Elsevier

Bone

Volume 39, Issue 5, November 2006, Pages 1107-1115
Bone

Osteoclasts play a part in pain due to the inflammation adjacent to bone

https://doi.org/10.1016/j.bone.2006.04.033Get rights and content

Abstract

Bone disorders with increased osteoclastic bone resorption are frequently associated with bone pain and inhibitors of osteoclasts reduce bone pain. Osteoclasts degrade bone minerals by secreting protons through the vacuolar H+-ATPase, creating acidic microenvironments. Because acidosis is a well-known cause of pain, we reasoned that osteoclasts cause pain through proton secretion. We explored this using an animal model in which a single subcutaneous injection of the complete Freund's adjuvant (CFA) in the hind-paw caused inflammatory hyperalgesia (hyper-responsiveness to noxious stimuli). Osteoclastic bone resorption was increased in the metatarsal bones in the CFA-injected hind-paws. CFA-induced hyperalgesia was significantly suppressed by the bisphosphonates, zoledronic acid (ZOL) and alendronate and osteoprotegerin. c-src-deficient mice in which osteoclasts are inherently dysfunctional exhibited reduced CFA-induced hyperalgesia. Repeated subcutaneous injections of parathyroid hormone-related protein into the hind-paw also induced hyperalgesia with increased osteoclastic bone resorption. The hyperalgesia was associated with increased mRNA expression of acid-sensing ion channel (ASIC) 1a, 1b and 3 in the ipsi-lateral dorsal root ganglions (DRGs) by RT-PCR and c-Fos in the ipsi-lateral spinal dorsal horn by immunohistochemistry. Of note, ZOL decreased the ASIC1a mRNA expression and c-Fos. Treatment of the DRG cell line F-11 with acid (pH5.5) increased ASIC1a, 1b and 3 mRNA expression and nuclear c-Fos expression. The ASIC blocker amiloride inhibited acid-induced c-Fos expression in F-11 cells. Moreover, F-11 cells transfected with the transient receptor potential channel vanilloid subfamily member 1 (TRPV1) showed increased acid-induced nuclear c-Fos expression compared with parental F-11 cells. Finally, bafilomycin A1, an inhibitor of the vacuolar H+-ATPase, reversed the hyperalgesia and down-regulated ASIC1a mRNA expression in the DRGs. These results led us to propose that osteoclasts play a part in CFA-induced inflammatory pain through an activation of the acid-sensing receptors including ASICs and TRPV1 by creating acidosis.

Introduction

Bone disorders with increased osteoclastic bone resorption such as metastatic bone diseases [1], Paget's disease of bone [2], osteoporosis [3], fibrous dysplasia [4] and osteogenesis imperfecta [5] are frequently associated with bone pain. Of note, bisphosphonates (BPs), potent and specific inhibitors of osteoclasts, have been shown to reduce bone pain complicated with these bone disorders [1], [2], [3], [4], [5]. BPs also diminish pain in rheumatoid arthritis [6] in which osteoclastic bone resorption is secondary to the neighboring synovial inflammation [7]. In addition, other types of inhibitors of osteoclasts including calcitonin [8], mithramycin [9] and gallium nitrate [10] that have different mechanism of action from BP are also shown to decrease bone pain. These results collectively led us to hypothesize that osteoclasts play a role in causing bone pain in the pathological conditions associated with increased bone resorption/destruction.

Here, we attempted to prove this notion and study the underlying mechanism using an animal model that has been widely used to study inflammatory pain and advanced our understanding of the mechanism of pain over the last 2 decades. A single bolus injection of the complete Freund's adjuvant (CFA) into the plantar surface of the unilateral hind-paw induced an intense and persistent inflammation characterized by the edema, redness and hyperalgesia (hyper-responsiveness to noxious stimuli) that appear within 2 h, peak at 6–24 h and last for approximately 2 weeks only on the CFA-injected hind-paw [11], [12]. These symptoms are in part attributable to increased production of pro-inflammatory cytokines such as inteleukin-1, interleukin-6 and tumor necrosis factor-α following CFA-injection [13], [14]. Of note, these pro-inflammatory cytokines are potent stimulators of osteoclastic bone resorption [15]. It therefore seems likely that increased osteoclastic bone resorption as a consequence of CFA-induced inflammatory reactions contributes to the CFA-induced hyperalgesia. However, the role of osteoclasts in the CFA-induced hyperalgesia is unknown.

