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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

To investigate the endogenous involvement of transient receptor potential vanilloid 1 (TRPV1) in a model of knee joint inflammation in the mouse.

Methods

Following characterization of wild-type (WT) and TRPV1-knockout mice, inflammation was induced via intraarticular (IA) injection of Freund's complete adjuvant (CFA). Knee swelling was assessed as diameter, and inflammatory heat hyperalgesia was determined using the Hargreaves technique, for up to 3 weeks. At 18 hours, acute hyperpermeability was measured with 125I-albumin, and cytokines and myeloperoxidase activity, a marker of neutrophils, were assayed in synovial fluid extracts. The possibility that exogenous tumor necrosis factor α (TNFα) was involved in influencing TRPV1 activation was investigated in separate experiments.

Results

Increased levels of knee swelling, hyperpermeability, leukocyte accumulation, and TNFα were found in WT mice 18 hours after IA CFA treatment compared with saline treatment. Significantly less knee swelling and hyperpermeability were found in TRPV1−/− mice, but leukocyte accumulation and TNFα levels were similar in WT and TRPV1−/− mice. Knee swelling in response to CFA remained significantly higher for a longer period in WT mice compared with TRPV1−/− mice, with thermal hyperalgesic sensitivity observed at 24 hours and at 1 week in WT, but not TRPV1−/−, mice. Knee swelling was attenuated (P < 0.05) in TRPV1−/− compared with WT mice 4 hours after IA administration of TNFα.

Conclusion

Our findings indicate that TRPV1 has a role in acute and chronic inflammation in the mouse knee joint. Thus, selective antagonism of TRPV1 should be considered as a potential target for treatment of acute and chronic joint inflammation.

Capsaicin, the active ingredient of hot peppers, selectively stimulates and then desensitizes a subpopulation of C-fiber polymodal nociceptive and Aδ sensory nerve fibers that are classified as capsaicin-sensitive afferents (1, 2). Capsaicin acts via transient receptor potential vanilloid 1 (TRPV1), which was cloned in 1997 and was formerly termed vanilloid receptor 1 (3). TRPV1 is a nonselective cation channel, which can be activated by noxious heat, protons, and vanilloids such as capsaicin, as well as a range of putative endogenous mediators (4, 5). The acute stimulation of sensory nerves by capsaicin is associated with acute pain and neurogenic inflammation (1, 2). Thermal nociceptive thresholds, observed after exposure to a hot plate or inflammatory insult in the paw, have been shown to be blunted in TRPV1-knockout mice, indicating the potential for TRPV1 in mediating inflammatory hyperalgesia (6, 7). The stimulation of TRPV1 leads to release of neuropeptides, including substance P and calcitonin gene-related peptide (CGRP) from sensory nerves, (2, 8, 9). Neuropeptides have the potential to contribute to inflammatory disease as the “neurogenic component” via a variety of mechanisms (10).

Levels of neuropeptides have been shown to be increased in samples of synovial fluid from patients with rheumatoid arthritis (11), and the ligation of sensory nerves in experimental joint inflammation is associated with a beneficial effect (12), providing evidence for involvement of sensory nerve activation in arthritis. Indeed, continued topical treatment with capsaicin has been shown to be beneficial in patients with hand osteoarthritis (13), and capsaicin creams are marketed for the alleviation of pain in diseases including both osteoarthritis and rheumatoid arthritis (14). There is evidence from rodent studies that TRPV1 activation can influence pathophysiologic events in arthritis. For example, capsaicin pretreatment depletes substance P, and this has been associated with attenuation of the severity of arthritis (15–18). However, it is difficult to utilize studies involving capsaicin to pinpoint the influence of endogenous activation of TRPV1 in vivo on joint inflammation, due to the capsaicin-stimulated events leading to both sensory nerve depletion and nonspecific desensitization. In addition, pharmacologic antagonists developed to date are, to our knowledge, unsuitable for studies of long-term responses (19). Thus, mice lacking TRPV1 present a distinct experimental advantage, since the influence of TRPV1 can be investigated in the absence of exogenously administered vanilloids.

