CC chemokine ligand 2 (CCL2) enhances TTX-sensitive sodium channel activity of primary afferent neurons in the complete Freud adjuvant-induced inflammatory pain model

Abstract

CC chemokine ligand 2 (CCL2) has been implicated in pathological pain, but the mechanism underlying the pronociceptive effect of CCL2 is not fully understood. Voltage-gated sodium (Nav) channels are important determinants of the excitability of sensory neurons. Hence we tested the hypothesis that CCL2 contributes to inflammatory pain via modulating Nav channel activity of primary afferent neurons. Chronic inflammatory pain was induced in rats by intraplantar injection of the complete Freud adjuvant (CFA) to one of the hind paws. Control rats received intraplantar injection of equal volume of saline. A significant increase of CCL2 mRNA and CCL2 receptor (CCR2) protein expression was detected in the ipsilateral dorsal root ganglion (DRG) in CFA-treated rats. Intraplantar injection of CCL2 protein in the control rats had minimal effect on the paw withdrawal threshold (PWT) in response to mechanical stimulation. However, in CFA-treated rats, intraplantar CCL2 led to an increase in pain responses. Patch-clamp recording of acutely dissociated DRG neurons revealed that CCL2 had minimum effect on the excitability of sensory neurons from control rats. However, CCL2 directly depolarized a large proportion of small to medium-sized sensory neurons from CFA-treated rats. In addition, CCL2 was found to enhance whole-cell TTX-sensitive sodium currents without significantly affecting the TTX-resistant sodium currents and the potassium currents. These results are in agreement with previous reports concerning the involvement of CCL2–CCR2 signaling in inflammatory hyperalgesia and further indicate that enhanced TTX-sensitive channel activity may partly underlie the pronociceptive effects of CCL2.

Introduction

CC chemokine ligand 2 (CCL2), also referred to as monocyte chemoattractant protein 1 (MCP1), is an important proinflammatory mediator which is able to induce migration and infiltration of monocytes and macrophages to injured tissues [1–3]. Apart from the well-established proinflammatory action, CCL2 and its cognate receptor CCR2 have also been implicated in pathological pain [4–6]. Upregulation of CCL2 and/or CCR2 in dorsal root ganglia (DRG) has been reported in different models of pathological pain [6]. In the hotplate test, CCR2-knockout and wild-type (WT) mice showed equivalent acute pain responses. However, CCR2-knockout mice had significantly reduced inflammatory and neuropathic pain compared with the WT mice [7], indicating that CCL2–CCR2 signaling may be essential in the development of pathological pain.

Increased excitability of peripheral nociceptors plays an important role in pathological pain. Some previous studies have examined the effects of CCL2 on the excitability of primary sensory neurons [5,8]. CCL2 directly depolarizes nociceptive neurons in the intact dorsal root ganglion (DRG) after CCD (chronic compression of DRG, an established model of neuropathic pain) and this is correlated with an upregulation of CCL2 and CCR2 in DRG [8].

Voltage-gated sodium (Nav) channels are major determinants of nociceptor excitability. Depending on the sensitivity to blockade by tetrodotoxin (TTX), sodium currents in sensory neurons can be divided into TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) currents [9,10]. In the peripheral nociceptive neurons, Nav1.7 mediates the TTX-S currents whilst Nav1.8 and Nav1.9 mediate the TTX-R currents [10]. Several elegant human genetic analyses have highlighted the crucial role of peripheral sodium channels in the pain process. For example, gain of function mutations in the SNC9A gene encoding hNav1.7 was shown to cause painful inherited neuropathies, whereas loss of function mutations result in congenital indifference to all forms of pain [11].

