Spinal cathepsin S and fractalkine contribute to chronic pain in the collagen-induced arthritis model

Authors


Abstract

Objective

The induction of rheumatoid arthritis (RA) by active and passive immunization of mice results in the development of pain at the same time as the swelling and inflammation, with both peripheral and central sensitization contributing to joint pain. The purpose of this study was to examine the development of pain in the rat model of collagen-induced arthritis (CIA) and to evaluate the contribution of neuroimmune interactions to established arthritis pain.

Methods

Mechanical hypersensitivity was assessed in female Lewis rats before and up to 18 days after induction of CIA by immunization with type II collagen. The effect of selective inhibitors of microglia were then evaluated by prolonged intrathecal delivery of a cathepsin S (CatS) inhibitor and a fractalkine (FKN) neutralizing antibody, from day 11 to day 18 following immunization.

Results

Rats with CIA developed significant mechanical hypersensitivity, which started on day 9, before the onset of clinical signs of arthritis. Mechanical hypersensitivity peaked with the severity of the disease, when significant microglial and astrocytic responses, alongside T cell infiltration, were observed in the spinal cord. Intrathecal delivery of microglial inhibitors, a CatS inhibitor, or an FKN neutralizing antibody attenuated mechanical hypersensitivity and spinal microglial response in rats with CIA.

Conclusion

The inhibition of microglial targets by centrally penetrant CatS inhibitors and CX3CR1 receptor antagonists represents a potential therapeutic avenue for the treatment of pain in RA.

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.

MATERIALS AND METHODS

Animals.

All experiments were carried out using adult female Lewis rats weighing 180–200 gm (Harlan). All experiments were undertaken with approval of the UK Home Office. Experimental study groups were randomized and blinded.

Induction of CIA.

Bovine type II collagen (4 mg/ml; MD Bioproducts) was dissolved in acetic acid (0.1M) and then emulsified with Freund's complete adjuvant (CFA; 1 mg/ml). To prepare the CFA, desiccated, killed Mycobacterium tuberculosis H37Ra was suspended in Freund's incomplete adjuvant (both from BD Biosciences). On day 0, rats were anesthetized with isoflurane and injected intradermally at the base of the tail with 200 μl of collagen/CFA emulsion (400 μg of collagen per rat).

For macroscopic assessment of arthritis, rats were scored for clinical signs on a scale of 0–3, where 0 = no inflammation, 1 = inflammation of the ankle joint, 2 = inflammation of the footpad, and 3 = inflammation of 1 or more digits. Each hind limb was graded (maximum score 6 per rat). Additionally, the thickness of each hind paw was measured using a pocket thickness gauge (Mitutoyo), and body weight was monitored throughout the study. As reported previously (19, 20), the first signs of arthritis were evident between days 11 and 14, with maximal inflammation occurring between days 16 and 18.

For histologic assessment, hind paws were removed on day 18 postimmunization, fixed overnight in 4% paraformaldehyde, decalcified for 7–10 days in 30% formic acid with 0.5M trisodium citrate, and embedded in paraffin. Longitudinal sections (5 μm) were cut from the center of the ankle joint in the sagittal plane and stained with hematoxylin and eosin. Sections were examined by light microscopy for cellular infiltration, synovitis, bone erosion, and structural integrity.

Behavioral testing.

Mechanical withdrawal thresholds were assessed by measuring the paw withdrawal threshold using a dynamic plantar anesthesiometer (Ugo Basile). Briefly, each animal was placed in a clear acrylic cubicle on top of a metal grid in a temperature-controlled room (∼22°C) and allowed to acclimate for 15 minutes before testing. The stimulus was applied via an actuator filament under computer control, which applied a linearly increasing force ramp to the plantar surface of the hind paw. The force necessary to elicit paw withdrawal was recorded. A cutoff of 50 gm was imposed to prevent any tissue damage. The withdrawal threshold of each hind paw was calculated as the average of 3 consecutive tests with at least 5 minutes between each test. Measurements were taken on 3 separate days prior to immunization and then throughout the disease process.

