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Does chloride channel accessory 3 have a role in arthritis pain? A study on murine antigen-induced arthritis
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Matthias Ebbinghaus a , Mieczyslaw Gajda b , Michael J. Holtzman c , Stefan Schulz d , Hans-Georg Schaible a,∗ a
Institute of Physiology I, University Hospital – Friedrich Schiller University Jena, Teichgraben 8, D-07743 Jena, Germany Institute of Pathology, University Hospital – Friedrich Schiller University Jena, Ziegelmühlenweg 1, D-07743 Jena, Germany c Pulmonary and Critical Care Medicine, Washington University School of Medicine, St. Louis, MI 63110, USA d Institute of Pharmacology and Toxicology, University Hospital – Friedrich Schiller University Jena, Drackendorfer Str. 1, D-07747 Jena, Germany b
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h i g h l i g h t s • • • • •
In DRGs, a 1724-fold up-regulation of mClca3 was shown in acute arthritis. mClca3 knock-out mice showed significantly less swelling in very acute AIA. No modification of mechanical hyperalgesia was seen in mClca3 knock-out mice. Pharmacological inhibition of CaCCs by niflumic acid did not alter AIA. mClca3 does not significantly contribute to arthritic pain.
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Article history: Received 8 April 2014 Received in revised form 23 May 2014 Accepted 27 May 2014 Available online xxx
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Keywords: Calcium-activated chloride channels Chloride channel accessory mClca3 AIA Arthritis Inflammation Pain
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1. Introduction
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Calcium-activated chloride channels (CaCCs) are thought to regulate neuronal excitability, and recently chloride (Cl− ) regulation in DRG neurons has attracted much attention in pain research. Furthermore, the activity of CaCCs is modified by a family of CLCA proteins. In acute antigen-induced arthritis (AIA), a remarkable up-regulation of the murine chloride channel accessory 3 (mClca3) was shown in dorsal root ganglion (DRG) neurons. Therefore we tested the hypothesis that mClca3 is involved in arthritic pain perception. In mClca3 knock-out mice and wild-type control mice, AIA was induced and measures of inflammation and pain were assessed. In the very acute phase of AIA, joint swelling was reduced in mClca3 knock-out mice. This effect disappeared during the course of AIA. We could not show significant differences in mechanical hyperalgesia between both groups of mice, neither at the acute nor at the chronic stage (21 days of AIA). Additional experiments on thermal hyperalgesia in wild-type and mClca3 knock-out mice in the first 3 days of AIA did not show a difference either. In addition, niflumic acid, an antagonist at CaCCs, did not significantly influence hyperalgesia during AIA. Thus, we were not able to provide evidence for a role of CaCCs, and in particular of mClca3, on the expression of arthritis or inflammation-evoked hyperalgesia. © 2014 Published by Elsevier Ireland Ltd.
Calcium-activated chloride channels (CaCCs) are a group of ubiquitously expressed ion channels. One important role is their
∗ Corresponding author at: Institute of Physiology I/Neurophysiology, Jena University Hospital – Friedrich Schiller University Jena, Teichgraben 8, D-07743 Jena, Germany. Tel.: +49 3641 938810; fax: +49 3641 938812. E-mail addresses: [email protected] (M. Ebbinghaus), [email protected] (M. Gajda), [email protected] (M.J. Holtzman), [email protected] (S. Schulz), [email protected] (H.-G. Schaible).
impact in neuronal excitability [1]. They are expressed in spinal cord neurons, autonomic neurons and dorsal root ganglion (DRG) neurons but the functions of CaCCs here remain poorly understood [2–5]. One member of the CaCC family (anoctamin 1) was recently identified to act directly as a heat sensor in mice [6]. CLCA proteins are a family of proteins which are associated with CaCCs. They are thought to act as chloride channel accessory molecules which can modify the currents through CaCCs [7]. In mouse medium-sized DRG neurons, calcium-activated chloride currents were reported, and subsequently transcripts of mClca1 and mClca5 were detected [2,8]. Using microarray analysis we recently detected mClca3 in the DRGs. This protein is the murine
http://dx.doi.org/10.1016/j.neulet.2014.05.051 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: M. Ebbinghaus, et al., Does chloride channel accessory 3 have a role in arthritis pain? A study on murine antigen-induced arthritis, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.051
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homolog for human CLCA1, also known as gob-5 [9]. In mice, mClca3 plays a critical role in allergen-induced airway hyperreactivity, goblet cell metaplasia and the regulation of tissue inflammation in the innate immune response [10–12]. Interestingly, we found a 1724-fold up-regulation of the mClca3 gene in the lumbar DRGs in the acute inflammatory phase of murine antigeninduced arthritis (AIA) [13]. Chloride (Cl− ) is in the focus of pain research. It was shown that the effect of GABA in the spinal cord depends on the intracellular Cl− concentration which is regulated by Cl− transporters such as NKCC1. If the intracellular Cl− concentration is elevated, GABA may cause rather an efflux than an influx of Cl− , and this changes the effect of GABA from inhibition to excitation. Also in DRG neurons changes in the Cl− regulation were shown to be important in nociceptive processing [14–16]. In vivo, NKCC1 knock-out mice show increased latencies to withdrawal from heat and a reduction in allodynia at the paws [17,18]. In DRG neurons, the chloride gradient is different from that in most other types of neurons such that calcium-activated Cl− current can be excitatory and that these channels are pronociceptive [1]. Consistent with this view the knockdown of anoctamin 1 (see above) reduced thermal pain [6]. It was suggested that inhibition of CaCCs may be a potential strategy for the development of analgesis [1]. Because we observed an upregulation of mClca3 in the DRGs of mice with AIA [13], we tested the hypothesis that mClca3 is involved in inflammatory pain perception in murine AIA. We used mClca3 knock-out mice and wild-type (WT) control mice, induced AIA and assessed inflammation and pain.