Given the crucial role of Nav channels in the control of nociceptor excitability, it is important to understand how CCL2–CCR2 signaling affects these channels. Indeed, several reports have revealed the impact of CCL2 on the sodium currents of DRG neurons. Kao et al. [12] reported that pretreatment of DRG neurons with CCL2 in culture increased the density of TTX-R sodium currents and this was associated with an increased transcription of Nav1.8 but not Nav1.9. Zhao et al. [13] further demonstrated that CCL2-induced upregulation of Nav1.8 current density in DRG neurons involves the PKC-NF-κB pathway, which promotes the phosphorylation of Nav1.8 and its expression. An earlier study demonstrated that acute application of CCL2 concentration-dependently increased TTX-R Nav1.8 current densities in small- and medium-sized sensory neurons [14]. Whereas these data support that CCL2–CCR2 elevates nociceptor excitability via up-regulating TTX-R Nav1.8 expression and function, few studies have directly investigated the impact of CCL2–CCR2 signaling on TTX-S Nav currents in peripheral nociceptors.

In the present study, we examined the expression pattern of CCL2 and CCR2 in the DRG of rats which had subjected to complete Freud adjuvant (CFA)-induced chronic inflammatory pain and further studied the effect of CCL2 on TTX-S Nav currents in DRG nociceptors. Our results support the involvement of CCL2–CCR2 signaling in inflammatory hyperalgesia and indicate that enhanced TTX-sensitive channel activity may partly underlie the pronociceptive effects of CCL2.

Materials and Methods

Animals

Male Sprague-Dawley rats (120–150 g) were obtained from Shanghai SLAC Laboratory Animal Co. (Shanghai, China) and housed in the institutional animal facility with a 12 h light dark cycle and free access to food and water. The protocols of the study were approved by the Institutional Ethical Committee for Animal Experiments of Shanghai Jiaotong University School of Medicine. All efforts were made to minimize the discomfort of the animals used in the study.

CFA-induced inflammatory pain model and behavior observations

Rats were injected with complete Freund’s adjuvant (CFA, 100 μl) in right hind paw under isoflurane anesthesia. Control rats were injected with equal volume of saline. Before intraplantar injection of CFA or saline, baseline threshold of the paw withdrawal response to mechanical probing of the hind paw was obtained. The rats were placed individually in a plexiglass box and allowed to acclimatize for 20 min. Then, the hind paws were stimulated with a set of von Frey filaments (5, 10, 20, 40, 60, 80, 100, and 120 mN) given in an ascending order. Each filament was applied for 1 s to each of the six designated sites of the plantar surface at intervals of 10–15 s. The threshold force, defined as the force corresponding to a 50% withdrawal, was determined by a Hill equation (Origin Version 6.0; Microcal Software, Malvern, UK). The PWT was daily examined following intraplantar injection of CFA or saline. On the fifth day, 25 μl of CCL2 (500 ng) or phosphate buffer saline (PBS) was subcutaneously injected into the plantar surface of the ipsilateral hind paw of each rat. After injection, rats were placed in plexiglass boxes and videotaped for 30 min to observe the flinching behavior (defined as the paw lift time per minute, PLTPM) and subsequently tested for PWT.

Detection of CCL2 mRNA expression in DRG

Total RNA was extracted from L4/L5 DRG using Trizol (Invitrogen, Grand Island, USA) according to the manufacturer’s guidelines. One microgram of total RNA was reverse-transcribed by using PrimeScript RT reagent kit (TaKaRa, Dalian, China). PCR was performed using SYBR Green PCR kit (Applied Biosystems, Foster City, USA) and ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). Primers used are: β-actin, forward 5’-ATGGTGGGTATGGGTCAGAAGG-3’, reverse 5’-TGGCTGGGGTGTTGAAGGTC-3’; CCL2, forward 5’-ACTTGACCCATAAAT CTGA-3’, reverse 5’-TGGAAGGGAATAGTGTAAT-3’. CCL2 mRNA expression level was normalized to that of the β-actin.

Immunofluorescent staining and western blot analysis of CCR2

Rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and perfused through the aorta with saline followed by 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4). The DRGs were dissected and post-fixed in 4% PFA in PBS (0.1 M, pH 7. 4) overnight, transferred to 25% sucrose in PBS in turn. Tissues were cut into 10-μm sections with a Leica cryostat (CM1900; Wetzlar, Germany) and thaw-mounted onto gelatin-coated slides. In order to test whether CCR2 is upregulated in nociceptors, we performed double immunostaining for CCR2 and P2X3, which is known to be expressed in small to medium-sized nociceptive neurons. The DRG sections were incubated with primary rabbit polyclonal antibody against CCR2 (1:400; Cell Signaling Technology, Beverly, USA) and primary guinea pig polyclonal antibody against P2X3 receptors (1:600; Chemicon International, Billerica, USA) for 24 h. After being washed with PBS, the sections were incubated with the secondary antibodies. The immunoreactivity was visualized by fluorescence microscopy.

