Rheumatoid arthritis (RA) and osteoarthritis (OA) are among the most frequent causes of chronic pain (1). It is now apparent that RA and OA pain involve substantial changes in the nociceptive (pain) system at all levels of the neuraxis, including the peripheral neurons and the central nervous system (2, 3). As a result, patients with advanced OA, for example, often report widespread pain beyond the affected joint (4, 5) and exhibit lower pressure pain thresholds in cutaneous and subcutaneous structures of the leg (4, 6). The brains of RA patients show strong activation of the so-called pain matrix (7), but atrophy of pain-related regions in the thalamus (8) and cortex (9) has also been reported, indicating severe alterations.
Widespread pain beyond the site of disease is attributed to “central sensitization,” a state of hyperexcitability of nociceptive neurons in the spinal cord (10). Indeed, patients with knee OA exhibit larger areas of referred pain and higher pain summation scores upon repetitive pressure on the knee than do controls, which indicates central sensitization (11). Spinal cord recordings in animals identified neuronal changes which are thought to underlie these findings in patients. Upon the development of inflammation in the joint, spinal cord neurons become more responsive to mechanical stimulation of the joint and, importantly, show enhanced responses to mechanical stimuli applied to healthy regions adjacent to and even remote from the inflamed joint (3). This spinal hyperexcitability is induced by sensory input from the inflamed joint. In addition, descending inhibitory systems from the brainstem may be less effective (12) and/or descending excitatory systems from the brainstem may be overactive (13, 14) in animals and humans with painful joint diseases. Spinal hyperexcitability also plays a role in the regulation of joint inflammation (15, 16).
Spinal hyperexcitability is elicited by changes in synaptic transmission mediated by the transmitter glutamate and the neuropeptides substance P and calcitonin gene-related peptide (3). More recently it has been recognized, mainly in studies on neuropathic pain (17), that mediators of the immune system, including interleukin-6 (IL-6) (18), modify the nociceptive processing in the spinal cord. Because the immune system plays a dominant role in painful RA, it is conceivable that the immune system contributes significantly to the generation of inflammatory joint pain. Indeed, both tumor necrosis factor (TNF) (19) and IL-6 (20) potently sensitize joint nociceptors. Because cytokines can also be produced in the spinal cord, e.g., by glial cells (17), the question of whether peripheral joint inflammation causes an increase in these mediators in the spinal cord and whether they contribute to central sensitization is intriguing. In fact, rats with articular Freund's complete adjuvant–induced inflammation and rats with generalized adjuvant-induced arthritis showed increased spinal levels of IL-6 (21, 22).
In the present study, we focused on the putative role of IL-6 signaling in the process of central sensitization in the rat spinal cord. In general, IL-6 can bind to the membrane-bound IL-6 receptor (IL-6R), which acts in cooperation with the transmembrane signal-transducing subunit glycoprotein 130 (gp130). Alternatively, IL-6 can bind to a soluble IL-6R (sIL-6R), and the IL-6/sIL-6R complex can bind to gp130 in cells that do not express the membrane-bound IL-6R, thus leading to IL-6 transsignaling (23, 24). Because the coadministration of IL-6 and sIL-6R caused stronger peripheral sensitization than IL-6 alone (20), in the present study we tested the effects of IL-6/sIL-6R on central sensitization. We recorded the responses of neurons in the exposed spinal cord in anesthetized rats and investigated first whether the injection of IL-6/sIL-6R into the knee joint is sufficient to induce central sensitization. We then examined whether application of IL-6/sIL-6R to the rat spinal cord changes the responsiveness of the neurons to stimulation of the joint. Finally, we investigated whether IL-6 is released from the spinal cord during the development of inflammation in the rat knee and whether the generation of central sensitization is attenuated by neutralizing spinal IL-6/sIL-6R.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
This study addressed the role of IL-6 in the generation of inflammation-evoked pain in the rat. The administration of IL-6/sIL-6R either into the rat knee joint or topically to the spinal cord caused an increase in the responses of spinal neurons to mechanical stimulation of the knee and other parts of the leg, including an expansion of the size of the receptive field of the neurons. Knee inflammation in the rat induced significant spinal release of IL-6. Spinal application of soluble gp130 prevented the increase in responses that occurred after spinal application of IL-6/sIL-6R and attenuated the generation of spinal hyperexcitability during the development of inflammation. However, the application of soluble gp130 to the rat spinal cord did not reverse established hyperexcitability after inflammation was fully developed.
IL-6 and sIL-6R are key players in systemic inflammation and arthritis (27, 28). RA patients exhibit enhanced levels of IL-6 (29, 30) and sIL-6R (31) in serum and synovial fluid, which facilitates inflammation (32, 33) and joint destruction (34). The neutralization of IL-6 with tocilizumab has become an option in RA therapy (35). Recent experiments showed that IL-6 is also a potent sensitizer of nociceptive sensory fibers (20). IL-6 is likely to have, at least in part, a direct effect on the sensory neurons because peripheral nerve fibers express the transmembrane signal-transducing subunit gp130 (36, 37). In behavioral experiments, soluble gp130 injection into the knee joint at the time of the induction of antigen-induced arthritis significantly reduced inflammation-evoked mechanical hyperalgesia, while joint swelling was not reduced at this stage (38).