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2. Materials and methods
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2.1. Animals
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Homozygous mClca3−/− mice were generated and kindly provided by Prof. M.J. Holtzman (Washington University School of Medicine, St. Louis, MI, USA) [9]. Female knock-out and WT C57BL/6J control mice (9–13 weeks) were bred and genotyped by the Animal Facility of the University Hospital Jena. All animal studies were approved by the local government commission for animal protection (No. 02-019/12).
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2.2. Arthritis induction and pharmacological treatment
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Mice were immunized twice at 21 and 14 days before AIA induction with subcutaneous injection of 100 g of methylated bovine serum albumin (mBSA) (Sigma–Aldrich, Taufkirchen, Germany), emulgated with 50 l of complete Freund’s adjuvant (CFA; Sigma–Aldrich), supplemented to 2 mg/ml Mycobacterium tuberculosis, strain H37Ra (Difco, Detroit, USA). Additionally, 5 × 108 heat-inactivated Bordetella pertussis germs (Chiron-Behring, Marburg, Germany) were applied intraperitoneally (i.p.). Monoarticular arthritis was induced by injection of 100 g mBSA in 25 l 0.9% NaCl into the right knee joint cavity on day 0. A flare-up reaction of inflammation was induced by a second intraarticular injection of 100 g mBSA in 25 l saline on day 42 after primary arthritis induction. In WT mice niflumic acid (NA), a potent blocker of CaCCs, was applied i.p. NA was dissolved in 0.4 M NaHCO3 in 5% glucose. Mice received either 10 mg or 30 mg NA/kg body weight one day before arthritis induction for 5 consecutive days as previously described [19].
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2.3. Assessment of AIA
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Swelling at the knee was assessed by measuring the mediolateral joint diameter using an Oditest vernier caliper (Kroeplin, Schlüchtern, Germany). For histopathological examination knee
joints were removed, fixed in toto in 4.5% formalin, decalcified in EDTA, embedded in paraffin and cut into 3 m frontal sections which were stained with hematoxylin–eosin. The pathologist who scored arthritis was unaware of the experimental groups. Acute inflammation (infiltration of the synovial membrane by granulocytes and exudation of granulocytes into the joint space) was scored 0–3: 0 = no, 1 = mild, 2 = moderate, and 3 = severe changes (+1 if fibrin exudation in the joint space). Chronic inflammation (hyperplasia of synovial lining cells, infiltration of the synovial membrane by mononuclear cells, fibrosis of the synovial membrane and the periarticular tissue) was also scored 0–3. Cartilage surface defects with cell necrosis were scored 0–4: 0 = no damage, 1 = 50% of the cartilage surface affected. Damage to bone was also evaluated: 0 = no, 1 = mild, 2 = medium, and 3 = severe damage (extensive area of deep invasive destruction of bone). 2.4. Pain-related behavior Mechanical hyperalgesia at the hindpaws as an indicator of secondary hyperalgesia remote from the inflamed knee joint was assessed before and until 21 days after induction of AIA. Mice were placed into the testing device, and after accommodation to the environment the pain threshold was determined. Two testings before arthritis induction defined the baseline (BL). For mechanical hyperalgesia a dynamic plantar aesthesiometer (Ugo Basile, Comerio, Italy) was used which applied increasing pressure (stimulus increase rate 1 g/s; cut-off value 10 g) to the paw. The latency of the elicited leg withdrawal which reflects the respective mechanical threshold was averaged from three consecutive stimuli. Data are given in gram as alteration related to BL (d(x)–BL). Gait abnormalities of the ipsilateral hindlimb were quantified in the guarding score: 0 = normal walking, 1 = slight limping, 2 = persistent severe limping (still touching floor), 3 = severe limping with partial guarding of ipsilateral hindlimb (sometimes not touching floor), 4 = mainly guarding of ipsilateral hindlimb (most times not touching floor), and 5 = no walking at all. In other groups of wild-type and mClca3−/− mice thermal hyperalgesia was assessed in the first 3 days of AIA using the Hargreaves plantar test (Ugo Basile) [20]. Two consecutive standardized heat stimuli were applied to the paw for evaluation of a mean latency (cut-off value 20 s). 2.5. Statistical analysis Data are expressed as mean ± SEM. Differences between groups were calculated using the two-tailed Student’s t-test for unpaired values. Arthritis score, guarding score and mechanical and thermal thresholds of experimental groups were compared using analysis of variance (ANOVA). Changes in responses within groups (baseline versus after AIA induction) were analyzed using Wilcoxon’s matched pairs signed rank test. Statistical significance was calculated with the SPSS software package (v.16.0, Chicago, USA) and accepted for p < 0.05.
