Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and joint destruction. The clinical signs of RA are accompanied by chronic pain, the management of which remains a difficult task. Models of acute pain and inflammation do not accurately replicate the complex mechanisms of a chronic inflammatory disorder such as RA. Collagen-induced arthritis (CIA) is widely used for studies of RA pathogenesis, and the model results in an immune response directed against the joints, which closely resembles many aspects of human RA (1, 2).
In DBA mice, CIA is associated with mechanical and thermal hypersensitivity in the hind paws from the day of disease onset (3). Furthermore, pain associated with passive arthritis induced by K/BxN serum transfer in mice has recently been characterized as a model of inflammatory pain with a late-developing neuropathic component (4).
At present, the contribution of central nervous system (CNS) changes in the CIA model is not fully understood, especially in relation to chronic inflammatory pain. While the cartilage is not innervated, it is the perturbation of the subchondral bone that likely leads to sensitization of primary afferent nociceptors of the joint and the sensitization of spinal cord neurons that underlie pain in the inflamed joint (5). Evidence of astrogliosis in established CIA (3) suggests that neuron–glial interactions may contribute to pain in arthritis.
Following a damaging stimulus to the peripheral nervous system, microglia enter a pain-related enhanced response state and contribute to enhanced nociceptive signaling (6). This microglial response is accompanied by infiltration of immune cells, such as T lymphocytes, into the dorsal horn (7–9). Accordingly, the inhibition of immune cell targets can reduce hypersensitivity in chronic pain models. In particular, the protease cathepsin S (CatS) is critically involved in chronic pain (10, 11). This microglial mediator exerts its pronociceptive effects via cleavage of the neuronal chemokine fractalkine (FKN) (10, 12).
Both FKN and CatS have been implicated in the peripheral destructive processes that give rise to RA. As an adhesion molecule and chemotactic factor, endothelial FKN mediates cellular infiltration in the synovium (13, 14). Independent evidence implicates CatS in both antigen presentation (15) and tissue destruction (16) associated with RA, with enhanced CatS activity observed in human arthritic joints (17). Importantly, CatS deficiency (15) and inhibition of FKN (18) are associated with reduced severity of CIA in mice. No connection between CatS activity and FKN cleavage in RA has been investigated, however. In the present study, we evaluated the contribution of microglial cells, particularly the CatS/FKN signaling pair, to the chronic pain associated with the CIA model in the rat.
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In this study, we showed that CIA represents a clinically relevant model of persistent inflammatory pain that is associated with an enhanced microglial response, astrogliosis, and T cell infiltration in the dorsal horn of the spinal cord. Specifically, CIA induces a robust mechanical hypersensitivity that develops before clinical signs of arthritis become apparent and peaks with the severity of the disease. Spinal inhibition of the CatS/FKN signaling pair in order to disrupt neuron–microglial communication is able to attenuate both established hypersensitivity and the CIA-induced enhanced microglial response without modifying the severity of joint inflammation.
Treatment of chronic pain in RA represents a major unmet clinical need. Despite the availability of disease-modifying agents that reduce the clinical signs of RA, the treatment of chronic pain remains poor (29–31). While inflammatory pain states associated with models of monarthritis have been extensively characterized, pain that occurs as a result of more clinically relevant polyarthritis models such as CIA has only recently been examined (3). Here we report that CIA in the rat represents a reliable model for the study of chronic arthritis pain, which is associated with significant immune response in the spinal cord. Notably, the development of pain in rats with CIA mirrors the sensory changes reported in models of other autoimmune disorders, such as multiple sclerosis, in which hypersensitivity is present before the onset of clinical features of the disease (32, 33). In murine active and passive models of RA, hypersensitivity in the hind paws is observed from the day of disease onset (3), and pain hypersensitivity develops alongside swelling (4). In both murine models, pain behaviors are sensitive to analgesics, including those used clinically for the treatment of RA, such as nonsteroidal antiinflammatory drugs and anti–tumor necrosis factor α (anti-TNFα) agents (3, 4), suggesting that a good correlation between the inflammatory pain in these models and pain in RA patients can be drawn.
Joint pain is the result of inflammation-induced sensitization of neurons innervating the joint whose cell bodies are in the DRG, as well as hyperexcitability of spinal cord neurons receiving input from the joint (5). We observed no significant changes in the expression of ATF-3, a marker of neuronal injury, in the DRG in rats with established CIA, which suggests that the mechanical hypersensitivity and spinal microglial response do not directly result from nerve damage in the joint. These data are in contrast to those of previous studies in active and passive CIA in mice, in which increased expression of ATF-3 indicates a contributory role of nerve injury (3, 4). However, the modest, yet significant, elevation in ATF-3 expression reported by Inglis and colleagues in mice with CIA (3) is similar to the level of 1% of total DRGs expressing ATF-3 reported in this study. Interestingly, an increased spinal microglial response is observed 4 days after intraplantar injection of CFA (34); however, this model of inflammation is not associated with altered ATF-3 expression in DRGs (35). In addition, brief activity of sensory neurons alone is sufficient driving force for the induction of a microglial response in the dorsal horn (27), suggesting that neuronal injury and microglial response occur independently under some circumstances.