For western blot analysis, L4/L5 DRGs were collected from rats which were deeply anesthetized, and the protein was extracted from DRG using a SDS sample buffer containing proteinase and phosphatase inhibitors. Each protein samples (50 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane which was blocked with 2% BSA in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature and then incubated with antibody against CCR2 (anti-rabbit, 1:50; Santa Cruz Biotechnology, Santa Cruz, USA) in 2% BSA in TBST at 4°C overnight. After three washes with TBST, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit antibody (Santa Cruz Biotechnology) and the antigen–antibody complexes were detected using enhanced chemoluminescence detection reagent (Amersham, Buckinghamshire, UK). The blots were scanned using a densitometer (GS-700; Bio-Rad, Hercules, USA), and quantification was performed using Multi-Analyst software (Bio-Rad).

Acute dissociation of DRG neurons

Rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). L4/L5 DRGs were dissected and digested at 37°C with collagenase A (1 mg/ml; Boehringer Mannheim, Ingelheim am Rhein, Germany) for 25 min followed by digestion with collagenase D (1 mg/ml; Boehringer Mannheim) and papain (30 units/ml; Worthington Biochemical, Lakewood, USA) for 25 min. The enzyme solutions were prepared in complete saline solution (CSS) containing: 137 mM NaCl, 5.3 mM KCl, 1 mM MgCl₂, 3 mM CaCl₂, 25 mM sorbitol, and 10 mM HEPES, adjusted to pH 7.2 with NaOH. The cells were dissociated by trituration in culture medium containing 1 mg/ml BSA and 1 mg/ml trypsin inhibitor (Boehringer Mannheim). DRG neurons were seeded onto cover slips coated with 0.1 mg/ml polyornithine and 1 mg/ml laminin (Boehringer Mannheim). The culture medium contained equal amounts of Dulbecco’s modified Eagle medium and F12 (Gibco, Grand Island, USA) with 1% penicillin (100 U/ml)/streptomycin (0.1 mg/ml; Life Technologies, Rockville, USA) and 10% fetal calf serum (HyClone Laboratories, Logan, USA). The DRG neurons were incubated for 12–18 h at 37°C, 95% air and 5% CO₂.

Electrophysiology

The coverslip with adherent DRG neurons was placed in a recording chamber mounted on the stage of an upright microscope (BX50-WI; Olympus Optical, Tokyo, Japan) and perfused continuously at a rate of 2–3 ml/min with a bath solution at room temperature. Electrophysiological recording was performed using Axonclamp 700B amplifier and pClamp 10 software (Axon Instruments, Jakarta, Indonesia) with borosilicate glass pipettes pulled on a PC-10 puller (Narishige, Tokyo, Japan). The impedance of a typical pipette was 2–4 M when filled with the pipette solution. Small to medium-sized DRG neurons (diameter <30 μm) were considered as primary nociceptors and were selected for membrane potential or membrane current recording. Electrophysiological signals were filtered at 3 kHz and digitized by A/D converter (Digidata 1322A; Axon Instruments) at 5 kHz.

For whole-cell current clamp recording of the membrane potential, bath solution contained: 145 mM NaCl, 3 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES and 10 mM glucose (pH 7.4, osmolarity 300–310 mOsm). Electrodes were filled with a pipette solution that contained: 120 mM K-gluconate, 10 mM KCl, 5 mM NaCl, 1 mM CaCl₂, 2 mM MgCl₂, 11 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP, and 1 mM Li-GTP (pH and osmolarity adjusted to 7.2 and 310 mOsm). To observe the effects of CCL2 on neuronal excitability, the agonist (100 nM) was applied via a micropipette ~100 μm from the cell.