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3.1. Slightly decreased severity of AIA in mClca3−/− mice
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After induction of AIA, joint swelling in the acute phase of AIA was significantly attenuated in mClca3−/− mice compared to control WT mice (ANOVA day 3: F [1,25] = 5.441; p = 0.028) (Fig. 1A). This difference between both groups of mice disappeared in the chronic phase of AIA from day 7 on. The second injection of mBSA at day 42 of AIA evoked a strong flare-up reaction in both WT and
Please cite this article in press as: M. Ebbinghaus, et al., Does chloride channel accessory 3 have a role in arthritis pain? A study on murine antigen-induced arthritis, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.051
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Fig. 1. Inflammation and pain-related behavior in acute AIA. (A) In mClca3−/− mice a significant reduction of joint swelling was observed on day 1 compared to C57BL/6J wild-type controls (n = 11/16 per group). (B) Histopathological examination of the inflamed knee joint at day 3 of flare-up AIA showed a significant difference only in the score for chronic inflammation (n = 7/8 per group). (C) The latency of mechanical withdrawal thresholds at the ipsilateral paw is given in gram as alteration related to the baseline (BL) (d(x)–BL). No difference was observed between mClca3−/− and wild-type mice in acute AIA (n = 14/15 per group). (D) No significant difference in the guarding score could be seen between both groups of mice (n = 9/7 per group). Values are mean ± SEM. *p < 0.05 for differences between groups at that time point.
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mClca3−/− but the swelling was not different in these experimental groups (data not shown). The histopathological arthritis score showed a significant difference between mClca3−/− and wild-type mice only for chronic inflammation parameters on day 3 of flare-up AIA (Fig. 1B). The total score for inflammation and destruction was only slightly decreased in mClca3−/− mice.
3.3. Pharmacological inhibition of CaCC did not alter AIA
3.2. Unchanged pain-related behavior in mClca3−/− mice
Although specific antagonists for CaCCs are lacking, niflumic acid (NA) is considered to be a potent blocker for CaCCs in mouse models [19]. Treatment of WT mice with NA in two concentrations did not alter joint swelling in the course of AIA, neither at 10 nor at 30 mg/kg, compared to untreated AIA controls (ANOVA day 7: F [2,27] = 1.896; p = 0.170) (Fig. 3A). On day 3 of AIA, both NAtreated (30 mg/kg) mice and untreated control mice showed similar
In the acute phase of AIA both mClca3−/− and WT mice showed a significant reduction of mechanical threshold for withdrawal at the ipsilateral paw compared to the baseline, indicating mechanical hyperalgesia (day 3: mClca3−/− p = 0.002; wild-type p = 0.004). No significant difference in the value of mechanical hyperalgesia could be observed in the two groups of mice (ANOVA day 7: F [1,27] = 0.203; p = 0.656) (Fig. 1C). The reduction of threshold disappeared in both groups in the chronic phase of AIA from day 7 on. In line with mechanical hyperalgesia, mClca3−/− and wild-type mice showed protective behavior (guarding) on the inflamed hind paw in acute AIA but the scores of both experimental groups did not differ significantly (ANOVA day 7: F [1,12] = 2.228; p = 0.161) (Fig. 1D). In other groups of wild-type and mClca3−/− mice we tested thermal hyperalgesia in the first 3 days of AIA. A reduction of thermal threshold for withdrawal at the ipsilateral paw was seen in both groups of mice (day 1: mClca3−/− p = 0.012; day 3: wild-type p = 0.028) (Fig. 2). No significant difference between the two groups was detected (ANOVA day 3: F [1,12] = 0.474; p = 0.504).
Fig. 2. Thermal hyperalgesia in acute AIA. In C57BL/6J control mice, mClca3−/− mice and niflumic acid (NA)-treated mice a significant reduction in the latency of thermal withdrawal thresholds at the ipsilateral paw was seen in the acute phase of AIA. Values are mean ± SEM (n = 6–9 per group). *p < 0.05 for changes within groups (baseline versus acute AIA).