Taken together, these data suggest that glial response in the dorsal horn following peripheral tissue damage and inflammation is not associated with neuropathy and is likely driven by the increased primary afferent inputs and release of glutamate, substance P, and brain derived neurotrophic factor (6, 26).
The contribution of CNS changes in the CIA model is not fully understood, especially in relation to CIA-induced chronic inflammatory pain. We observed astrogliosis in the lumbar dorsal horn, as was previously reported in both active (3) and passive (4) models of RA. We also observed extensive infiltration of T cells in the dorsal horn following CIA. Both human RA and CIA are heavily dependent on T cells (36), and administration of anti-CD3 antibodies is able to reduce the severity of CIA in mice (37). Infiltration of T cells is also associated with neuropathic pain behaviors following peripheral nerve injury (7–9, 38). However, it is unclear at present if changes in astrocyte and T cell activity contribute to arthritis-induced hypersensitivity. We observed significant increases in the microglial response in the dorsal horn of animals with CIA, a finding supported by the results of previous studies of passive RA (4) and adjuvant-induced arthritis (39), which suggests that microglial cells and their mediators may contribute to pain in models of arthritis. The temporal sequence of glial cell activation and T cell infiltration from immunization will be established in future studies.
It is well known that both pharmacologic and genetic inhibition of microglial targets can reduce hypersensitivity in models of chronic pain. In particular, our previous work determined that the lysosomal cysteine protease CatS is critically involved in chronic pain (10, 11). This microglial mediator exerts its pronociceptive effects via cleavage of the neuronal chemokine FKN (10, 12). In the present study, we observed that prolonged intrathecal treatment with either the CatS inhibitor LHVS or an anti-FKN neutralizing antibody was able to reverse established pain behaviors in rats with CIA, but did not slow the development of the clinical signs of arthritis, suggesting that the inflammatory pain can be uncoupled from the disease process itself. Accordingly, in RA patients, TNFα neutralization was shown to inhibit pain before reducing inflammation in the joint (40), possibly through inhibition of central sensitization. Indeed, the murine CIA model successfully predicted the therapeutic effects of human TNFα blockade in RA (41, 42). In murine CIA, systemic treatment with anti-TNFα beginning on the day of disease onset is able to attenuate disease severity (3, 41) as well as pain behaviors (3). In this study, spinal inhibition of CatS/FKN signaling beginning after the onset of CIA-associated pain behaviors was unable to alter the progression of CIA. Preventative inhibition of these proinflammatory agents was not examined, however.
Systemic inhibition of CatS and FKN, in order to attenuate the peripheral destructive processes occurring in the joint in RA, may be necessary for disease prevention, as has been reported with anti-FKN in murine CIA (18). The antihyperalgesic effects of both CatS and FKN inhibition are mediated via a reduction in the activity levels of microglial cells in the spinal cord of animals with CIA, as is the case in other pain models (10, 28), suggesting that the response state of these cells is key for the maintenance of CIA-induced inflammatory pain.
Both CatS and FKN have been independently associated with the peripheral immunopathologic processes that cause RA. CatS is critical for the process of antigen presentation (43); hence, compounds that inhibit the proteolytic activity of CatS reduce the severity and/or delay the onset of experimental arthritis via an impairment of antigen presentation (16, 44, 45). In addition, CatS-null mice have decreased susceptibility to CIA, with reductions in clinical scores as compared to their wild-type littermates (15). CatS may also play an extracellular role in joint degradation during RA, as enhanced enzymatic activity of cysteine proteases is significantly elevated in the inflamed joints of rats (16), as well as in the synovial fluid from arthritic joints of humans (17). The role of FKN in RA has been attributed to its chemotactic and adhesion properties (46, 47). In particular, FKN mediates cellular infiltration into the inflamed joint (13, 14) and systemic administration of anti-FKN reduces the severity of CIA in mice (18).
In summary, this study is the first to show the active contribution of spinal microglial cells to the chronic inflammatory pain associated with the CIA model of RA. In particular, a critical role of the microglial protease CatS and its neuronal signaling partner FKN demonstrate the vital contribution of neuroimmune communication in this model. We suggest that the inhibition of microglial targets by centrally penetrant CatS inhibitors and CX3CR1 receptor antagonists represents a novel therapeutic avenue for the treatment of pain in RA.