β2- and β3-adrenergic receptors drive COMT-dependent pain... : PAIN (original) (raw)
1. Introduction
A growing literature demonstrates that catecholamines and pathways regulating their bioavailability influence pain. Patients with chronic pain conditions, including fibromyalgia and temporomandibular disorders (TMD), exhibit increased levels of the catecholamines epinephrine and norepinephrine [19,45,69,78] and decreased levels of the enzyme catechol-O-methyltransferase (COMT) [16,26,80], which metabolizes epinephrine and norepinephrine [49]. Consistent with these findings, animal studies show that epinephrine administration [11,36,37] or COMT inhibition [34,52] increases mechanical and thermal hyperalgesia. Pharmacologic studies reveal that COMT-dependent pain, defined as increased pain after COMT inhibition, is mediated via β2- and β3-adrenergic receptors (β2- and β3ARs). Antagonism of both β2- and β3ARs are required to completely block acute COMT-dependent pain, because antagonism of either β2- or β3ARs alone only produces a partial blockade [52].
β2ARs and β3ARs are G-protein–coupled receptors expressed in peripheral, spinal, and supraspinal sites involved in pain transmission. Stimulation of β2- or β3ARs on peripheral afferents sensitizes nociceptors [2,36] and produces allodynia [35] through activating intracellular kinases. Additionally, stimulation of β2- or β3ARs indirectly enhance pain transmission through the release of proinflammatory molecules including nitric oxide (NO) and cytokines [1,7,21–23,28,48,74,76].
Nitric oxide is a gaseous molecule whose production by NO synthases can be induced by stimulation of β2ARs on endothelial cells, smooth muscle, sympathetic afferent neurons, and macrophages [1,21,28] or stimulation of β3ARs on adipocytes and fibroblasts [7,23]. After release, NO lowers nociceptor firing thresholds [3,5] to enhance experimental inflammatory and neuropathic pain [29,40,58]. Furthermore, NO can stimulate release of additional molecules involved in nociception, including proinflammatory cytokines [9,29].
Proinflammatory cytokines linked to pain include tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), interleukin 6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2, MCP-1). β2- and β3AR stimulation promotes the production and release of TNFα, IL-1β, IL-6, and CCL2 [22,48,62,74,76], which act to lower nociceptor firing thresholds and enhance pain [4,14,33,56,57,72].
Of note, NO and cytokines influence one another's release. NO drives the production and release of cytokines including TNFα and IL-1β [9,13,32,82], whereas cytokines upregulate NO synthase expression and promote NO release [25,41,73,77]. This positive feedback loop may contribute to the development and/or maintenance of pain [13]. Although NO and cytokines are released after β2- and β3AR stimulation and linked with pain, their role in COMT-dependent pain has not been established.
To investigate the role of NO and cytokines in COMT-dependent pain mediated by β2- and β3ARs, we measured plasma NO and cytokines after administration of a COMT inhibitor in the presence or absence of β2- and β3AR antagonists. Additionally, we measured mechanical and thermal pain sensitivity after COMT inhibition in the presence or absence of an NO synthase inhibitor or TNFα, IL-1β, IL-6, or CCL2 neutralizing antibodies. Results demonstrate that (1) COMT-dependent pain is accompanied by increases in peripheral NO derivatives and cytokines mediated by β2- and β3ARs, (2) inhibition of NO synthesis and neutralization of the innate immunity cytokines TNFα, IL-1β, IL-6 block COMT-dependent pain, and (3) NO and cytokines potentiate one another's biosynthesis: NO promotes TNFα, IL-1β, IL-6, and CCL2 release, whereas TNFα and IL-6 promote NO release.
2. Materials and methods
2.1. Subjects
Adult male Sprague-Dawley rats (Charles River Laboratories, Raleigh, NC) were used in all experiments. Rats weighed between 215 and 265g for β2- and β3AR antagonism and NO synthase inhibition experiments and between 315 and 360g for cytokine neutralization experiments.