Please cite this article in press as: M. Ebbinghaus, et al., Does chloride channel accessory 3 have a role in arthritis pain? A study on murine antigen-induced arthritis, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.051
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Fig. 3. Acute AIA after treatment with niflumic acid (NA) in wild-type mice. After pharmacological inhibition of CaCCs, no significant differences in joint swelling (A), mechanical withdrawal thresholds at the ipsilateral paw (B) and in the guarding score (C) could be observed between NA-treated and untreated control mice. BL = baseline. Values are mean ± SEM (n = 9–10 per group).
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mechanical hyperalgesia at the ipsilateral paw when compared to the mechanical threshold for withdrawal at the contralateral paw (NA-treated mice: p = 0.012; control mice: p = 0.004). Between both groups no significant difference was observed for comparison of the threshold of the ipsilateral paw (ANOVA day 7: F [1,16] = 0.094; p = 0.763) (Fig. 3B). The value of guarding also did not differ either in both groups in the course of AIA (ANOVA day 7: F [1,15] = 1.302; p = 0.272) (Fig. 3C). NA-treated and control mice also showed a significant reduction of thermal threshold for withdrawal at the ipsilateral paw at day 3 of AIA compared to the baseline (NA-treated mice p = 0.015; control mice p = 0.028) (Fig. 2, columns on the right). At this time point in AIA no difference between both groups was detected (ANOVA day 7: F [1,13] = 0.202; p = 0.660).
The present study addressed the hypothesis that mClca3 knock-out mice and WT mice differ in the expression of inflammation-evoked hyperalgesia during antigen-induced arthritis (AIA). However, the development and magnitude of mechanical hyperalgesia was similar in mClca3 knock-out mice and WT mice throughout AIA. Thermal hyperalgesia, tested in the first 3 days of AIA, was also similar in mClca3 knock-out mice and WT mice. Only joint swelling was slightly reduced in mClca3 knock-out mice in the very acute phase of AIA. The CaCC blocker niflumic acid did not significantly alter mechanical or thermal hyperalgesia either. Thus, we did not find any evidence that mClca3 is critically involved in arthritic pain although we found a substantial upregulation of the mClCa3 gene in DRGs in the acute inflammatory phase [13]. The slight reduction of swelling in the acute phase of AIA was significant. These data support a role of mClca3 in inflammatory processes. However, in the swelling during the flare-up reaction upon the second injection of mBSA into the knee joint, which is significantly stronger than the initial swelling, such a difference between mClca3 knock-out and WT mice was no longer observed. We exclude, therefore, that mClca3 has an important role in the expression of arthritis. The assessment of behavior was focused on the measurement of mechanical threshold and gait analysis because arthritis produces mainly mechanical hyperalgesia [21,22]. In these tests, we could not find any significant difference between mClca3 knock-out and WT mice suggesting that mechanical hyperalgesia does not depend on mClca3. It should be noted that mechanical hyperalgesia in AIA is significantly reduced by neutralizing interleukin-17 [23,24], by somatostatin [25] and endothelin receptor antagonists [13] showing that AIA is a model suitable for the testing of mechanical hyperalgesia. We did not test for thermal hyperalgesia beyond 3 days of AIA. During this acute phase the reduction of thermal threshold was similar in WT and mClca3 knock-out mice. Although we cannot exclude that thermal hyperalgesia is different in WT and mClca3 knock-out mice at later stages of AIA we consider this unlikely because the time course of mechanical and thermal hyperalgesia in mouse AIA was found to be parallel [13]. In a more general approach we aimed to test whether CaCCs are at all critically involved in arthritic pain, and therefore we treated arthritic WT mice with niflumic acid. This compound is considered a potent blocker for CaCCs in mouse models [19]. However, we found no influence of niflumic acid on AIA and mechanical and thermal hyperalgesia. Thus we could not identify an important role of CaCCs in arthritis and inflammation-evoked hyperalgesia. The negative effect on a putative role of mClca3 in the present study does not exclude that CaCCs play a role in other models of pain, either in acute models of arthritis or in models of cutaneous and visceral pain; e.g. Liu et al. described a role of CaCC in bradykinin-induced pain [26]. At this stage we do not know the relative importance of bradykinin in AIA pain, compared to other mediators.
5. Conclusions Although we found a significant upregulation of the mClca3 gene in the DRGs of mice with AIA [13] we were not able to demonstrate an important consequence on the expression of arthritis and hyperalgesia which could be attributed to mClca3. The lack of effect of knocking-out mClca3 and of the CaCC blocker niflumic acid lead us to conclude that neither mClca3 nor CaCCs significantly contribute to arthritic pain.
