies toward cytokines, glia, or infiltrating immune cells is a new approach for pain therapy, although it has already been employed with some success in oncology and rheumatology.
Immunosuppression In immune mediated diseases it is important to suppress the pathologic elevations of cytokines rather than affecting a complete blockade. This approach is known as immunomodulation and serves to restore the normal balance in immune function. Immunosuppressive agents such as methotrexate (49) and leflunomide (50) attenuate tactile hypersensitivity in rodent radiculopathy and neuropathy models. Leflunomide is approved for clinical use in rheumatoid arthritis and has several anti-inflammatory actions, including inhibition of IL-1 , TNF , and the expression of nitric oxide and COX-2 genes. However no clinical studies in chronic neuropathic pain conditions have been reported. Caution is advised as general immunosuppressants can increase the risk and reduce the resolution of infection.

Nutrition and fatty acid therapies. A somewhat unexpected drug class for immunomodulation comprises the statins or 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase inhibitors. Following reports that statin treatment could produce improvement in a model of multiple sclerosis (51), great interest is now being directed toward this class of drugs (52). Treatment with atorvastatin induced the secretion of anti-inflammatory cytokines (IL-4, IL-5, and IL-10) and inhibited the secretion of Th-1 pro-inflammatory cytokines (IL-2, IL-12, IFN , and TNF ). Another statin, lovastatin, inhibited the expression of TNF , IL-1 , and IL-6 in rat astrocytes, microglia, and macrophages (53). Statins decreased the expression of inflammatory mediators in the CNS, including TNF . The potential benefit of statins in neuropathic pain is unexplored, but these agents may be effective in preemptive use. How many postoperative or traumatic neuropathic pain states have been avoided by concomitant use of statins? Future studies may provide some guidance for these agents.

Shir and colleagues have reported that dietary fat can reduce the neuropathic pain-related behaviors resulting from partial sciatic nerve ligation (55). The consumption of unsaturated corn or soy oils suppressed tactile allodynia and heat hyperalgesia, and this effect was accentuated by dietary protein from multiple sources (55). Dietary fats can modulate both innate and adaptive immune responses through Toll-like receptor-4 (TLR-4) receptors. TLR-4 functions in the innate immune system as a stable pattern recognition receptor for the invariant structures of pathogens. TLR-4 occurs exclusively on microglia in the rat CNS (56). TLR-4 can be activated by bacterial wall molecules such as endotoxin or lipopolysaccharide and by endogenous ligands such as heat shock proteins, proteoglycans, and saturated fatty acids released after neural injury and degeneration (57,6). Saturated fatty acids activate Toll-like receptors, but omega-3 polyunsaturated fatty acids inhibit agonist-induced TLR activation (58). Partial but significant reduction in hyperalgesia and allodynia behavior can be accomplished by interfering with the function of TLR-4 in microglia (59). This mechanism raises intriguing possibilities but much work remains.

Inhibitors of cytokine production and function. Glucocorticoids have been used for many years to treat CRPS and inflammatory diseases. They can modulate the immune system and inhibit the production of a wide range of inflammatory mediators and stimulate the production of anti-inflammatory agents. Glucocorticoid utility for chronic diseases is severely compromised by a wide range of adverse effects including diabetes, impaired wound healing and susceptibility to infections, metabolic problems and bone demineralization (60). The search for safer and more effective inhibitors of inflammatory mediators has yielded several new therapeutic agents. Successful use of TNF monoclonal antibodies or TNF -receptor fusion protein has changed the therapy for rheumatoid arthritis and several other chronic inflammatory diseases. Open-label clinical reports have claimed rapid resolution of acute sciatica (involving spinal root irritation) using TNF inhibitors infliximab (61) or entanercept (62). These findings however were not supported by a larger controlled study of acute radiculopathy pain (63). There are no reports of these agents being used in trials of CRPS therapy. In one small experimental study elevated interstitial cytokine levels from CRPS affected region are markedly reduced by infliximab treatment coincident with a reduction in clinical symptoms (64). Anakinra is a recombinant human IL-1-receptor antagonist approved for use in rheumatoid arthritis (65). No studies have looked at its ability to alter the development or maintenance of other chronic pain states.

Thalidomide was developed as a sedative and antinausea drug, but its teratogenic effects and propensity to cause peripheral neuropathy with prolonged use have limited its utility. It is orally active and functions as an immunomodulator by inhibiting the production of a broad range of pro-inflammatory mediators including TNF , IL-1 , and IL-6 and by increasing the level of IL-10, IL-2, and IFN . It also inhibits the production of TNF from human microglial cells (67). Clinically, thalidomide has been reported to reduce pain and hyperalgesia in complex regional pain syndrome (CRPS) type I (68 - 71).

Medicinal chemistry efforts have produced several generations of immunomodulatory agents derived from thalidomide with decreased toxicity. Lenalidomide is a novel immunomodulatory drug or IMiD with anti-inflammatory properties, potently inhibits the secretion of pro-inflammatory cytokines (e.g. TNF , IL-1 and IL-6) and stimulates the secretion of anti-inflammatory cytokines (e.g. Il-10). Lenalidomide is approved for the treatment of patients with transfusion-dependent anemia due to low- or intermediate-1-risk myelodysplastic syndromes with a deletion 5q cytogenetic abnormality with or without additional cytogenetic abnormalities. Based upon reports of symptomatic improvement of CRPS in response to treatment with thalidomide, and upon the pharmacological properties of lenalidomide, a pilot study was undertaken to assess the safety and preliminary efficacy of lenalidomide in subjects with unilateral CRPS Type 1.
Lenalidomide was used in an open label study of 40 patients with unilateral CRPS of at least 1year duration, optimal conventional therapy and with entry pain levels at least 4 on a 0-10 pain scale (72). The patients had high levels of pain on average at baseline [7.1 (SD 1.3)] and were taking, on average 4.2 concomitant CRPS pain medications. Despite this high level of treatment, over one third of subjects reported at least 30% improvement and one-half reported a 20% improvement in pain levels as well as broad and significant reductions in CRPS symptoms and sleep disturbance. Of the original 40 subjects 28 continued into an extension phase and 14 are still in the study with continued benefit after over two years on the study drug. Data from this study demonstrated a good overall safety profile, however as the study was uncontrolled, it is difficult to interpret the precise relationship of adverse events to study treatment. Most adverse events were mild to moderate in severity and many events were attributed to the disease rather than to a reaction to the study drug. Few subjects required dose reductions or discontinuation due to adverse events. The most frequent adverse events were rash and pruritus, followed by dizziness and headache. All drug-related adverse events that led to the discontinuation of lenalidomide, except for an episode of DVT, were mild or moderate in severity. Four serious adverse events were reported and of these, only the DVT was suspected as related to the study drug. The results of this study demonstrate a level of safety and efficacy justifying additional study for CRPS. Controlled studies are currently underway in CRPS subjects.

We need to find new approaches to the treatment of chronic neuropathic pain and CRPS. By changing our perspective and looking beyond traditional neuroanatomy and neurophysiology to understand the body's response to injury, we may uncover new therapeutic strategies. In this short article adequate coverage of the extensive literature supporting immune and nervous system interaction especially in pain states cannot be provided. An appreciation of the role played by the immune system in injury-induced pain states, as summarized in this article, represents a new opportunity. Currently available immunomodulators and immunosuppressive agents need to be cautiously evaluated for their pain-modulating ability. The results of these initial studies will certainly foster more extensive therapeutic development efforts.

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