2.2. Drugs and chemicals
As described in Nackley et al., 2007 [52], OR486 was dissolved in DMSO and diluted in 0.9% saline (3:2). ICI118, 551, SR59230A, and L-NAME were dissolved in DMSO and 0.9% saline (1:4). Functional-grade antibodies against tumor necrosis factor α (α-TNFα), interleukin-1β (α-IL-1β), interleukin-6 (α-IL-6), chemokine (C-C motif) ligand 2 (α-CCL2), or immunoglobulin (Ig) G control were dissolved in 0.9% saline. OR486, ICI118,551, and SR59230A were purchased from Tocris (Ellisville, MO). L-NAME was purchased from Sigma-Aldrich (St. Louis, MO). Neutralizing antibodies against TNFα, IL-1β, CCL2 and Armenian hamster IgG controls were purchased from eBiosciences (San Diego, CA), and the antibody against IL-6 (polyclonal goat IgG) was purchased from R&D Systems (Minneapolis, MN).
2.3. General experimental conditions
Animals were handled and habituated for 4 days before testing day. On testing day, animals were habituated to the environment for 10 to 15minutes, and then stable baseline responses to mechanical or thermal stimuli were established in separate groups of rats. After baseline testing, animals were randomly assigned to drug treatment group (Fig. 1) and behavior was reassessed. Responses to mechanical stimuli were reassessed at 30, 75, and 120minutes after OR486 and responses to thermal heat were reassessed at 120minutes after OR486. The experimenter was blinded to drug treatment group.
Timeline of administered treatments used in this study. The COMT inhibitor OR486 or vehicle was administered in the presence or absence of the β2- and β3-adrenergic receptor antagonists ICI118,551 and SR59320A, the NOS inhibitor L-NAME, or neutralizing antibodies against TNFα, IL-1β, IL-6, or CCL2. COMT, catechol-O-methyltransferase; L-NAME, L-NG-nitroarginine methyl ester; TNF, tumor necrosis factor; NOS, nitric oxide synthase; IL, interleukin; CCL2, chemokine (C-C motif) ligand 2; AR, adrenergic receptor.
We first sought to determine whether COMT-dependent pain is accompanied by increases in NO and cytokines and whether this was mediated by β2- and β3ARs. Separate groups of animals received intraperitoneal (i.p.) ICI118,551 (0.5mg/kg) together with SR59230A (5.0mg/kg) or vehicle 30minutes before i.p. OR486 (30mg/kg) or vehicle.
We then sought to elucidate the role of NO and cytokines in driving COMT-dependent pain. To determine whether NO production was required for the development of COMT-dependent pain, separate groups of animals received i.p. L-NAME (30mg/kg) or vehicle 30minutes before i.p. OR486 (30mg/kg) or vehicle. L-NAME dosage was based on that used in Kuboyama et al., 2011 [40]. To determine whether cytokine action was required for the development of COMT-dependent pain, separate groups of animals received intravenous (i.v.) α-TNFα (75μg), α-IL-1β (75μg), α-IL-6 (75μg), α-CCL2 (75μg) or IgG control (75μg) dissolved in 250μL 0.9% saline 2hours before i.p. OR486 (30mg/kg) or vehicle. Dosages of neutralizing antibody were determined by 2 sources: previous reports using neutralizing antibodies [8,46] and the effective neutralizing dose that would neutralize cytokines at the average dosages we observed at 180minutes after OR486 administration. We chose to administer the antibodies by i.v. injection to optimize the circulation of the antibody in a relatively short amount of time.
Finally, we sought to establish whether NO and cytokines influenced one another's biosynthesis. To determine whether NO synthesis was required for cytokine release, plasma collected from animals in the L-NAME experiments was measured for levels of TNFα, IL-1β, IL-6, and CCL2. To determine whether cytokine action was required for NO release, plasma from animals receiving neutralizing antibodies against TNFα, IL-1β, IL-6, and CCL2 was measured for levels of total nitrite (nitrite and nitrate).