Voltage-clamp recording of sodium currents (Nav currents) was performed under a reduced sodium gradient to reduce space clamp problems. The bath solution contained: 35 mM NaCl, 105 mM TEA, 4 mM 4-aminopyridine (4-AP), 2 mM BaCl₂, 0.1 mM CaCl₂, 0.1 mM CdCl₂, 10 mM HEPES and 10 mM glucose (pH 7.4, osmolarity 300–310 mOsm). The pipette solution contained: 140 mM CsF, 10 mM NaCl, 10 mM EGTA, and 10 mM HEPES (pH 7.2, osmolarity 290–300 mOsm). Cells were stimulated with depolarizing step pulses from the holding potential (−60 mV) to 60 mV (10 mV increments at 1 s interval, 15 ms pulse duration) to elicit the voltage-gated sodium currents (total Nav currents). TTX (0.3 μM) was applied via a micropipette ~100 μm from the cell. TTX-sensitive Nav currents were obtained by subtracting the remaining currents in the presence of TTX (designated as TTX-resistant Nav currents) from the total Nav currents in the absence of TTX. This protocol was repeated in the presence of CCL2 (100 nM) in order to observe its effect on TTX-S and TTX-R Nav currents.

For potassium current recordings, the bath solution contained: 145 mM N-methyl-d-glucamine, 3 mM KCl, 2.5 mM CdCl₂, 0.6 mM MgCl₂, 10 mM HEPES and 10 mM glucose (adjusted to pH 7.4 using HCl) to suppress the sodium and calcium currents. Potassium currents were elicited by applying voltage steps from −120 mV to 60 mV (10 mV increments, 500 ms pulse duration).

Statistical analysis

All data were presented as the mean ±  SEM. Statistical significance was determined using Origins 8.0 using Two-tailed Student’s t-test (paired or unpaired) or ANOVA with appropriate post-test for comparison of the mean. P values <0.05 were considered as statistically significant differences.

Results

CCL2 increased the nociceptive behavior of CFA-inflamed rats

As shown in Fig. 1A, following intraplantar injection of CFA, the PWT was significantly decreased on the ipsilateral but not on the contralateral side. On the fifth day following CFA or vehicle injection, CCL2 or PBS was intradermally injected into the ipsilateral paw. CCL2 did not alter the PWT of vehicle-treated (control) rats, but caused a further decrease in PWT of CFA-treated rats (Fig. 1B). In addition, CCL2 also caused acute spontaneous nocifensive behavior in CFA-treated rats, characterized by frequent flinching (Fig. 1C) and prolonged paw lifting time (Fig. 1D). These results are in agreement with previous reports that CCL2 exerts a pronociceptive effect in pathological conditions.

CCL2 and CCR2 expressions were increased after CFA injection

Quantitative real-time PCR analysis of L4/L5 DRG revealed a greater increase of CCL2 transcripts in ipsilateral than that in contralateral DRG on Day 1, Day 3 and Day 5 after CFA treatment (Fig. 2A). The most significant increase of CCL2 transcripts occurred on the first day after CFA injection (Fig. 2B, values normalized to vehicle-treated rats, using GAPDH as the internal control).

Immunohistochemistry was conducted to examine the expression of CCR2 in L4/L5 DRG. An increased CCR2 immunofluorescence was detected in DRG from CFA-treated rats when compared with that of vehicle-treated (control) rats. Double-labeling analysis showed that P2X3⁺ small to medium-sized neurons were positive for CCR2 immunoreactivity (Fig. 2C). Western blot analysis further indicated that the expression of CCR2 in L4/L5 DRG was significantly increased in CFA-treated rats compared with that of the control rats (Fig. 2D).

Acute application of CCL2 increased the excitability of primary sensory neurons of CFA-treated rats

We then evaluated the effect of CCL2 on the excitability of acutely-dissociated small to medium-sized DRG neurons (diameter <30 μm, with a mean membrane capacitance 30.20 ± 4.01 pF) from the CFA (CFA neurons) or vehicle-treated rats (control neurons). Of the 44 CFA neurons tested, 19 (43.2%) were depolarized (>2 mV, mean = 13.8 ± 1.9 mV) in the presence of CCL2 (100 nM) for 1 min (Fig. 3A,B). Nine of the 19 responsive CFA neurons (47%) fired action potentials during CCL2 application. By contrast, CCL2 only depolarized a minor proportion (4 of 38, 10.5%) of control neurons.