Intrathecal pumps and drug treatment.

For pharmacologic experiments, intrathecal cannulas were implanted as previously described (10, 11). Two weeks prior to induction of CIA, with the rat under anesthesia, a small laminectomy was made at the sixth or seventh thoracic vertebra, and a flexible cannula (external diameter 1.14 mm; Merck) was inserted under the dura mater, such that the tip rested at the lumbar enlargement. The opposite end of the cannula was placed subcutaneously for delayed drug treatment. Eleven days after immunization, rats that had developed mechanical allodynia were anesthetized, and an osmotic minipump (Alzet model 2001) was inserted subcutaneously and connected to the cannula.

For the first pharmacologic experiment, rats received the irreversible CatS inhibitor morpholinurea-leucine-homophenylalanine-vinyl sulfone-phenyl (LHVS) (30 nmoles/day; NeoMPS) or vehicle (20% Cremophor EL/saline; Sigma) for 7 days beginning on day 11 postimmunization. For the second pharmacologic experiment, rats received either an FKN neutralizing antibody (1 μg/day of anti-FKN AF537) or normal goat IgG (both from R&D Systems) for 7 days beginning on day 11 postimmunization. Doses of pharmacologic agents were determined based on previous studies (10–12) in order to inhibit CatS enzymatic activity and interfere with FKN signaling in the spinal cord, respectively.

Immunohistochemistry.

On day 18 postimmunization, rats were placed under pentobarbital anesthesia and transcardially perfused with 0.9% saline solution followed by 4% paraformaldehyde in 0.1M phosphate buffer. The lumbar spinal cord and L4 and L5 dorsal root ganglia (DRGs) were excised, postfixed for 4 hours in the perfusion fixative, cryoprotected in 20% sucrose in 0.1M phosphate buffer (72 hours at 4°C), and frozen in OCT embedding compound (VWR). Transverse sections of spinal cord (20 μm) and DRGs (15 μm) were cut with a cryostat and thaw-mounted onto glass slides. Spinal cord sections were incubated overnight with primary antibody solution for rabbit anti–ionized calcium–binding adapter molecule 1 (IBA-1) (1:1,000 dilution; Wako), mouse anti-CD11b (OX42) (1:100 dilution; Serotec), rabbit anti–glial fibrillary acidic protein (anti- GFAP) (1:1,000 dilution; DakoCytomation), or mouse anti-CD3 (1:50 dilution; BD PharMingen), followed by secondary antibody solution (Alexa Fluor 488–conjugated or Alexa Fluor 546–conjugated IgG; Molecular Probes). For counterstaining of the microglial cell population, rabbit anti–phospho-p38 MAPK (1:100 dilution; Cell Signaling Technology) was visualized with ExtrAvidin-FITC (1:500 dilution; Sigma) following signal amplification with avidin–biotin–peroxidase (Vector) and biotinyl tyramide (NEN Life Science Products), as previously described (21, 22).

Sections were then incubated with primary antibody for IBA-1, followed by secondary antibody solution (Alexa Fluor 546–conjugated goat anti-rabbit IgG) (1:1,000 dilution; Molecular Probes). DRG sections were incubated overnight with primary antibody solution for rabbit anti–activating transcription factor 3 (anti–ATF-3) (1:500 dilution; Santa Cruz Biotechnology) and mouse anti–β3-tubulin (1:1,000 dilution; Promega), followed by the appropriate secondary antibodies. In control experiments, primary antibody was omitted from some sections of spinal cord and DRGs, whereby staining was completely abolished. Slides were coverslipped with Vectashield mounting medium (Vector) and visualized under a Zeiss LSM 710 confocal microscope.

Quantitative assessment.

Quantitative assessment of immunostaining in spinal cord sections was carried out by counting the number of positive profiles within a fixed area of the dorsal horn (10–12, 21–23). A box measuring 104 μm2 was placed onto areas of the lateral, central, and medial dorsal horn, and the number of profiles positive for each marker within this area were counted. These measurement protocols were carried out on three L5 spinal sections from each animal. For quantitative assessment of immunostaining in DRG sections, the number of neurons positive for ATF-3 was expressed as a percentage of total neurons expressing β3-tubulin.