2.4. Assessment of mechanical allodynia and mechanical hyperalgesia
Paw withdrawal threshold was measured using the von Frey up-down method, as described in Nackley et al., 2007, and later. Nine calibrated von Frey monofilaments (bending forces of 0.40, 0.68, 1.1, 2.1, 3.4, 5.7, 8.4, 13.2, and 25.0 g; Stoelting, Wood Dale, IL) with equal logarithmic spacing between filaments were applied to the plantar surface of the hind paw. A series of 6 applications of monofilaments with varying gram forces was applied for 3seconds to the plantar surface of the hindpaw. Testing began with the middle filament in the series (3.4 g). If the response included the withdrawal of the hindpaw, an incrementally lower filament was applied. In the absence of a paw withdrawal, an incrementally higher filament was applied. These data were entered into Paw Flick module within the National Instruments LabVIEW 2.0 (Austin, TX) software. A logarithmic algorithm accounted for the order and number of withdrawal responses as well as the gram force of the final filament to calculate mechanical threshold, the gram force that would elicit paw withdrawal in 50% of trials (10[Xf+kδ]/10,000, where Xf=value [in log units] of the final von Frey hair used; k=tabular value of positive and negative responses, and δ=mean difference [in log units] between stimuli). Mechanical allodynia was defined as a heightened response to a normally innocuous stimulus and was determined as a significant decrease in paw withdrawal threshold from baseline.
After determining paw withdrawal threshold, paw withdrawal frequency to a noxious von Frey monofilament was assessed. The highest gram force filament (25.0 g) was applied to the hind paw 10 times. Stimulus was applied for 1second followed by a 1-second interval without a stimulus. The number of paw withdrawals was recorded for each hindpaw. Mechanical hyperalgesia was defined as an increase in the number of paw withdrawals to a noxious mechanical stimulus from baseline.
2.5. Assessment of thermal hyperalgesia
Thermal hyperalgesia was measured using the radiant method by applying radiant heat to the hind paw as described in Hargreaves et al., 1988 [27]. Animals were placed in individual Plexiglas chambers and habituated for approximately 10minutes. After habituation, a radiant beam of light was applied to the plantar surface of the rat hind paw through a glass floor heated to 30°C. Latencies of paw withdrawal from the heat stimulus were recorded in duplicate. If the second paw withdrawal latency was not within ±4seconds of the first withdrawal latency, then a third measure was recorded. The 2 latencies closest in value were averaged and included in the analysis. Thermal hyperalgesia was defined as a decrease in paw withdrawal latency to a noxious thermal stimulus compared to baseline.
2.6. Tissue collection
After behavioral testing, animals were euthanized by injection of 0.5mL Fatal-Plus (Vortech Pharmaceuticals, Dearborn, MI). Arterial blood was collected and placed in EDTA plasma tubes, then centrifuged for 15minutes at 15,000 g. After collection, plasma was stored at −80°C.
2.7. Measurement of NO derivatives
To measure nitrite, NO in blood plasma was assessed using the Griess Reaction (Promega, Madison, WI). To measure total nitrite (nitrite and nitrate), NO in blood plasma was assessed by kit from R&D Systems (Minneapolis, MN).
2.8. Measurement of cytokines
To determine whether COMT inhibition raised TNFα plasma levels downstream of β2- and β3AR stimulation, plasma TNFα was measured by the UNC Proteomics/Immunotechnologies Core using ELISA kits from Biosource (Camarillo, CA). To determine whether COMT inhibition raised TNFα plasma levels downstream of NO production, plasma TNFα was measured by chemiluminescent ELISA (Life Technologies Carlsbad, CA) due to discontinuation of aforementioned Biosource kit. IL-1β was measured by the UNC Cytokine Analysis Facility using the Luminex Rat Cytokine Multiplex Array from R&D Systems (Minneapolis, MN). IL-6 and CCL2 were measured by ELISA (eBioscience, San Diego, CA; R&D Systems, Minneapolis, MN, respectively). Selected ELISAs and multiplex were based on minimum assay range and analyte sensitivity. All plasma samples were diluted at 2×.