CCL2 also caused a decrease in the current threshold of CFA neurons, defined as the minimal current for generation of action potentials. To measure the current threshold before and after exposure to CCL2 in CFA neurons, a series of increasing current steps, each of 500 ms duration, were delivered in increments of 10 pA starting from −50 pA (Fig. 3C). Prior to CCL2 exposure, current threshold was 61.4 ± 3.7 pA (n = 28). After exposure to CCL2 for 1 min, the mean current threshold for the same set of neurons was decreased to 46.8 ± 3.6 pA (Fig. 3D). Furthermore, the number of action potentials evoked in response to the threshold depolarizing currents increased by 6.4 folds during CCL2 application (n = 19; Fig. 3E,F). These results showed that CCL2 may increase the excitability of peripheral nociceptors in the inflamed rats.

TTX-sensitive sodium currents were enhanced by CCL2

Voltage-gated sodium and potassium channels are crucial determinants of neuronal excitability [15]. Enhanced excitability of DRG neurons after local or peripheral inflammation is closely associated with the changes in sodium and potassium channels [16,17]. We therefore tested the effect of CCL2 on sodium and potassium currents in L4/L5 DRG neurons from CFA-treated rats.

To investigate the effect of CCL2 on sodium channel activation, CFA neurons were subject to depolarizing voltage steps (−60 to +60 mV in 10 mV increments, 15 ms pulse duration) to elicit total sodium currents. Thereafter, TTX (0.3 μM) was applied and the remaining Nav currents were designated as the TTX-resistant sodium current (TTX-R I_Na). The TTX-sensitive sodium current (TTX-S I_Na) was obtained by subtracting the TTX-R I_Na from the total sodium current. In the 25 CFA neurons recorded, TTX-R I_Na was the predominant current. The ratio of TTX-R to total sodium current (measured at −20 mV) was 0.73 ± 0.03. After treatment with CCL2 for 1 min, this fraction was significantly decreased to 0.59 ± 0.05 (n = 25, P < 0.01, paired t-test). The decrease was not due to the inhibition of TTX-R I_Na but due to the increase of TTX-S I_Na in the presence of CCL2. Prior to CCL2 exposure, the peak current density of TTX-S I_Na had an average value of −27.8 ± 4.3 pA/pF. In the presence of CCL2 for 1 min, the peak current density of TTX-S I_Na was significantly enhanced to −49.1 ± 5.9 pA/pF (Fig. 4B; P < 0.01, RM ANOVA, Tukey post hoc test). The increase in the TTX-S I_Na induced by CCL2 was accompanied by a leftward shift in the activation curve to more hyperpolarized potentials, suggesting that the midpoint of activation (estimated by a Boltzmann function) for the TTX-S I_Na was significantly more negative during CCR2 activation than before. In contrast, the peak current density of TTX-R I_Na (measured at −10 mV) was not significantly affected by exposure to CCL2 (Fig. 4B; 44.0 ± 4.0 versus 39.9 ± 5.0 pA/pF, n = 25, P > 0.05, RM ANOVA, Tukey post hoc test). The voltage-gated potassium currents (slow decaying A-type K⁺ currents) in CFA neurons were also unaffected by CCL2 (Fig. 5A,B). These results suggest that enhancement of TTX-S sodium channel activity may partly underlie the pronociceptive role of CCL2 in CFA-induced chronic inflammatory pain.

Discussion

There has been a continuing interest in the role of CCL2 and its cognate receptor CCR2 in pathological pain [18–20]. In agreement with previous reports, the present study revealed an elevated expression of CCL2 and CCR2 in DRG of the intraplantar CFA-induced inflammatory pain model. Application of CCL2 in vivo was found to potentiate CFA-induced hyperalgesia, without significantly affecting the mechanical pain threshold in vehicle-treated rats. Furthermore, we found that acute application of CCL2 increased the excitability of primary nociceptors from CFA-treated rats partly through enhancing TTX-S sodium but not TTX-R sodium or the slow decaying A-type potassium currents.