Please cite this article in press as: M. Ebbinghaus, et al., Does chloride channel accessory 3 have a role in arthritis pain? A study on murine antigen-induced arthritis, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.051
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Acknowledgements The authors thank Konstanze Ernst and Cornelia Hüttich for excellent technical assistance.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Does chloride channel accessory 3 have a role in arthritis pain? A study on murine antigen-induced arthritis
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Matthias Ebbinghaus a , Mieczyslaw Gajda b , Michael J. Holtzman c , Stefan Schulz d , Hans-Georg Schaible a,∗ a
Institute of Physiology I, University Hospital – Friedrich Schiller University Jena, Teichgraben 8, D-07743 Jena, Germany Institute of Pathology, University Hospital – Friedrich Schiller University Jena, Ziegelmühlenweg 1, D-07743 Jena, Germany c Pulmonary and Critical Care Medicine, Washington University School of Medicine, St. Louis, MI 63110, USA d Institute of Pharmacology and Toxicology, University Hospital – Friedrich Schiller University Jena, Drackendorfer Str. 1, D-07747 Jena, Germany b
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In DRGs, a 1724-fold up-regulation of mClca3 was shown in acute arthritis. mClca3 knock-out mice showed significantly less swelling in very acute AIA. No modification of mechanical hyperalgesia was seen in mClca3 knock-out mice. Pharmacological inhibition of CaCCs by niflumic acid did not alter AIA. mClca3 does not significantly contribute to arthritic pain.
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Article history: Received 8 April 2014 Received in revised form 23 May 2014 Accepted 27 May 2014 Available online xxx
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Keywords: Calcium-activated chloride channels Chloride channel accessory mClca3 AIA Arthritis Inflammation Pain
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1. Introduction
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Calcium-activated chloride channels (CaCCs) are thought to regulate neuronal excitability, and recently chloride (Cl− ) regulation in DRG neurons has attracted much attention in pain research. Furthermore, the activity of CaCCs is modified by a family of CLCA proteins. In acute antigen-induced arthritis (AIA), a remarkable up-regulation of the murine chloride channel accessory 3 (mClca3) was shown in dorsal root ganglion (DRG) neurons. Therefore we tested the hypothesis that mClca3 is involved in arthritic pain perception. In mClca3 knock-out mice and wild-type control mice, AIA was induced and measures of inflammation and pain were assessed. In the very acute phase of AIA, joint swelling was reduced in mClca3 knock-out mice. This effect disappeared during the course of AIA. We could not show significant differences in mechanical hyperalgesia between both groups of mice, neither at the acute nor at the chronic stage (21 days of AIA). Additional experiments on thermal hyperalgesia in wild-type and mClca3 knock-out mice in the first 3 days of AIA did not show a difference either. In addition, niflumic acid, an antagonist at CaCCs, did not significantly influence hyperalgesia during AIA. Thus, we were not able to provide evidence for a role of CaCCs, and in particular of mClca3, on the expression of arthritis or inflammation-evoked hyperalgesia. © 2014 Published by Elsevier Ireland Ltd.
Calcium-activated chloride channels (CaCCs) are a group of ubiquitously expressed ion channels. One important role is their
∗ Corresponding author at: Institute of Physiology I/Neurophysiology, Jena University Hospital – Friedrich Schiller University Jena, Teichgraben 8, D-07743 Jena, Germany. Tel.: +49 3641 938810; fax: +49 3641 938812. E-mail addresses: [email protected] (M. Ebbinghaus), [email protected] (M. Gajda), [email protected] (M.J. Holtzman), [email protected] (S. Schulz), [email protected] (H.-G. Schaible).
impact in neuronal excitability [1]. They are expressed in spinal cord neurons, autonomic neurons and dorsal root ganglion (DRG) neurons but the functions of CaCCs here remain poorly understood [2–5]. One member of the CaCC family (anoctamin 1) was recently identified to act directly as a heat sensor in mice [6]. CLCA proteins are a family of proteins which are associated with CaCCs. They are thought to act as chloride channel accessory molecules which can modify the currents through CaCCs [7]. In mouse medium-sized DRG neurons, calcium-activated chloride currents were reported, and subsequently transcripts of mClca1 and mClca5 were detected [2,8]. Using microarray analysis we recently detected mClca3 in the DRGs. This protein is the murine
http://dx.doi.org/10.1016/j.neulet.2014.05.051 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: M. Ebbinghaus, et al., Does chloride channel accessory 3 have a role in arthritis pain? A study on murine antigen-induced arthritis, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.051
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homolog for human CLCA1, also known as gob-5 [9]. In mice, mClca3 plays a critical role in allergen-induced airway hyperreactivity, goblet cell metaplasia and the regulation of tissue inflammation in the innate immune response [10–12]. Interestingly, we found a 1724-fold up-regulation of the mClca3 gene in the lumbar DRGs in the acute inflammatory phase of murine antigeninduced arthritis (AIA) [13]. Chloride (Cl− ) is in the focus of pain research. It was shown that the effect of GABA in the spinal cord depends on the intracellular Cl− concentration which is regulated by Cl− transporters such as NKCC1. If the intracellular Cl− concentration is elevated, GABA may cause rather an efflux than an influx of Cl− , and this changes the effect of GABA from inhibition to excitation. Also in DRG neurons changes in the Cl− regulation were shown to be important in nociceptive processing [14–16]. In vivo, NKCC1 knock-out mice show increased latencies to withdrawal from heat and a reduction in allodynia at the paws [17,18]. In DRG neurons, the chloride gradient is different from that in most other types of neurons such that calcium-activated Cl− current can be excitatory and that these channels are pronociceptive [1]. Consistent with this view the knockdown of anoctamin 1 (see above) reduced thermal pain [6]. It was suggested that inhibition of CaCCs may be a potential strategy for the development of analgesis [1]. Because we observed an upregulation of mClca3 in the DRGs of mice with AIA [13], we tested the hypothesis that mClca3 is involved in inflammatory pain perception in murine AIA. We used mClca3 knock-out mice and wild-type (WT) control mice, induced AIA and assessed inflammation and pain.