2.9. Statistical analysis
All behavioral data were analyzed using a t test to verify that there were no significant differences in baseline values. Baseline mechanical allodynia values did differ in 2 groups and were normalized using the following formula: D=(Average baseline for all groups) – (average baseline for specific group). Value, D, was then added to each animal's threshold value at all time points. Mechanical allodynia and hyperalgesia data were analyzed by 2-way analysis of variance (ANOVA). Thermal hyperalgesia and molecular data were analyzed using a 1-way ANOVA. Post-hoc comparisons were performed using the Bonferroni test and were corrected for multiple testing. P<.05 was considered to be statistically significant.
3. Results
3.1. COMT inhibition results in increased pain sensitivity and production of proinflammatory mediators via β2- and β3ARs
To recapitulate our laboratory's previous results showing that acute COMT-dependent pain is mediated by both β2- and β3ARs, we measured pain behavior in animals receiving the β2AR antagonist ICI118,551 together with the β3AR antagonist SR59320A before the COMT inhibitor OR486. As expected, animals receiving OR486 showed mechanical allodynia (F3,137=9.223, P<.0001; Fig. 2A), mechanical hyperalgesia (F3,139=11.45, P<.0001; Fig. 2B), and thermal hyperalgesia (F3, 54=5.336, P<.003; Fig. 2C) compared with those receiving vehicle. COMT-dependent increases in pain sensitivity were observed 30 to 120minutes after drug administration and were completely blocked by coadministration of β2- and β3AR antagonists.
COMT inhibition increases pain, NO derivatives, and cytokines via β2- and β3ARs. Animals receiving OR486 (30 mg/kg) exhibit (A) mechanical allodynia, (B) mechanical hyperalgesia, and (C) thermal hyperalgesia, as well as increased circulating levels of (D) nitrite, (E) TNFα, (F) IL-1β, (G) IL-6, and (H) CCL2. COMT-dependent increases in pain, nitrite, and cytokines were completely blocked by coadministration of ICI118,551 (0.5 mg/kg) and SR59320A (5.0 mg/kg). N = 6 to 10 per group. Data are mean ± SEM. * P < .05, ** P < .01, *** P < .001 different from Veh/Veh, # P < .05 different ICI+SR/Veh and ICI+SR/OR486. COMT, catechol-O-methyltransferase; TNF, tumor necrosis factor; NO, nitric oxide; IL, interleukin; CCL2, chemokine (C-C motif) ligand 2; AR, adrenergic receptor.
After the conclusion of behavioral experiments, blood plasma was collected to measure circulating levels of NO derivatives, TNFα, IL-1β, IL-6, and CCL2. Animals receiving OR486 showed increased levels of nitrite (F3, 23=3.929, P<.03; Fig. 2D), TNFα (F2,18=5.663, P<.02; Fig. 2E), IL-1β (F3,27=3.428, P<.04; Fig. 2F), IL-6 (F3,19=1.354, _P_=.2; Fig. 2G), and CCL2 (F3,27=3.569, P<.03; Fig. 2H). COMT-dependent increases in nitrite and cytokines were completely blocked by coadministration of ICI118,551 and SR59320A.
3.2. NO synthase inhibition and cytokine neutralization prevent COMT-dependent pain
As NO and cytokines are released after stimulation of β2- and β3ARs and have been implicated in the development of pain in other models, we sought to determine their role in the development of acute COMT-dependent pain. To first evaluate the contribution of NO synthesis, we measured pain behavior in separate groups of animals that received the NO synthase inhibitor L-NAME or vehicle 30minutes before OR486. Administration of L-NAME before OR486 blocked the development of mechanical allodynia (F3,138=5.195, P<.003; Fig. 3A), mechanical hyperalgesia (F3,138=5.195, P<.003; Fig. 3B), and thermal hyperalgesia (F3,54=6.337, P<.001; Fig. 3C). Therefore, NO production by NO synthases is required for the development of COMT-dependent increases in mechanical and thermal pain.