Increased expressions of CCL2 and CCR2 have been reported in sensory ganglion, the dorsal horn of the spinal cord and discrete brain regions including the rostral ventromedial medulla in different models of chronic pain [4,19,21,22]. Importantly, CCR2-deficient mice are insensitive to neuropathic pain [7] and intrathecal injection of a CCR2 antagonist (AZ889) induces a dose-dependent analgesia in the chronic constriction injury (CCI) model [23]. Intrathecal administration of the dicer substrate small interfering RNA (DsiRNA) targeting CCR2 reduced spinal microglia activation in CCL2-induced mechanical hypersensitivity and hypernociceptive responses were observed in the CFA-induced inflammatory chronic pain model [24]. In agreement with previous reports, our results showed that CCL2 transcription and protein expression were significantly increased in ipsilateral DRG following intraplantar injection of CFA and that application of CCL2 potentiated CFA-induced hyperalgesia. These results indicate that CCL2/CCR2 signaling is an important mediator of neuropathic and inflammatory pain. However, the mechanisms underlying the pronociceptive role of CCL2/CCR2 are not fully understood.

Nav channels are critically important for the generation and conduction of impulses in primary sensory neurons [10]. Primary sensory neurons, particularly the nociceptive small neurons, express multiple sodium channel isoforms, with Nav1.8 and Nav1.9 mediating the TTX-R currents, and Nav1.7, Nav1.6, Nav1.5, Nav1.3, Nav1.2 and Nav1.1 mediating the TTX-S currents [9,10]. Nav channels are dynamically regulated after nerve injury and/or inflammation and there is strong evidence that TTX-S Nav1.7 and TTX-R Nav1.8 and Nav1.9 are crucial in inflammatory and probably neuropathic pain [25,26]. Therefore, it is interesting to test whether CCL2/CCR2 signaling can modulate the excitability of primary nociceptive neurons via affecting these channels.

CCL2/CCR2 signaling reportedly increases the excitability of peripheral nociceptors after nerve injury [8,27–29]. Here, we found that acute application of CCL2 increases the excitability of DRG nociceptors from CFA-treated rats, with only minor effect on nociceptors from control rats. This result is consistent with a previous report showing that acute application of CCL2 depolarizes DRG neurons from CCD rats but not from naive rats [8]. It has been reported that CCL2 increases nociceptor excitability through up-regulating the expression of TTX-R Nav1.8 [12,13]. Intriguingly, however, TTX-R and TTX-S Na⁺ currents in the small DRG neurons were found to be significantly down-regulated after chronic compression of DRG, an established model of neuropathic pain [30]. Hence, CCL2/CCR2 may directly modulate the activity of Nav channels, an aspect which has not been vigorously tested.

We further tested the effect of acute CCL2 treatment on voltage-gated sodium currents of DRG nociceptors (small to medium-sized neurons) from CFA-treated rats, and found that CCL2 caused a significant increase in TTX-S Nav currents without significantly affecting the amplitude of TTX-R Nav currents or voltage-gated potassium currents. This result contrasts with the findings of Belkouch et al. [14] that acute treatment with CCL2 increases TTX-R Nav1.8 channel activity of DRG neurons from naive rats. This discrepancy suggests that CFA-induced chronic inflammation may alter downstream signaling of CCL2/CCR2 in sensory neurons.

In summary, in the present study we provided additional evidence that CCL2/CCR2 is upregulated in peripheral nociceptors under inflammatory conditions and acute application of CCL2 potentiates CFA-induced hyperalgesia in vivo and increases the excitability of DRG neurons of CFA-treated rats in vitro. In addition, this study also suggests that CCL2/CCR2 may enhance the activity of TTX-S sodium channels.

Funding

This work was supported by the grant from the National Natural Science Foundation of China (No. 81270464).

References

Author notes