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2. Materials and methods
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2.1. Animals
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Homozygous mClca3−/− mice were generated and kindly provided by Prof. M.J. Holtzman (Washington University School of Medicine, St. Louis, MI, USA) [9]. Female knock-out and WT C57BL/6J control mice (9–13 weeks) were bred and genotyped by the Animal Facility of the University Hospital Jena. All animal studies were approved by the local government commission for animal protection (No. 02-019/12).
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2.2. Arthritis induction and pharmacological treatment
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Mice were immunized twice at 21 and 14 days before AIA induction with subcutaneous injection of 100 g of methylated bovine serum albumin (mBSA) (Sigma–Aldrich, Taufkirchen, Germany), emulgated with 50 l of complete Freund’s adjuvant (CFA; Sigma–Aldrich), supplemented to 2 mg/ml Mycobacterium tuberculosis, strain H37Ra (Difco, Detroit, USA). Additionally, 5 × 108 heat-inactivated Bordetella pertussis germs (Chiron-Behring, Marburg, Germany) were applied intraperitoneally (i.p.). Monoarticular arthritis was induced by injection of 100 g mBSA in 25 l 0.9% NaCl into the right knee joint cavity on day 0. A flare-up reaction of inflammation was induced by a second intraarticular injection of 100 g mBSA in 25 l saline on day 42 after primary arthritis induction. In WT mice niflumic acid (NA), a potent blocker of CaCCs, was applied i.p. NA was dissolved in 0.4 M NaHCO3 in 5% glucose. Mice received either 10 mg or 30 mg NA/kg body weight one day before arthritis induction for 5 consecutive days as previously described [19].
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2.3. Assessment of AIA
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Swelling at the knee was assessed by measuring the mediolateral joint diameter using an Oditest vernier caliper (Kroeplin, Schlüchtern, Germany). For histopathological examination knee
joints were removed, fixed in toto in 4.5% formalin, decalcified in EDTA, embedded in paraffin and cut into 3 m frontal sections which were stained with hematoxylin–eosin. The pathologist who scored arthritis was unaware of the experimental groups. Acute inflammation (infiltration of the synovial membrane by granulocytes and exudation of granulocytes into the joint space) was scored 0–3: 0 = no, 1 = mild, 2 = moderate, and 3 = severe changes (+1 if fibrin exudation in the joint space). Chronic inflammation (hyperplasia of synovial lining cells, infiltration of the synovial membrane by mononuclear cells, fibrosis of the synovial membrane and the periarticular tissue) was also scored 0–3. Cartilage surface defects with cell necrosis were scored 0–4: 0 = no damage, 1 = 50% of the cartilage surface affected. Damage to bone was also evaluated: 0 = no, 1 = mild, 2 = medium, and 3 = severe damage (extensive area of deep invasive destruction of bone). 2.4. Pain-related behavior Mechanical hyperalgesia at the hindpaws as an indicator of secondary hyperalgesia remote from the inflamed knee joint was assessed before and until 21 days after induction of AIA. Mice were placed into the testing device, and after accommodation to the environment the pain threshold was determined. Two testings before arthritis induction defined the baseline (BL). For mechanical hyperalgesia a dynamic plantar aesthesiometer (Ugo Basile, Comerio, Italy) was used which applied increasing pressure (stimulus increase rate 1 g/s; cut-off value 10 g) to the paw. The latency of the elicited leg withdrawal which reflects the respective mechanical threshold was averaged from three consecutive stimuli. Data are given in gram as alteration related to BL (d(x)–BL). Gait abnormalities of the ipsilateral hindlimb were quantified in the guarding score: 0 = normal walking, 1 = slight limping, 2 = persistent severe limping (still touching floor), 3 = severe limping with partial guarding of ipsilateral hindlimb (sometimes not touching floor), 4 = mainly guarding of ipsilateral hindlimb (most times not touching floor), and 5 = no walking at all. In other groups of wild-type and mClca3−/− mice thermal hyperalgesia was assessed in the first 3 days of AIA using the Hargreaves plantar test (Ugo Basile) [20]. Two consecutive standardized heat stimuli were applied to the paw for evaluation of a mean latency (cut-off value 20 s). 2.5. Statistical analysis Data are expressed as mean ± SEM. Differences between groups were calculated using the two-tailed Student’s t-test for unpaired values. Arthritis score, guarding score and mechanical and thermal thresholds of experimental groups were compared using analysis of variance (ANOVA). Changes in responses within groups (baseline versus after AIA induction) were analyzed using Wilcoxon’s matched pairs signed rank test. Statistical significance was calculated with the SPSS software package (v.16.0, Chicago, USA) and accepted for p < 0.05.