Inhibition of NO synthesis prevents COMT-dependent pain. Administration of the universal nitric oxide synthase inhibitor L-NAME (30 mg/kg) before OR486 (30 mg/kg) normalized (A) mechanical allodynia, (B) mechanical hyperalgesia, and (C) thermal hyperalgesia. N = 8 to 10 per group. Data are mean ± SEM. * P < .05, ** P < .01, *** P < .001 different from Veh/Veh. COMT, catechol-O-methyltransferase; L-NAME, L-NG-nitroarginine methyl ester; NO, nitric oxide.
To next evaluate the individual contributions of TNFα, IL-1β, IL-6, and CCL2 to acute COMT-dependent pain, we measured pain behavior in separate groups of animals receiving neutralizing antibodies against TNFα, IL-1β, IL-6, and CCL2 or control IgG before OR486. Results show that neutralization of the innate immunity cytokines (TNFα, IL-1β, and IL-6), but not CCL2, prevented OR486-dependent increases in mechanical and thermal pain. Administration of α-TNFα (F3,84=10.71, P<.0001; Fig. 4A), α-IL-1β (F3,83=19.34, P<.0001; Fig. 4D), and α-IL-6 (F3,87=10.96, P<.0001; Fig. 4G) blocked mechanical allodynia. Additionally, pretreatment with α-TNFα (F3,89=30.95, P<.0001; Fig. 3B), α-IL-1β (F3,89=29.72, P<.0001; Fig. 4E), and α-IL-6 (F3,93=23.33, P<.0001; Fig. 4H) blocked mechanical hyperalgesia. Finally, α-TNFα (F3,47=5.312, P<.004; Fig. 4C), α-IL-1β (α-IL-1β: F3,49=5.639, P<.002; Fig. 4F), and α-IL-6 (F3,48=3.339, P<.003; Fig. 4I) blocked thermal hyperalgesia at 120minutes. However, α-CCL2 was not effective at blocking mechanical allodynia (Fig. 4J), mechanical hyperalgesia (Fig. 4K) or thermal hyperalgesia (Fig. 4L). Therefore, the innate immunity cytokines TNFα, IL-1β, and IL-6 are required for the development of COMT-dependent pain.
Neutralization of TNFα, IL-1β, and IL-6, but not CCL2, blocks COMT-dependent pain. Administration of α-TNFα (75 μg), α-IL-1β (75 μg), or α-IL-6 (75 μg) before OR486 (30 mg/kg) normalized (A, D, G) mechanical allodynia, (B, E, H) mechanical hyperalgesia, and (C, F, I) thermal hyperalgesia. (J–L) Administration of α-CCL2 failed to block OR486-induced increases in mechanical and thermal pain. N = 6 to 8 per group. Data are mean ± SEM. * P < .05, ** P < .01, *** P < .001 different from control IgG/Veh. # P < .05 different from α-TNFα/Veh and α-TNFα/OR486. COMT, catechol-O-methyltransferase; TNF, tumor necrosis factor; IL, interleukin; CCL2, chemokine (C-C motif) ligand 2.
3.3. Interplay between NO and cytokine protein expression in COMT-dependent pain
We then sought to determine whether these proinflammatory molecules could influence the synthesis and release of one another downstream of β2- and β3AR stimulation. Blood plasma was collected from animals that received L-NAME or cytokine-neutralizing antibodies before OR486 and peripheral levels of NO derivatives and cytokines were measured. In NO inhibition experiments, levels of TNFα, IL-1β, IL-6, and CCL2 were elevated in animals receiving vehicle before OR486. Preadministration of L-NAME blocked OR486-mediated increases in TNFα (F3,39=0.2989, P<.83; Fig. 5A), IL-1β (F3,27=3.255, P<.04; Fig. 5B), IL-6 (F3,18=1.354, P<.3; Fig. 5C), and CCL2 (F3,27=2.761, _P_=.06; Fig. 5D).