Statistical analysis.

For analysis of behavioral data, we used two-way repeated-measures analysis of variance followed by Tukey's post hoc test. For analysis of immunohistochemical data, Student's t-test was used. P values less than 0.05 were considered significant.

RESULTS

CIA induces marked mechanical hypersensitivity throughout the disease time course.

In this study, we examined mechanical pain thresholds throughout the time course of CIA in the rat. As previously reported (19, 20), clinical signs of arthritis, as evidenced by the clinical scores (Figure 1A) and paw swelling (Figure 1B), began to develop between days 11 and 14 following immunization and were augmented throughout the course of the disease. Histologic examination of the ankles on day 18 postimmunization revealed joint degradation, which paralleled increases in the clinical scores (Figures 1E and F). Mechanical withdrawal thresholds in rat hind paws remained unaltered from preimmunization thresholds in rats with CIA and in control rats until day 7. However, animals with CIA exhibited a significant reduction in mechanical thresholds from day 9 until day 18 as compared to control animals (Figure 1C). The severity of mechanical hypersensitivity progressed throughout the study and was maximal on day 18 following immunization, corresponding to the peak of disease. In addition, area under the curve (AUC) analysis of paw withdrawal thresholds revealed significant mechanical hypersensitivity in animals with CIA as compared to controls (Figure 1D).

Figure 1.

Collagen-induced arthritis (CIA) produces mechanical hypersensitivity. A and B, Clinical signs of arthritis, including development of clinical features (A) and paw swelling (B), are evident beginning on day 11 postimmunization. C and D, Rats develop significant mechanical hypersensitivity, as determined by paw withdrawal thresholds (PWTs) (C) and the area under the curve analysis of paw withdrawal thresholds (D), following induction of CIA, which precedes clinical signs of arthritis. Values in A–D are the mean ± SEM of 13 rats per CIA group and 6 rats per control group. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus baseline and † = P < 0.05; ††† = P < 0.001 versus controls, by two-way repeated-measures analysis of variance, with Tukey's post hoc test. E and F, Representative photomicrographs of hematoxylin and eosin–stained sections of paws with low versus high scores. Paws with a clinical score of 0 (E) had a smooth articular surface with no signs of inflammation, which is in contrast to the synovial hyperplasia (arrow), aggressive invading pannus (asterisks), and overall loss of articular integrity in paws with a clinical score of 3 (F). Fields are centered on the tarsal joints. Bar = 300 μm.

CIA enhances glial response and induces T lymphocyte infiltration in the dorsal horn.

Multiple mechanisms operating both peripherally and centrally contribute to chronic pain behaviors in rodent models (24, 25). In the dorsal horn, the microglia and astrocytes respond to remote damage by entering a pain-related enhanced response state, which promotes lymphocyte infiltration and augments nociceptive signaling (6). We therefore used immunohistochemical analyses to examine the response to CIA of glial cells, as well as the extent of T lymphocyte infiltration, in the dorsal horn of the spinal cord. As with the clinical signs of arthritis, mechanical hypersensitivity peaked on day 18 postimmunization (Figure 1). Thus, we selected this time point to examine possible changes that may underpin mechanical hypersensitivity in CIA.

We initially examined the number of IBA-1+ cells in order to determine the total numbers of microglial cells within the dorsal horn. Low numbers of IBA-1+ cells were observed in the dorsal horn of control rats (Figure 2B). Eighteen days postimmunization, animals with CIA exhibited a significant increase in the numbers of IBA-1+ microglia in the dorsal horn (Figure 2A), reaching a mean ± SEM of 161 ± 3% of control levels (7.9 ± 0.2 cells/104 μm2 in control rats [n = 6] and 12.8 ± 0.2 cells/104 μm2 in rats with CIA [n = 13]; P < 0.001 by Student's t-test).

Figure 2.