In the present study, TRPV1−/− mice were used in protocols involving recently developed techniques designed to enable correlation of knee swelling and inflammatory edema (20). Unilateral adjuvant-induced arthritis was elicited by intraarticular (IA) injection of Freund's complete adjuvant (CFA) into the knee joint. The objective was to investigate the sensory neurogenic involvement through use of methods developed to facilitate the analysis of microvascular events in addition to the measurement of nociceptive behavior. The results provide evidence that the deletion of TRPV1 led to reduced acute (18 hours) and chronic knee swelling. Acute knee swelling was associated with TRPV1-sensitive hyperpermeability in the knee joint. Knee swelling was also reduced long term in TRPV1−/− mice, and reduced nociceptive responses were observed. The techniques were also used to evaluate the role of TRPV1 in the local generation of, and acute effects of, tumor necrosis factor α (TNFα). Our findings provide evidence for a key role of TRPV1 in mediating ongoing, in addition to acute, joint inflammation.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Preparation of animals.

Male and female C57BL6/129SVJ mice (25–35 gm, at least 12 weeks of age), either genetically unaltered (wild-type [WT]) or lacking the gene for TRPV1, were bred in-house from WT and TRPV1−/− breeding colonies that were donated by Dr. S. Boyce (Merck, Sharp & Dohme, Harlow, UK). Breeding pairs were generated as described previously (6) and supplied by D. Julius (University of California, San Francisco). Mice were kept in a climatically controlled environment and had access to food and water ad libitum. Experiments were carried out in accordance with the 1986 UK Home Office Animals (Scientific Procedures) Act. TRPV1−/− mice bred normally and were matched by sex and weight with WT mice for all procedures. All recovery procedures were performed using isofluorane anesthesia (2%), and all nonrecovery procedures were carried out using urethane anesthesia (2.5 mg/gm intraperitoneally); 30-gauge needles were used for all intraarticular injections (Micro-Fine insulin syringes; 0.3 ml) (BD Medical, Franklin Lakes, NJ). All reagents were obtained from Sigma-Aldrich (Dorset, UK), unless otherwise stated.

Techniques used to characterize mice.

Capsaicin-induced ear plasma extravasation was examined after 125I-albumin (45 kBq; MP Biomedicals, London, UK) was injected intravenously (IV) into anesthetized mice, and 5 minutes later capsaicin in ethanol (20 μl of 10 mg/ml) was applied externally to both surfaces of one ear, and ethanol (vehicle control) to the contralateral ear (9). Edema was allowed to develop for 60 minutes. A blood sample was collected by cardiac puncture, the mice were killed, and then ears were removed, weighed, and gamma activity counted for analysis of plasma extravasation. In separate experiments, the hot plate (55°C) test was used to assess nociceptive responses in WT compared with TRPV1−/− naive mice. Mice were placed individually on the hot plate and the time taken to respond to the heat was recorded. Responses noted were licking or shaking of a hind limb or jumping from the hot plate. This assay was used for naive mice, but responses obtained after induction of unilateral joint/paw inflammation were nonreproducible, presumably due to the unilateral nature of the model. Carrageenan-induced paw inflammation was induced by intraplantar injection of carrageenan (500 μg/50 μl). Paw swelling was measured using calipers, and thermal nociceptive responses were examined over a 4-hour experimental period using the Hargreaves technique (see below).

The genotype of founder animals and of offspring from subsequent generations was determined by polymerase chain reaction (PCR) analysis of genomic DNA extracted from tail biopsy specimens. TRPV1 WT primers (5′-CGAGGATGGGAAGAATAACTCACTG and 5′-GGATGATGAAGACAGCCTTGAAGTC) were used to amplify a PCR product with predicted size of 100 bp, corresponding to the WT gene. Neomycin gene primers (5′-TTTTGTCAAGACCGACCTGTCC and 5′-CCCTCAGAAGAACTCGTCAAGAAG) were used to amplify a PCR product with predicted size of 700 bp, corresponding to the neomycin construct found in the disrupted gene in the knockout mice. PCR products were analyzed by agarose gel electrophoresis (through Merck, Sharp & Dohme, in association with Taconic Biotechnology, Germantown, NY).

Induction of joint inflammation and measurement of swelling.