Inhibition of NOS prevents COMT-dependent increases in cytokines. Administration of the nitric oxide synthase inhibitor L-NAME (30 mg/kg) before OR486 (30 mg/kg) blocked increases in circulating levels of (A) TNFα, (B) IL-1β, (C) IL-6, and (D) CCL2. N = 6 to 10 per group. * P < .05 different from Veh/Veh. COMT, catechol-O-methyltransferase; L-NAME, L-NG-nitroarginine methyl ester; TNF, tumor necrosis factor; NOS, nitric oxide synthase; IL, interleukin; CCL2, chemokine (C-C motif) ligand 2.
In cytokine neutralization experiments, total nitrite (nitrite+nitrate) concentrations in blood plasma were elevated in animals receiving control IgG before OR486. Preadministration of α-TNFα (F3,21=3.230, P<.05; Fig. 6A) or α-IL-6 (F3,22=3.772, P<.03; Fig. 6C) before OR486 blocked elevations in total nitrite. However, preadministration of α-Il-1β (Fig. 6B) or α-CCL2 (Fig. 6D) failed to block OR486-mediated increases in total nitrite levels. Thus, NO and cytokines drive one another's biosynthesis.
Neutralization of TNFα and IL-6 prevents COMT- dependent increases in NO. OR486-induced increases in total nitrite (nitrite and nitrate) were blocked by pretreatment with (A) α-TNFα (75 μg) or (C) α-IL-6 (75 μg), but not (B) α-IL-1β (75 μg) or (D) α-CCL2 (75 μg). N = 6 to 8 per group. Data are mean ± SEM. % P < .05 different from α-TNFα/Veh, # P < .05 different from α-IL-6/Veh and α-IL-6/OR486. COMT, catechol-O-methyltransferase; TNF, tumor necrosis factor; NO, nitric oxide; IL, interleukin; CCL2, chemokine (C-C motif) ligand 2; Ig, immunoglobulin.
4. Discussion
Our laboratory previously demonstrated that COMT inhibition produces remarkable increases in mechanical and thermal pain sensitivity through stimulation of both β2- and β3ARs [52]. However, the molecular mechanisms whereby these receptors drive COMT-dependent pain have remained unknown. Here, we identify NO, TNFα, IL-1β, and IL-6 as molecules downstream of β2- and β3AR stimulation that are critical for the development of pain associated with decreased COMT activity. Furthermore, we demonstrate that NO and cytokines act in a positive feedback loop to induce one another's biosynthesis.
4.1. Role of nitric oxide in COMT-dependent pain
NO is a paracrine signaling molecule produced by 3 different nitric oxide synthase isoforms: neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3). Although previous studies have linked NO to inflammatory and neuropathic pain, here we provide the first demonstration that NO contributes to COMT-dependent pain. Specifically, we found that stimulation of β2- and β3ARs after COMT inhibition resulted in increased levels of NO derivatives and that inhibition of NO synthesis with L-NAME prevented the development of COMT-dependent mechanical allodynia, mechanical hyperalgesia, and thermal hyperalgesia. These findings are in line with results from clinical and animal studies showing that NO is upregulated after injury and inflammation [9,13,29,40,51,58,65] and that genetic or pharmacologic blockade of NO can suppress pain in these models [9,29,40,50,58,61].
NO is able to produce pain through several mechanisms, including the canonical stimulation of cyclic guanosine monophosphate (cGMP), which can enhance activity of Ca2+-activated K+ channels, and thus the firing rate of nociceptors. NO also can stimulate cyclic adenosine monophosphate (cAMP)–mediated production of pro-pain prostaglandins (PGE2) that sensitize primary afferents [3,5]. Furthermore, NO can stimulate cAMP production through S-nitrosylation of adenylate cyclase and the phosphorylation of cAMP response element binding (CREB) protein by cGMP. Activation of CREB leads to enhanced expression of cytokines such as IL-1β and TNFα [9,32,82]. Although others have linked NO production with β2- and β3AR stimulation in the context of inflammation [1,21,28,74], this is the first demonstration that NO synthesis is critical for COMT-dependent pain and cytokine production.