Collagen-induced arthritis (CIA) produces an enhanced microglial response in the dorsal horn. A and B, Ionized calcium–binding adapter molecule 1 (IBA-1) immunoreactivity in the dorsal horn of rats with CIA (A) and control rats (B). C and D, Colocalization of IBA-1 (red) and phospho-p38 (green) immunoreactivity (yellow) in the dorsal horn of rats with CIA (C) and control rats (D). E, Quantification of IBA-1+ and phospho-p38+ microglia in the dorsal horn of rats with CIA and control rats. F and G, Colocalization of IBA-1 (red) and OX42 (green) immunoreactivity (yellow) in the dorsal horn of rats with CIA (F) and control rats (G). H, Quantification of IBA-1+ and OX42+ microglia in CIA and control dorsal horn. Bars in A and C = 100 μm. Values are the mean ± SEM of 6–9 rats per group in E and 6–11 rats per group in H. ∗∗∗ = P < 0.001 versus controls, by Student's t-test.

Next, we examined the expression of 2 classic markers of enhanced microglial response, phospho-p38 and OX42, within the IBA-1 cell population. Phosphorylation of p38 MAPK occurs exclusively in microglia in a number of nociceptive models and represents a robust early marker of cell activation (21, 22, 26). Double-immunohistochemistry studies revealed that CIA induced a significant increase in the number of microglial cells exhibiting phospho-p38 (Figures 2C–E). In dorsal horn from control rats, a mean ± SEM of 51 ± 6% of IBA-1+ cells also exhibited immunoreactivity for phospho-p38, whereas following CIA induction, 90 ± 1% of IBA-1+ cells were also positive for phospho-p38 (Figures 2C–E).

The expression of the complement receptor CD11b (OX42) is extensively up-regulated by microglia and is used as a marker of enhanced microglial response (23). A second immunohistochemical study revealed that CIA induced a significant increase in the number of OX42+ microglial cells (Figures 2F–H). In dorsal horn from control rats, 64 ± 1% of IBA-1+ cells also exhibited immunoreactivity for OX42, whereas following CIA induction, 91 ± 1% of IBA-1+ cells were positive for OX42 (Figures 2F–H). These data suggest that the inflammatory pain evident during CIA is associated with an enhanced response of microglia within the dorsal horn of the spinal cord. Microglial cells, the first sentinel to remote damage in the periphery, can attract lymphocytes into the CNS, which is generally followed by activation of astrocytes.

Eighteen days postimmunization, we observed a significant increase in the intensity of GFAP immunoreactivity (Figures 3A–C) as well as an increase in the number of CD3+ T cells (Figures 3D–F) in the dorsal horn of animals with CIA as compared to control animals, indicating that CIA is associated with both gliosis and T cell infiltration in the spinal cord.

Figure 3.

Collagen-induced arthritis (CIA) produces astrogliosis and T cell infiltration in the dorsal horn. A and B, Glial fibrillary acidic protein (GFAP) immunoreactivity in the dorsal horn of rats with CIA (A) and control rats (B). C, Quantification of GFAP intensity in the dorsal horn of rats with CIA and control rats. D and E, CD3 immunoreactivity in the dorsal horn of rats with CIA (D) and control rats (E). F, Quantification of CD3+ T cells in the dorsal horn of rats with CIA and control rats. Bar in A = 100 μm. Values are the mean ± SEM of 6–11 rats per group in C and 5–6 rats per group in F. ∗ = P < 0.05; ∗∗∗ = P < 0.001 versus controls, by Student's t-test.

Although an increased primary afferent barrage can drive the microglial response in the dorsal horn (27), we examined whether CIA was associated with peripheral neuron injury by determining the expression level of ATF-3 in L4 and L5 DRGs. There was no significant change in the percentage of DRG cells expressing ATF-3 in rats with CIA as compared to control rats (1.15 ± 0.03% versus 1.22 ± 0.09% [n = 4 rats per group]; P > 0.05 by Student's t-test), which excludes the possibility that damage to the peripheral terminals of sensory neurons in the inflamed joint contributes to pain hypersensitivity and microglial response in CIA.