For induction of joint inflammation, the hind knees of the mice were injected IA with 10 μg CFA (10 μl in the ipsilateral joint) and saline (0.9% sodium chloride, pyrogen free; Baxter Healthcare, Thetford, UK) (10 μl in the contralateral joint) and inflammation was allowed to develop for varying periods of up to 3 weeks. The inflammatory effects of 10 pmoles recombinant murine TNFα (10 μl IA) were examined over a 4-hour period, after which synovial neutrophil accumulation, plasma extravasation, intravascular volume, and hyperalgesia were determined. The diameter of knee joints was measured with calipers (Mitutoyo, Kanagawa, Japan) and used as an index of knee swelling.

Measurement of hyperpermeability in and around the joint.

Hyperpermeability was determined based on extravascular accumulation of IV-injected 125I-albumin (45 kBq), administered 1 hour prior to the end of the experimental period. Mice were killed by cervical dislocation at the end of the accumulation period, and the skin overlying the knee was excised. 125I-albumin accumulation in the joint was then determined using a collimated gamma probe (Europrobe; Bright Technologies, Sheffield, UK) (see ref. 20). Briefly, the head of the probe was held against the joint region and the counts per minute detected in the joint were recorded. Plasma extravasation was expressed as a percentage of cpm detected in the agent-treated joint compared with the saline-treated joint, to allow for unavoidable differences in the quantity of radioactivity injected IV. A percentage of 100 indicates that the same quantity of 125I-albumin is present in both joints, and thus, that there is no plasma extravasation. A percentage of >100 shows that more 125I-albumin is present in the test joint compared with the control joint. The larger the percentage, the greater the amount of plasma extravasation. Alternatively, to ensure that the quantity of 125I-albumin was not merely due to increased intravascular volume, 125I-albumin was injected 2 minutes prior to the end of the experimental period. Thus, it was distributed throughout the cardiovascular system for 2 minutes, but with minimal time for plasma extravasation to take place.

Collection of synovial fluid.

Mice were killed and the skin overlying the knee was excised. The patellar ligament was then carefully dissected to expose the synovial membrane. A 30-gauge needle (Micro-Fine insulin syringe; 0.3 ml) was inserted through the membrane, and the synovial cavity was washed by injecting and immediately aspirating 25 μl of heparinized saline (5 units/ml) to obtain the synovial lavage material. This was repeated once (total 50 μl of synovial lavage sample). The collected fluid was snap-frozen in liquid nitrogen and stored at −20°C prior to assay.

Measurement of neutrophil accumulation and cytokines.

A spectrophotometric assay was used to measure myeloperoxidase (MPO) activity, as described previously (21). The quantity of MPO in mouse joint synovial lavage fluid is expressed in units/ml. For the cytokine bead array, a commercial kit was used (Mouse Th1/Th2 Cytokine CBA kit; BD Biosciences, San Diego, CA).

Hargreaves test.

Hind paw thermal nociceptive thresholds were determined before and at the stated times after administration of test agents, according to the Hargreaves method (21) adapted for mice (22). Briefly, the mice were housed in a behavior box on a glass platform and allowed to acclimatize for 30 minutes before readings. The nociceptive threshold was measured with an automatic heat source (50W, 10V) directed onto the plantar surface of the paw and a timer linked to a light sensor. Withdrawal of the paw produced a change in the measured light and stopped the timer and heat source. A cutoff time of 22 seconds was used to avoid tissue damage to the hind paw. Measurements were obtained in triplicate, and the mean value was recorded as the nociceptive threshold. Results were measured as paw withdrawal latency.

Statistical analysis.

Results are expressed as the mean ± SEM. Statistical analysis was carried out using Student's paired or unpaired 2-sample t-test or two-way analysis of variance, followed by the Bonferroni post hoc test for comparison of means as necessitated by the data. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Functional characterization of WT and TRPV1−/− mice.

In initial studies, the TRPV1−/− mice were characterized by comparison with paired WT mice. The vascular response was examined after topical application of capsaicin to the ear. Acute edema formation was observed in WT mice and was absent in TRPV1−/− mice after topical application of capsaicin (Figure 1A), as expected from previous studies (7, 9). In separate studies, nociceptive activity was found to be similar to that observed in previous experiments (6, 7), i.e., TRPV1−/− mice were less responsive to heat when compared with WT mice in the thermal hot plate test and also in the Hargreaves assay of thermal nociceptive threshold in carrageenan-induced inflammation (Figures 1B and C). In comparison, paw swelling in response to carrageenan was similar in WT and TRPV1−/− mice, as previously shown (7) (ipsilateral paw volume expressed as a percentage of saline-injected contralateral paw volume 147.9 ± 9.1% in WT mice and 146.3 ± 3.4% in TRPV1−/− mice) (mean ± SEM; n = 6–9 per group). Proof of genotype is shown in Figure 1D.