4.2. Role of proinflammatory cytokines in COMT-dependent pain
TNFα, IL-1β, and IL-6 are innate immunity cytokines, considered to be the first responders to injury or proinflammatory events. In an acute setting, these cytokines convey a protective advantage by promoting wound healing [17]. However, sustained elevations of these cytokines can promote tissue damage and pain. Here, we found that COMT inhibition led to the release of TNFα, IL-1β, IL-6, and CCL2 mediated by β2- and β3ARs. We also found that neutralization of the innate immunity cytokines TNFα, IL-1β, and IL-6, but not CCL2, prevented COMT-dependent mechanical and thermal sensitivity.
Stimulation of β2- and β3ARs located on cells in the periphery and central nervous system can enhance production of TNFα, IL-1β, IL-6, and CCL2 [30,31,44,53,74,76,81,83], which can then enhance pain sensitivity. Elevations in these cytokines have been found in local synovial joint fluid from patients with TMD [39] and in blood from patients with fibromyalgia and migraine [64,67,79]. Neutralization of TNFα, IL-1β, and IL-6 reduces the development of allodynia and hyperalgesia in models of neuropathic pain [4,46,56,68], suggesting that these cytokines are critical for pain.
Cytokines downstream of β2- and β3AR stimulation likely drive COMT-dependent pain through direct and indirect mechanisms. Previous studies have demonstrated that TNFα, IL-1β, and IL-6 can bind to their respective receptors on nerve terminals to directly sensitize peripheral nociceptors [4,14,56,57]. TNFα also can drive sensitization of nociceptors through receptor-independent increases in the production of other proinflammatory cytokines. Cunha et al. found that α-TNFα blocked CFA-induced increases in pain and IL-1β production [12]. They speculated that TNFα acts as the first cytokine in the cascade to stimulate the sequential release of IL-6, IL-1β, and PGE2.
In contrast to the innate immunity cytokines, administration of α-CCL2 did not prevent the development of COMT-dependent pain. This may be due to 1 of 2 possibilities: that CCL2 is critical for the maintenance vs the development of pain or that higher dosages of α-CCL2 may reduce COMT-dependent pain and NO release. Previous studies have shown that CCL2 recruitment of monocytes and neutrophils to the site of injury occurs at later time points after 2hours [59]. Furthermore, CCL2 is released from spinal dorsal horn astrocytes, which are glial cells involved in the maintenance of pain states [24].
4.3. Interplay between NO and cytokines in COMT-dependent pain
Mounting evidence suggests that a positive feedback loop exists between NO and cytokines, such that they can induce one another's biosynthesis. Here, we found that inhibition of NO synthesis effectively blocked COMT-dependent increases in TNFα, IL-1β, IL-6, and CCL2, whereas neutralization of TNFα and IL-6 blocked COMT-dependent increases in the production of NO derivatives. Disruption of NO, TNFα, or IL-6 signaling reduces the proinflammatory feedback mechanism important for COMT-dependent pain. This synergistic relationship between NO and cytokines has been observed as a key characteristic of inflammation. NO has long been known to act as a putative molecule dictating macrophage trafficking [5] and cytokine production and release [9,29,40]. Furthermore, NO can influence the transcription of cytokines such as TNFα [32] and IL-1β [82]. Cytokines can also influence NO synthesis, as TNFα, IL-1β, and IL-6 have been found to increase NOS transcription by directly binding to the promoter or by stimulating p38-MAPK [41,47,73]. The collective work from our laboratory and others demonstrates that NO and cytokines influence one another's biosynthesis and suggest that it is the net effect of these molecules that ultimately influences pain.