Osteoclasts degrade bone minerals by secreting protons through the vacuolar H+-ATPase, making neighboring microenvironments acidic [15], [16]. Acid is a well-known cause of pain [17], [18], [19]. Recently, the two main classes of acid-sensing nociceptors, the acid-sensing ion channels (ASICs) and the transient receptor potential channel vanilloid subfamily member 1 (TRPV1), are found to be expressed in the sensory neurons innervating bone [20]. ASICs and TRPV1 elicit pain signals upon activation by acid [17], [18], [19], [21]. We reasoned that the acidic microenvironment created by bone-resorbing osteoclasts activates these acid-sensing nociceptors, causing bone pain. To examine this, we investigated the expression and activation of ASICs and TRPV1 in the primary sensory neurons in the dorsal root ganglions (DRGs). Activation of these acid-sensing receptors was evaluated by examining nuclear c-Fos expression in the sensory neurons in the spinal dorsal horn. Earlier studies showed that c-Fos expression in the spinal dorsal horn was up-regulated following CFA injection into the hind-paw [22], [23]. Elevated nuclear c-Fos expression in the spinal dorsal horn in response to noxious stimuli has been used as a molecular indicator of neural activation [24], [25], [26]. Subsequently, we attempted to study the activation of these acid-sensing nociceptors by acid using the F-11 rat DRG cells in culture [27], [28]. Finally, we examined the effects of bafilomycin A1, an inhibitor of the vacuolar H+-ATPase [16], [29], on the CFA-induced hyperalgesia in this model.

Our results are consistent with the notion that the acidosis created by osteoclasts plays a part in causing inflammatory pain adjacent to bone through an activation of acid-sensing receptors including ASICs and TRPV1.

Section snippets

Reagents

ZOL (2-(imidazol-1-yl)-1-hydroxyethylidene-1,1-bisphosphonate) and ALN (4-amino-1-hydroxybutylidene bisphosphonate) were provided as dehydrated disodium or monosodium salt by Novartis Pharma AG (Basel, Switzerland) and Merck and Co., Inc. (Rahway, NJ), respectively. Recombinant human OPG (rhOPG) was a generous gift from Amgen Inc. (Thousand Oaks, CA). Synthetic human parathyroid hormone-related protein (PTH-rP), bafilomycin A1 and amiloride hydrochloride hydrate were purchased from Peptide

CFA-induced osteoclastic bone resorption and hyperalgesia

As previously described [12], inflammation and swelling became evident in the CFA-injected ipsi-lateral hind-paws and the volume of the inflamed paw was apparently larger than that of the non-CFA-injected contra-lateral paw 24 h after the injection (day 1) (data not shown). Of note, histological and histomorphometrical examinations showed that an injection of CFA stimulated bone resorption with increased TRAP-positive osteoclasts in the metatarsal bones (Fig. 1). Hyperalgesia determined by the

Discussion

Here, we conducted in vivo and in vitro studies to obtain experimental evidence for the involvement of osteoclasts in the pathophysiology of pain due to the inflammation adjacent to bone and to determine the role of acidosis created by osteoclasts in causing pain.

Using a well-characterized animal model [11], [12], [31], we verified that CFA caused inflammatory pain. In addition, our data show that CFA increased osteoclastic bone resorption at the injected site. Of note, ZOL, ALN and OPG, of

Acknowledgments

This study was supported in part by Grants in aid 14771020, 16791123 (to T.H.), 12137205 and 21st century Center of Excellence (COE) program (to T.Y.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, Senri Life Science Foundation Research Grant (to T.H.) and Osaka Cancer Society Research Grant (to T.H.).

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