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Figure 1. Characterization of transient receptor potential vanilloid 1 (TRPV1)–knockout (KO) mice, determined based on comparisons with wild-type (WT) mice. A, Effect of 1% capsaicin on ear edema formation over a 1-hour period. Values are the mean and SEM (n = 9–10 per group). ∗∗∗ = P < 0.001 versus ethanol-treated ears of WT mice; ### = P < 0.001 versus capsaicin-treated ears of WT mice. B, Hot plate sensitivity. The time to response to heat at 55°C in the hot plate test is shown. Values are the mean ± SEM (n = 6 per group). ∗∗∗ = P < 0.001 versus WT mice. C, Carrageenan-induced thermal hyperalgesia. Values are the mean ± SEM (n = 8–11 per group). ∗ = P < 0.05 versus pretreatment value in the same joints or in saline-treated joints of WT mice. D, Polymerase chain reaction analysis of mouse tail DNA, showing absence of the predicted reaction at 700 bp but presence of the predicted reaction at 100 bp in a WT mouse, and presence of the predicted reaction at 700 bp but absence of the predicted reaction at 100 bp in a TRPV1−/− mouse (homozygote [HOM]). DNA size markers are shown on the left.

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Involvement of TRPV1 in acute knee swelling and hyperpermeability in adjuvant-induced arthritis.

The role of TRPV1 in mediating joint inflammation was investigated in a model of unilateral CFA-induced arthritis. Based on time course data from an earlier study (20), we assessed acute inflammation at the 18-hour time point. Knee diameter, used to assess knee swelling, was substantially increased in response to CFA, with less swelling observed in the TRPV1−/− mice. The diameter of the contralateral saline-injected joints was identical in WT and TRPV1−/− mice (Figure 2A). Hyperpermeability, determined by local extravasation of IV-injected 125I-albumin in the last hour (i.e., from 17 to 18 hours), was increased by ∼40% in WT mice compared with the contralateral saline-injected joint in the same mice, with significantly less intense edema formation observed in TRPV1−/− mice. The contribution of intravascular volume to these results (measured over the shortest possible time [2 minutes] in order to provide an assessment of blood volume in the joint at that time point) was minimal and was similar in WT and TRPV1−/− mice (Figure 2B). MPO activity, measured to assess polymorphonuclear leukocyte accumulation (20), was similar in WT and TRPV1−/− mice (Figure 2C).

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Figure 2. Effects of intraarticular injection of complete Freund's adjuvant (CFA) (10 μg in 10 μl) or vehicle in WT and TRPV1−/− mice, 18 hours after injection. A, Knee swelling. B, Hyperpermeability. 125I-albumin plasma extravasation in the joints of WT and TRPV1−/− mice was measured from 17 hours to 18 hours after CFA administration. Intravascular volume was determined after 125I-albumin circulated for 2 minutes before the 18-hour time point. C, Neutrophil accumulation, assessed by myeloperoxidase activity of synovial lavage fluid collected from the knee joints of WT and TRPV1−/− mice. Values are the mean ± SEM (n = 13 per group in A, 5–10 per group in B, and 6 per group in C). ∗ = P < 0.05 versus WT mice. See Figure 1 for other definitions.

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Persistently elevated levels of TNFα in the synovial fluid of both WT and TRPV1−/− mice.

The levels of 5 different cytokines, TNFα, interferon-γ (IFNγ), interleukin-5 (IL-5), IL-4, and IL-2, were measured in synovial samples 18 hours after IA administration of CFA. The pattern of results was similar in WT and TRPV1−/− mice (Table 1). The most striking finding was that TNFα levels were high in all CFA-treated joints, regardless of the presence or absence of TRPV1 in the mice. Furthermore, although levels of IFNγ, IL-4, and IL-2 were low, IL-5 levels were modestly, but significantly increased.