Mechanical hypersensitivity following CIA is dependent on spinal CatS and FKN.

In order to delineate possible pathways for neuronal–microglial communication in the dorsal horn during CIA, we examined the possible role of microglial CatS/neuronal FKN signaling which is critically involved in the full expression of chronic pain following peripheral nerve injury and acute peripheral inflammation (10, 12, 28). Induction of CIA resulted in mechanical hypersensitivity, as indicated by a significant reduction in hind paw mechanical withdrawal thresholds beginning on day 9 postimmunization (Figures 4A and D). Following behavioral testing on day 11, prior to overt clinical signs of arthritis, animals displaying mechanical hypersensitivity began to receive drug treatment over the lumbar spinal cord. Continuous intrathecal administration of the CatS inhibitor LHVS from day 11 to day 18 following immunization significantly attenuated mechanical hypersensitivity beginning on day 12, as compared to administration of vehicle (Figure 4A). Disease progression was not altered by spinal CatS inhibition, since clinical signs of arthritis, such as the clinical score (Figure 4B) and paw swelling (Figure 4C), in both LHVS-treated and vehicle-treated animals were comparable.

Figure 4.

Spinal inhibition of cathepsin S (CatS) (A–C) or fractalkine (FKN) (D–F) attenuates mechanical hypersensitivity following collagen-induced arthritis (CIA). A, Long-term intrathecal administration of the CatS inhibitor morpholinurea-leucine-homophenylalanine-vinyl sulfone-phenyl (LHVS), as compared with vehicle administration, significantly reverses established mechanical hypersensitivity in rats with CIA, as determined by paw withdrawal thresholds (PWTs). Thick bar above the x-axis indicates the treatment duration. B and C, Intrathecal LHVS administration does not modify the clinical score (B) or paw swelling (C) in rats with CIA. Values are the mean ± SEM of 6 rats per group treated with vehicle and 7 rats per group treated with LHVS. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus baseline and ††† = P < 0.001 versus vehicle-treated rats with CIA, by two-way repeated-measures analysis of variance, with Tukey's post hoc test. D, Long-term intrathecal administration of an FKN neutralizing antibody (anti-FKN), as compared with IgG administration, significantly reverses established mechanical hypersensitivity in rats with CIA, as determined by paw withdrawal thresholds. Thick bar above the x-axis indicates the treatment duration. E and F, Intrathecal anti-FKN administration does not modify the clinical score (E) or paw swelling (F) in rats with CIA. Values are the mean ± SEM of 8 rats per group treated with IgG and 9 rats per group treated with anti-FKN. ∗ = P < 0.05; ∗∗∗ = P < 0.001 versus baseline and ††† = P < 0.001 versus IgG-treated rats with CIA, by two-way repeated-measures analysis of variance, with Tukey's post hoc test.

Similarly, continuous intrathecal infusion of an FKN neutralizing antibody (anti-FKN) from day 11 to day 18 following immunization significantly attenuated mechanical hypersensitivity beginning on day 12, as compared to IgG (Figure 4D). Disease progression was not altered by inhibition of spinal FKN signaling, since clinical signs of arthritis (Figures 4E and F) were comparable between the 2 groups. AUC analysis revealed that both LHVS and anti-FKN treatment reversed mechanical hypersensitivity to a similar extent (mean ± SEM AUC for LHVS was 124 ± 3% of vehicle levels and for anti-FKN was 120 ± 4% of IgG levels). These data suggest that spinal CatS and FKN contribute to CIA-induced chronic pain behaviors. Interestingly, the clinical signs of arthritis were not modified by either pharmacologic treatment, which suggests that the chronic inflammatory pain component of the CIA model can be uncoupled from disease progression.

Enhanced spinal microglial response in CIA is attenuated by spinal CatS and FKN inhibition.

Since both CatS and the CX3CR1 receptor for FKN are expressed by microglia, we examined whether attenuation of CIA-induced hypersensitivity by inhibition of CatS and FKN signaling was associated with a reduction in the spinal microglial response in this disease model.