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3.1. Slightly decreased severity of AIA in mClca3−/− mice
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After induction of AIA, joint swelling in the acute phase of AIA was significantly attenuated in mClca3−/− mice compared to control WT mice (ANOVA day 3: F [1,25] = 5.441; p = 0.028) (Fig. 1A). This difference between both groups of mice disappeared in the chronic phase of AIA from day 7 on. The second injection of mBSA at day 42 of AIA evoked a strong flare-up reaction in both WT and
Please cite this article in press as: M. Ebbinghaus, et al., Does chloride channel accessory 3 have a role in arthritis pain? A study on murine antigen-induced arthritis, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.051
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Fig. 1. Inflammation and pain-related behavior in acute AIA. (A) In mClca3−/− mice a significant reduction of joint swelling was observed on day 1 compared to C57BL/6J wild-type controls (n = 11/16 per group). (B) Histopathological examination of the inflamed knee joint at day 3 of flare-up AIA showed a significant difference only in the score for chronic inflammation (n = 7/8 per group). (C) The latency of mechanical withdrawal thresholds at the ipsilateral paw is given in gram as alteration related to the baseline (BL) (d(x)–BL). No difference was observed between mClca3−/− and wild-type mice in acute AIA (n = 14/15 per group). (D) No significant difference in the guarding score could be seen between both groups of mice (n = 9/7 per group). Values are mean ± SEM. *p < 0.05 for differences between groups at that time point.
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mClca3−/− but the swelling was not different in these experimental groups (data not shown). The histopathological arthritis score showed a significant difference between mClca3−/− and wild-type mice only for chronic inflammation parameters on day 3 of flare-up AIA (Fig. 1B). The total score for inflammation and destruction was only slightly decreased in mClca3−/− mice.
3.3. Pharmacological inhibition of CaCC did not alter AIA
3.2. Unchanged pain-related behavior in mClca3−/− mice
Although specific antagonists for CaCCs are lacking, niflumic acid (NA) is considered to be a potent blocker for CaCCs in mouse models [19]. Treatment of WT mice with NA in two concentrations did not alter joint swelling in the course of AIA, neither at 10 nor at 30 mg/kg, compared to untreated AIA controls (ANOVA day 7: F [2,27] = 1.896; p = 0.170) (Fig. 3A). On day 3 of AIA, both NAtreated (30 mg/kg) mice and untreated control mice showed similar
In the acute phase of AIA both mClca3−/− and WT mice showed a significant reduction of mechanical threshold for withdrawal at the ipsilateral paw compared to the baseline, indicating mechanical hyperalgesia (day 3: mClca3−/− p = 0.002; wild-type p = 0.004). No significant difference in the value of mechanical hyperalgesia could be observed in the two groups of mice (ANOVA day 7: F [1,27] = 0.203; p = 0.656) (Fig. 1C). The reduction of threshold disappeared in both groups in the chronic phase of AIA from day 7 on. In line with mechanical hyperalgesia, mClca3−/− and wild-type mice showed protective behavior (guarding) on the inflamed hind paw in acute AIA but the scores of both experimental groups did not differ significantly (ANOVA day 7: F [1,12] = 2.228; p = 0.161) (Fig. 1D). In other groups of wild-type and mClca3−/− mice we tested thermal hyperalgesia in the first 3 days of AIA. A reduction of thermal threshold for withdrawal at the ipsilateral paw was seen in both groups of mice (day 1: mClca3−/− p = 0.012; day 3: wild-type p = 0.028) (Fig. 2). No significant difference between the two groups was detected (ANOVA day 3: F [1,12] = 0.474; p = 0.504).
Fig. 2. Thermal hyperalgesia in acute AIA. In C57BL/6J control mice, mClca3−/− mice and niflumic acid (NA)-treated mice a significant reduction in the latency of thermal withdrawal thresholds at the ipsilateral paw was seen in the acute phase of AIA. Values are mean ± SEM (n = 6–9 per group). *p < 0.05 for changes within groups (baseline versus acute AIA).