Table 1. Cytokines measured in synovial fluid extracts from WT or TRPV1−/− mice, 18 hours after intraarticular administration of CFA*
Mice, treatmentTNFαIFNγIL-5IL-4IL-2
  • *

    Values are the mean ± SEM pg/ml exudates (n = 6–7 per group). WT = wild-type; TRPV1 = transient receptor potential vanilloid 1; CFA = Freund's complete adjuvant; TNFα = tumor necrosis factor α; IFNγ = interferon-γ; IL-5 = interleukin-5.

  • P < 0.01 versus the contralateral saline-treated joint.

  • P < 0.05 versus the contralateral saline-treated joint.

WT, saline13.6 ± 2.27.65 ± 1.017.65 ± 1.0110.05 ± 1.863.93 ± 0.6
WT, CFA72.1 ± 14.1912.6 ± 3.5225.79 ± 7.5412.16 ± 0.985.5 ± 0.25
TRPV1−/−, saline12.7 ± 1.697.66 ± 0.526.6 ± 0.769.27 ± 2.074.24 ± 0.73
TRPV1−/−, CFA92.24 ± 24.3911.36 ± 2.527.66 ± 6.7914.37 ± 1.445.77 ± 0.72

Link between TNFα and TRPV1 activation.

TNFα was administered IA and knee swelling measured after 4 hours, by which time the characteristic leukocyte accumulation observed in response to TNFα was high. The possibility that TNFα can influence TRPV1-mediated responses was examined in 2 ways. First, knee swelling induced by TNFα was measured and found to be significantly higher in WT than in TRPV1−/− mice (Figure 3A), providing evidence of a link between TNFα and TRPV1. Second, plasma extravasation 3–4 hours after IA TNFα administration was assessed. While there was a trend toward a lower amount of edema formation during this period, this did not reach statistical significance (Figure 3B).

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Figure 3. Effects of intraarticular administration of tumor necrosis factor α (TNFα) (10 pmoles in 10 μl) or vehicle in WT and TRPV1−/− mice, after 4 hours. A, Knee swelling. ∗ = P < 0.05 versus WT mice. B, Hyperpermeability. 125I-albumin extravasation in the joints of WT and TRPV1−/− mice was measured from 3 hours to 4 hours after TNFα administration. C, Neutrophil accumulation, assessed by myeloperoxidase activity of synovial lavage fluid collected from the knee joints of WT and TRPV1−/− mice. Values are the mean ± SEM (n = 12–14 per group in A, 7–8 per group in B, and 6 per group in C). See Figure 1 for other definitions.

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Influence of TRPV1 on knee swelling and hyperalgesia in chronic conditions.

The effect of CFA on joint inflammation was studied over longer periods in order to further characterize the influence of endogenous activation of TRPV1 in chronic conditions. At 24 hours, WT and TRPV1−/− mice exhibited a significant increase in knee swelling in the CFA-treated joint compared with the saline-injected contralateral joint. This significant increase had disappeared by 1 week in the TRPV1−/− mice but was still apparent in the WT mice (Figure 4), suggesting that the absence of TRPV1 led to a quicker resolution of the response. By 3 weeks, the knee swelling had reduced to control levels in WT as well as TRPV1−/− mice. The difference between CFA-treated joints of WT mice and CFA-treated joints of TRPV1−/− mice was not significant at any time point.

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Figure 4. Time course of the effect of TRPV1 on knee swelling after intraarticular injection of Freund's complete adjuvant (CFA) (ipsilateral joint, 10 μg in 10 μl) or saline vehicle (10 μl) in WT and TRPV1−/− mice. Values are the mean ± SEM (n = 11–15 per group). ∗∗∗ = P < 0.001 versus saline-treated WT mice; ∗∗ = P < 0.01 versus saline-treated WT mice; ## = P < 0.01 versus saline-treated TRPV1−/− mice. See Figure 1 for other definitions.