As illustrated above, on day 18 following immunization, animals with CIA treated with vehicle exhibited significant numbers of IBA-1+ microglial cells in the dorsal horn of the spinal cord (Figures 5 and 6). Following prolonged intrathecal delivery of LHVS, the numbers of IBA-1+ microglial cells were significantly reduced as compared to vehicle-treated animals (Figure 5). In addition, the number of microglial cells exhibiting OX42 (Figures 5A–F and 5M) or phospho-p38 (Figures 5G–L and 5N) immunoreactivity was significantly attenuated by LHVS treatment. In the dorsal horn of animals with CIA treated with vehicle, 96 ± 1% and 91 ± 3% of IBA-1+ cells also exhibited immunoreactivity for OX42 and phospho-p38, respectively. This is in contrast to animals with CIA treated with LHVS, where only 78 ± 2% and 75 ± 5% of IBA-1+ cells in the dorsal horn were also positive for OX42 and phospho-p38, respectively.

Figure 5.

Spinal inhibition of cathepsin S attenuates the microglial response following collagen-induced arthritis (CIA). A–F, Colocalization of ionized calcium–binding adapter molecule 1 (IBA-1) (red) and OX42 (green) immunoreactivity (yellow in merge images) in the dorsal horn of rats with CIA treated with morpholinurea-leucine-homophenylalanine-vinyl sulfone-phenyl (LHVS) or vehicle. G–L, Colocalization of IBA-1 (red) and phospho-p38 (green) immunoreactivity (yellow in merge images) in the dorsal horn of rats with CIA treated with LHVS or vehicle. Bars = 100 μm. M and N, Quantification of IBA-1+ and OX42+ microglia (M) and IBA-1+ and phospho-p38+ microglia (N) in the dorsal horn of rats with CIA. Values are the mean ± SEM of 6–7 rats per group in M and 8–9 rats per group in N. ∗ = P < 0.05; ∗∗ = P < 0.01 versus vehicle-treated rats with CIA, by Student's t-test.

Figure 6.

Spinal inhibition of fractalkine (FKN) attenuates the microglial response following collagen-induced arthritis (CIA). A–F, Colocalization of ionized calcium–binding adapter molecule 1 (IBA-1) (red) and OX42 (green) immunoreactivity (yellow in merge images) in the dorsal horn of rats with CIA treated with anti-FKN or IgG. G–L, Colocalization of IBA-1 (red) and phospho-p38 (green) immunoreactivity (yellow in merge images) in the dorsal horn of rats with CIA treated with anti-FKN or IgG. Bars = 100 μm. M and N, Quantification of IBA-1+ and OX42+ microglia (M) and IBA-1+ and phospho-p38+ microglia (N) in the dorsal horn of rats with CIA. Values are the mean ± SEM of 6–7 rats per group in M and 8–9 rats per group in N. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus IgG-treated rats with CIA, by Student's t-test.

Similarly, prolonged intrathecal treatment with anti-FKN significantly attenuated the microglial response as compared to IgG treatment in animals with CIA (Figure 6). In the dorsal horn of animals with CIA treated with IgG, 95 ± 1% and 92 ± 1% of IBA-1+ cells also exhibited immunoreactivity for OX42 (Figures 6A–C) and phospho-p38 (Figures 6G–I), respectively. However, in the dorsal horn of animals with CIA treated with anti-FKN, 78 ± 3% and 68 ± 4% of IBA-1+ cells in the dorsal horn were also positive for OX42 (Figures 6D–F) and phospho-p38 (Figures 6J–L), respectively. These data suggest that spinal microglial cells are key contributors to inflammatory pain following CIA and that inhibition of the enhanced microglia response may represent a potential therapeutic avenue for the treatment of pain in RA.

DISCUSSION

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.

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. Malcangio 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. Clark, Malcangio.

Acquisition of data. Clark, Grist, Al-Kashi.

Analysis and interpretation of data. Clark, Perretti, Malcangio.

Ancillary