Please cite this article in press as: M. Ebbinghaus, et al., Does chloride channel accessory 3 have a role in arthritis pain? A study on murine antigen-induced arthritis, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.051
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Fig. 3. Acute AIA after treatment with niflumic acid (NA) in wild-type mice. After pharmacological inhibition of CaCCs, no significant differences in joint swelling (A), mechanical withdrawal thresholds at the ipsilateral paw (B) and in the guarding score (C) could be observed between NA-treated and untreated control mice. BL = baseline. Values are mean ± SEM (n = 9–10 per group).
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mechanical hyperalgesia at the ipsilateral paw when compared to the mechanical threshold for withdrawal at the contralateral paw (NA-treated mice: p = 0.012; control mice: p = 0.004). Between both groups no significant difference was observed for comparison of the threshold of the ipsilateral paw (ANOVA day 7: F [1,16] = 0.094; p = 0.763) (Fig. 3B). The value of guarding also did not differ either in both groups in the course of AIA (ANOVA day 7: F [1,15] = 1.302; p = 0.272) (Fig. 3C). NA-treated and control mice also showed a significant reduction of thermal threshold for withdrawal at the ipsilateral paw at day 3 of AIA compared to the baseline (NA-treated mice p = 0.015; control mice p = 0.028) (Fig. 2, columns on the right). At this time point in AIA no difference between both groups was detected (ANOVA day 7: F [1,13] = 0.202; p = 0.660).
The present study addressed the hypothesis that mClca3 knock-out mice and WT mice differ in the expression of inflammation-evoked hyperalgesia during antigen-induced arthritis (AIA). However, the development and magnitude of mechanical hyperalgesia was similar in mClca3 knock-out mice and WT mice throughout AIA. Thermal hyperalgesia, tested in the first 3 days of AIA, was also similar in mClca3 knock-out mice and WT mice. Only joint swelling was slightly reduced in mClca3 knock-out mice in the very acute phase of AIA. The CaCC blocker niflumic acid did not significantly alter mechanical or thermal hyperalgesia either. Thus, we did not find any evidence that mClca3 is critically involved in arthritic pain although we found a substantial upregulation of the mClCa3 gene in DRGs in the acute inflammatory phase [13]. The slight reduction of swelling in the acute phase of AIA was significant. These data support a role of mClca3 in inflammatory processes. However, in the swelling during the flare-up reaction upon the second injection of mBSA into the knee joint, which is significantly stronger than the initial swelling, such a difference between mClca3 knock-out and WT mice was no longer observed. We exclude, therefore, that mClca3 has an important role in the expression of arthritis. The assessment of behavior was focused on the measurement of mechanical threshold and gait analysis because arthritis produces mainly mechanical hyperalgesia [21,22]. In these tests, we could not find any significant difference between mClca3 knock-out and WT mice suggesting that mechanical hyperalgesia does not depend on mClca3. It should be noted that mechanical hyperalgesia in AIA is significantly reduced by neutralizing interleukin-17 [23,24], by somatostatin [25] and endothelin receptor antagonists [13] showing that AIA is a model suitable for the testing of mechanical hyperalgesia. We did not test for thermal hyperalgesia beyond 3 days of AIA. During this acute phase the reduction of thermal threshold was similar in WT and mClca3 knock-out mice. Although we cannot exclude that thermal hyperalgesia is different in WT and mClca3 knock-out mice at later stages of AIA we consider this unlikely because the time course of mechanical and thermal hyperalgesia in mouse AIA was found to be parallel [13]. In a more general approach we aimed to test whether CaCCs are at all critically involved in arthritic pain, and therefore we treated arthritic WT mice with niflumic acid. This compound is considered a potent blocker for CaCCs in mouse models [19]. However, we found no influence of niflumic acid on AIA and mechanical and thermal hyperalgesia. Thus we could not identify an important role of CaCCs in arthritis and inflammation-evoked hyperalgesia. The negative effect on a putative role of mClca3 in the present study does not exclude that CaCCs play a role in other models of pain, either in acute models of arthritis or in models of cutaneous and visceral pain; e.g. Liu et al. described a role of CaCC in bradykinin-induced pain [26]. At this stage we do not know the relative importance of bradykinin in AIA pain, compared to other mediators.
5. Conclusions Although we found a significant upregulation of the mClca3 gene in the DRGs of mice with AIA [13] we were not able to demonstrate an important consequence on the expression of arthritis and hyperalgesia which could be attributed to mClca3. The lack of effect of knocking-out mClca3 and of the CaCC blocker niflumic acid lead us to conclude that neither mClca3 nor CaCCs significantly contribute to arthritic pain.
Please cite this article in press as: M. Ebbinghaus, et al., Does chloride channel accessory 3 have a role in arthritis pain? A study on murine antigen-induced arthritis, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.051
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Acknowledgements The authors thank Konstanze Ernst and Cornelia Hüttich for excellent technical assistance.
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