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Thermal hyperalgesic thresholds, measured using the Hargreaves technique, were stable in WT and TRPV1−/− mice, with an expected higher threshold in the TRPV1−/− mice (see Figure 1 and refs. 6 and7). Twenty-four hours after induction of knee joint inflammation by IA administration of CFA, a significant decrease in the thermal hyperalgesic threshold was observed in the paws on the limbs of both the saline-treated contralateral and the CFA-treated ipsilateral knee joints in the WT mice, but not the TRPV1−/− mice (Figure 5A). By 1 week, the hyperalgesic threshold in the contralateral hind paws of the WT mice had returned to basal levels, whereas there was still a significant decrease in the ipsilateral paws (Figure 5B). To ensure that this effect at 24 hours on the contralateral paw was not due to the trauma of IA injection, tests were performed on mice in which 10 μl saline had been injected IA into one knee joint and the other joint left untreated. At 24 hours, neither hind paw exhibited a change in thermal hyperalgesic threshold (data not shown), thus suggesting that the observed change seen in the contralateral paw at 24 hours may have been due to bilateral hyperalgesia (23–25). Throughout the time course, the TRPV1−/− mice exhibited no change in thermal hyperalgesic thresholds from baseline levels, whereas it was not until 2 weeks that the threshold returned to normal in the paws of the CFA-treated WT mice (Figure 5).

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Figure 5. Thermal nociceptive thresholds over time in WT and TRPV1−/− mice after intraarticular injection of Freund's complete adjuvant (CFA) (ipsilateral joint, 10 μg in 10 μl) or saline vehicle (10 μl). The Hargreaves test was used to determine the time (seconds) to paw withdrawal before and after treatment. Values are the mean ± SEM (n = 7–15 per group). ∗ = P < 0.05 versus pretreatment value in the same joint. See Figure 1 for other definitions.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In this study we demonstrated a pivotal role of TRPV1 in mediating vascular changes that occur in both acute and chronic joint inflammation. Results from studies of acute effects (at 18 hours) showed that knee swelling is reduced in TRPV1−/− mice compared with WT mice. Furthermore, this decreased swelling is directly related to attenuated plasma extravasation, suggesting that TRPV1 participates in influencing hyperpermeability for at least 18 hours after induction of joint inflammation. Despite this, TNFα levels and polymorphonuclear leukocyte levels were found to be similar in WT and TRPV1−/− mice at this time point, providing evidence that TRPV1 acts as a modulatory, rather than a primary, mediator. Studies with TNFα administered IA over 4 hours, by which time TNFα-induced synovial leukocyte accumulation is high, revealed a link between knee swelling and the presence of TRPV1, since TNFα-induced knee swelling was significantly greater in WT compared with TRPV1−/− mice.

Importantly, when the chronic effects of CFA-induced joint inflammation were observed over a 3-week period, knee swelling in TRPV1−/− mice showed an altered profile compared with that in WT mice. The responses in WT mice remained significantly increased for 1 week, while those in the TRPV1−/− mice were lower throughout, with a nonsignificant increase observed at 1 week. These results implicate TRPV1 in maintaining the inflammatory response. Furthermore, measurement of thermal nociceptive thresholds revealed a stable level in CFA- and vehicle-treated limbs except for a significantly decreased nociceptive threshold at 24 hours and at 1 week in WT, but not TRPV1−/−, mice. Taken together these results indicate an essential role of TRPV1 in the ongoing as well as the acute pathophysiologic events in joint inflammation.

An important aspect of sensory nerve biology is that, soon after the onset of inflammation, the different classes of primary sensory neurons and their dorsal root ganglion (DRG) cell bodies exhibit phenotypic changes. Findings in some rodent studies have suggested that these include up-regulation of TRPV1. For example, an increase in TRPV1 protein (but not messenger RNA) in DRG neurons at 2 days after induction of CFA inflammation, which resulted in an increase in TRPV1 levels in peripheral nociceptor terminals, has been reported (26). The notion of TRPV1 up-regulation during inflammation is further supported by findings in studies of CFA-induced inflammation (27, 28). In comparison, a negligible increase in the number of TRPV1-positive L1–L5 ganglia receptors was found in a model of CFA-induced monarthritis in rat knees examined at 3 days (acute response) and 21 days (chronic response). The variation in these results highlights the importance of analyzing functional, in addition to histochemical and molecular, components. Altered activation of the receptor leading to a functional response does not appear to be necessarily associated with up-regulation of TRPV1.