In the present study, the injection of IL-6/sIL-6R into the rat knee joint was sufficient to elicit a typical pattern of inflammation-evoked spinal hyperexcitability consisting of enhanced responses to both knee and ankle stimulation (25, 26) (Figure 1C). Such a pattern most likely results from the increased responsiveness of spinal neurons generated by intraspinal mechanisms (3, 10). However, the increase in responses was smaller than that usually seen during the development of inflammation (compare Figures 1C and 3C), presumably because IL-6/sIL-6R sensitizes only C fibers and not Aδ fibers (20) and because other mediators, such as TNF (19), contribute to inflammatory sensitization.
An increase in the responses to peripheral stimulation in the rat, including an expansion of the receptive fields of the spinal neurons toward the ankle or the paw, was also generated by the spinal application of exogenous IL-6/sIL-6R, showing that IL-6 also acts centrally. In fact, gp130, the signal-transducing complex and target of IL-6/sIL-6R, is abundant in neurons and glial cells of the spinal cord, suggesting different target sites of IL-6 (39, 40). The membrane receptor IL-6R (also called gp80) is mainly found in glial and endothelial cells and is sparse in neurons of the central nervous system (41). In this study, sensitization by application of IL-6/sIL-6R to the rat spinal cord was prevented by concomitant application of soluble gp130, which abolishes transsignaling (23, 24). It is likely that the application of both IL-6 and sIL-6R is necessary, since in a previous study the spinal application of IL-6 alone did not enhance spinal hyperexcitability (42). The precise mechanism of the sensitization is not known. The effects of IL-6 may depend on a decrease in inhibitory currents (43) and/or changes in transmitter release.
This is the first study to show that IL-6 is released from the spinal cord in the initial hours of peripheral inflammation in the rat. We cannot exclude the possibility that the high initial level of IL-6 was partly due to the preceding surgery (but see also ref.44). While microglia and astroglia are known sources of IL-6 (21), we also found IL-6–like immunoreactivity in a substantial proportion of DRG neurons in the rat, suggesting that significant amounts of IL-6 may actually be released from primary afferent fibers. In fact, cultured DRG neurons release IL-6 upon stimulation (Ebersberger A, et al: unpublished observations). The attenuation of spinal hyperexcitability by soluble gp130 strongly suggests that IL-6 release is important for the generation of full spinal hyperexcitability. Although a number of other cytokines share the transmembrane receptor gp130 with IL-6 (40), the effect of soluble gp130 is quite specific for IL-6/sIL-6R transsignaling (23).
The early release of IL-6 after induction of inflammation and the effect of soluble gp130 are strongly coincident. However, soluble gp130 did not fully prevent the development of hyperexcitability. This incomplete effect may have several causes. First, IL-6 is a modulator of synaptic transmission, whereas glutamate is the key mediator of synaptic transmission (10, 25), and neuropeptides and spinal prostaglandins (3, 26, 45) also contribute to spinal hyperexcitability. Second, soluble gp130 prevents transsignaling (implying that endogenous sIL-6R must be present) but not the direct effects of IL-6 on membrane-bound IL-6R. Substantial levels of IL-6R have been found in the DRGs and in cortical tissue under pathophysiologic conditions (41, 46, 47), indicating that the expression of IL-6R may limit the ability of soluble gp130 to block IL-6 signaling as a whole.
The application of soluble gp130 to the rat spinal cord 7–11 hours after the induction of inflammation and spinal hyperexcitability did not reduce the responses to stimulation of the inflamed knee joint and the adjacent and remote areas. An attractive hypothesis is that IL-6 triggers persistent effects and that after the induction of hyperexcitability, the continuous presence of IL-6 is no longer necessary to maintain it. The findings of previous studies support this idea. In peripheral nociceptors, soluble gp130 prevented the sensitization caused by IL-6/sIL-6R but did not reverse established mechanical sensitization (20). A single application of IL-6 primed muscle nociceptors for prostaglandin effects for at least 24 hours (48). The preventive application of soluble gp130 during the induction of antigen-induced arthritis reduced mechanical hyperalgesia, but systemic treatment with soluble gp130 after the onset of antigen-induced arthritis did not reduce mechanical hyperalgesia in the first 2 weeks (38). We propose, therefore, that IL-6 may be a key player in the induction of chronic pain in the deep somatic tissue, and quick pain relief may not be expected from the neutralization of IL-6 during established disease.
In summary, IL-6–deficient mice have been shown to exhibit less hyperalgesia in response to mechanical and thermal stimulation after subcutaneous injection of carrageenan than wild-type mice (49), and the up-regulation of spinal IL-6 in models of inflammation (21, 22) and models of neuropathic pain (50) suggested a role of spinal IL-6 in pain. The present study demonstrated a significant role of peripheral and spinal IL-6 signaling in the generation of inflammation-evoked central sensitization and hyperalgesia in the rat.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Ebersberger had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Vazquez, Kahlenbach, Segond von Banchet, König, Schaible, Ebersberger.
Acquisition of data. Vazquez, Kahlenbach, Segond von Banchet, König, Schaible, Ebersberger.
Analysis and interpretation of data. Vazquez, Kahlenbach, Segond von Banchet, König, Schaible, Ebersberger.