4.4. Potential site of action
β2- and β3ARs are expressed on cells in peripheral, spinal, and central sites where they could potentially mediate pain sensitivity. In the periphery, β2ARs are located on mononuclear leukocytes [42], adipocytes [38], and vascular, uterine, and airway smooth muscle cells [18], whereas β3ARs are expressed in brown and white adipose tissue [71]. In the central nervous system, β2ARs are located on thalamic, cerebellar [54,60], and spinal dorsal horn neurons [55] as well as glial cells [63,70], whereas β3ARs are located on dorsal root ganglia [35]. In the present study, we found that COMT-dependent β2- and β3AR stimulation resulted in the release of proinflammatory molecules circulating in the periphery. Another recent study by our group shows that adrenalectomized rats, lacking peripheral epinephrine, fail to develop increased mechanical and thermal pain sensitivity after sustained COMT inhibition, thus providing further evidence for a peripheral contribution of adrenergic systems to COMT-dependent pain. [10]. Additional work is required to determine the relative contributions of peripheral, spinal, and supraspinal β2- and β3ARs to COMT-dependent pain.
4.5. Greater implications and clinical relevance
As observed here, decreased COMT activity enhances pain by increasing the production of NO and cytokines via β2- and β3ARs. Genetic variants resulting in decreased COMT activity have been associated with chronic pain conditions such as fibromyalgia [24] and TMD [16], which are linked to increased levels of catecholamines [19,78] and production of proinflammatory molecules [6,15,43]. Specifically, patients with fibromyalgia [6,43] and TMD [20,39,66,67] show higher levels of NO derivatives (eg, nitrite and nitrate) and cytokines such as TNFα, IL-1β, IL-6, and CCL2. Recent reports suggest that β-adrenergic mechanisms involved in COMT-dependent pain may overlap with those observed in complex regional pain syndrome [44], which is also linked to stimulation of βARs and increased production of proinflammatory cytokines. Thus, βAR antagonist therapy used to mitigate catecholamine signaling and alleviate pain in patients with fibromyalgia and TMD [45,69,75,84] may benefit other patient populations suffering from pain conditions of shared etiology. Future studies will use a more clinically relevant model of sustained COMT inhibition to evaluate the efficacy of βAR antagonists in reversing COMT-dependent pain after its induction.
4.6. Conclusions
In conclusion, these findings elucidate the molecules downstream of β2- and β3ARs that drive acute COMT-dependent pain. Elevated levels of norepinephrine/epinephrine, resulting from decreased COMT activity, stimulate β2- and β3ARs to promote the release of NO and the innate immunity cytokines TNFα, IL-1β, and IL-6, which in turn produce heightened pain sensitivity. The chemokine CCL2 was elevated in COMT-deficient animals, but its blockade did not prevent the development of acute COMT-dependent pain. Additionally, we found that NO and innate immunity cytokines function in a positive feedback loop to strengthen their own biosynthesis. This amplification mechanism may form the basis for the development of prolonged hypersensitive pain states. Finally, these data suggest that patients suffering from pain conditions associated with abnormalities in catecholamine signaling may benefit from therapeutic agents that selectively regulate the activity of β2- and β3ARs and downstream effectors.
Conflict of interest statement
The authors declare no conflicts of interest.
Acknowledgements
The authors thank the UNC Cytokine Core for their assistance with multiplex cytokine experiments and members of the Nackley laboratory and the Center for Pain Research and Innovation for their helpful feedback and support. This work was funded by the NIH/NINDS R01 NS072205 to A.G.N.
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Keywords:
Allodynia; Catecholamines; Chemokine (C-C motif) ligand 2 (CCL2); Epinephrine; Hyperalgesia; Inflammation; Interleukin 1 beta (IL-1β); Interleukin 6 (IL-6); Monocyte chemotactic protein 1 (MCP-1); Nitrite; Nitrate; Norepinephrine; Tumor necrosis factor alpha (TNFα)
© 2014 International Association for the Study of Pain