In our study, bilateral hyperalgesia was seen at 24 hours postinjection in the WT mice, but this had disappeared by 1 week. There is evidence that dorsal root reflexes occur bilaterally in fine articular afferents in CFA-induced arthritis in the rat (24). Dorsal root reflexes are action potentials that originate in afferent fibers at the spinal cord entry point and propagate to the periphery (24). In addition, bilateral up-regulation of the expression of neurokinin 1 and bradykinin 2 receptors during acute inflammation in the DRG neurons of rats with antigen-induced arthritis has been demonstrated (29). Both the central nervous system and local inflammatory processes have been shown to be involved in bilateral inflammation (30). As discussed above, phenotypic changes occur in sensory neurons during inflammation. It may be that activity from the central nervous system influences phenotypic changes in the DRG neurons in the periphery, thus leading to bilateral hyperalgesia. Our results observed in WT but not TRPV1−/− mice suggest an essential involvement of TRPV1 in this process. However, TRPV1 protein was not seen to be increased in the contralateral paw at 48 hours after CFA injection in the rat (28).

The finding of similar levels of TNFα in synovial fluid extracts from WT and TRPV1−/− mice led us to examine the possibility that TNFα may influence TRPV1 activity due to an action of TNFα upstream from TRPV1. Expression of TNF receptor subtypes occurs on cultured neonatal DRG cells (31, 32), and TNFα can influence neuropathic pain via a p38 MAPK–dependent mechanism (33). Furthermore, TNFα and TNF receptor 1 are suggested to play a prominent role in priming sensory nerve endings in mouse peripheral airways, leading to potentiation of a sensory nerve–mediated tracheal hyperpermeability response (34). The finding that TNFα-induced knee swelling is greater in WT mice compared with TRPV1−/− mice is consistent with the concept that TNFα can prime TRPV1 sensory nerve–mediated swelling in the knee joint, via as-yet-uncharacterized mechanisms.

The most common hypothesis is that the inflammatory events following TRPV1 activation are mediated by release of neuropeptides, principally substance P (acting via its vasoactive neurokinin 1 receptor) and CGRP, which are up-regulated in clinical and experimental joint inflammation (29, 35–38). Substance P stimulates acute plasma protein extravasation when injected into joints (39, 40), although its possible role in more severe and chronic phases of disease is unclear (40, 41). However, we have recently shown that there was no difference in responses in the CFA-induced model of joint inflammation, used in the present study, in WT versus neurokinin 1–knockout mice after 18 hours (20). Thus, one cannot assume a pivotal involvement of tachykinins or the major vasoactive tachykinin neurokinin 1 receptor in this model of arthritis. Results of clinical studies with neurokinin 1 antagonists do not suggest that they have a beneficial effect, at least in osteoarthritis (42), although there has been some debate regarding the potency of neurokinin 1 antagonists used in clinical trials to date (43).

The role of CGRP is less well understood. It is a potent vasodilator that can potentiate joint plasma extravasation (44), and it has been suggested to influence the inflammatory process in the joint (45–48) and contribute to hyperalgesia (49). A CGRP antagonist has recently been shown to have a favorable effect in the treatment of pain associated with migraine (50), and it is possible that CGRP is responsible for mediating some of the proinflammatory effects observed after TRPV1 activation. However, sensory nerve–mediated events are more complex in that activated sensory nerves also release antiinflammatory neuropeptides, in particular, somatostatin (51), in and around the joint. It should be noted that if the sum effect of TRPV1 activation is antiinflammatory, one would not expect the deletion of TRPV1 to act in the beneficial manner observed in the present study.

In summary, we propose that TRPV1 is involved in mediating joint swelling at both the acute and chronic stages and is essential in the development of a bilateral decreased threshold of thermal nociception at 24 hours, which persists for 1 week on the arthritic ipsilateral side. We conclude that TRPV1 does not influence the acute generation of the prominent inflammatory cytokine TNFα. However, this cytokine may be involved in stimulating or potentiating TRPV1-mediated responses. These results are consistent with the possibility that TRPV1 antagonists may have therapeutic potential for both acute and chronic inflammatory joint disease. Indeed, it has been recently shown that a novel TRPV1 antagonist, AMG 9810, reverses CFA-induced thermal and mechanical hyperalgesia in the rat paw (52), illustrating the potential of TRPV1 antagonists in the treatment of inflammatory disease.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES