Antibody-induced pain-like behavior and bone erosion: links to subclinical inflammation, osteoclast activity, and acid-sensing ion channel 3-dependent sensitization.

ABSTRACT: Several bone conditions, eg, bone cancer, osteoporosis, and rheumatoid arthritis (RA), are associated with a risk of developing persistent pain. Increased osteoclast activity is often the hallmark of these bony pathologies and not only leads to bone remodeling but is also a source of pronociceptive factors that sensitize the bone-innervating nociceptors. Although historically bone loss in RA has been believed to be a consequence of inflammation, both bone erosion and pain can occur years before the symptom onset. Here, we have addressed the disconnection between inflammation, pain, and bone erosion by using a combination of 2 monoclonal antibodies isolated from B cells of patients with RA. We have found that mice injected with B02/B09 monoclonal antibodies (mAbs) developed a long-lasting mechanical hypersensitivity that was accompanied by bone erosion in the absence of joint edema or synovitis. Intriguingly, we have noted a lack of analgesic effect of naproxen and a moderate elevation of few inflammatory factors in the ankle joints suggesting that B02/B09-induced pain-like behavior does not depend on inflammatory processes. By contrast, we found that inhibiting osteoclast activity and acid-sensing ion channel 3 signaling prevented the development of B02/B09-mediated mechanical hypersensitivity. Moreover, we have identified secretory phospholipase A2 and lysophosphatidylcholine 16:0 as critical components of B02/B09-induced pain-like behavior and shown that treatment with a secretory phospholipase A2 inhibitor reversed B02/B09-induced mechanical hypersensitivity and bone erosion. Taken together, our study suggests a potential link between bone erosion and pain in a state of subclinical inflammation and offers a step forward in understanding the mechanisms of bone pain in diseases such as RA.

Identification of a Neuronal Receptor Controlling Anaphylaxis.

Allergic reactions can in severe cases induce a state of circulatory shock referred to as anaphylaxis. Histamine, the primary mediator of this condition, is released from immune cells, and, therefore, anaphylaxis has so far been considered an immune system disorder. However, we here show that the glutamatergic receptor mGluR7, expressed on a subpopulation of both peripheral and spinal cord neurons, controls histamine-induced communication through calcium-dependent autoinhibition with implications for anaphylaxis. Genetic ablation of mGluR7, and thus altered regulation of histamine-sensing neurons, caused an anaphylaxis-like state in mGluR7(-/-) mice, which could be reversed by antagonizing signaling between neurons and mast cells but not by antagonizing a central itch pathway. Our findings demonstrate the vital role of nervous system control by mGluR7 in anaphylaxis and open up possibilities for preventive strategies for this life-threatening condition.

Autoantibodies to citrullinated proteins induce joint pain independent of inflammation via a chemokine-dependent mechanism.

OBJECTIVE: An interesting and so far unexplained feature of chronic pain in autoimmune disease is the frequent disconnect between pain and inflammation. This is illustrated well in rheumatoid arthritis (RA) where pain in joints (arthralgia) may precede joint inflammation and persist even after successful anti-inflammatory treatment. In the present study, we have addressed the possibility that autoantibodies against citrullinated proteins (ACPA), present in RA, may be directly responsible for the induction of pain, independent of inflammation. METHODS: Antibodies purified from human patients with RA, healthy donors and murinised monoclonal ACPA were injected into mice. Pain-like behaviour was monitored for up to 28 days, and tissues were analysed for signs of pathology. Mouse osteoclasts were cultured and stimulated with antibodies, and supernatants analysed for release of factors. Mice were treated with CXCR1/2 (interleukin (IL) 8 receptor) antagonist reparixin. RESULTS: Mice injected with either human or murinised ACPA developed long-lasting pronounced pain-like behaviour in the absence of inflammation, while non-ACPA IgG from patients with RA or control monoclonal IgG were without pronociceptive effect. This effect was coupled to ACPA-mediated activation of osteoclasts and release of the nociceptive chemokine CXCL1 (analogue to human IL-8). ACPA-induced pain-like behaviour was reversed with reparixin. CONCLUSIONS: The data suggest that CXCL1/IL-8, released from osteoclasts in an autoantibody-dependent manner, produces pain by activating sensory neurons. The identification of this new pain pathway may open new avenues for pain treatment in RA and also in other painful diseases associated with autoantibody production and/or osteoclast activation.

VGLUT2 controls heat and punctuate hyperalgesia associated with nerve injury via TRPV1-Cre primary afferents.

Nerve injury induces a state of prolonged thermal and mechanical hypersensitivity in the innervated area, causing distress in affected individuals. Nerve injury-induced hypersensitivity is partially due to increased activity and thereby sustained release of neurotransmitters from the injured fibers. Glutamate, a prominent neurotransmitter in primary afferents, plays a major role in development of hypersensitivity. Glutamate is packed in vesicles by vesicular glutamate transporters (VGLUTs) to enable controlled release upon depolarization. While a role for peripheral VGLUTs in nerve injury-induced pain is established, their contribution in specific peripheral neuronal populations is unresolved. We investigated the role of VGLUT2, expressed by transient receptor potential vanilloid (TRPV1) fibers, in nerve injury-induced hypersensitivity. Our data shows that removal of Vglut2 from Trpv1-Cre neurons using transgenic mice abolished both heat and punctuate hyperalgesia associated with nerve injury. In contrast, the development of cold hypersensitivity after nerve injury was unaltered. Here, we show that, VGLUT2-mediated glutamatergic transmission from Trpv1-Cre neurons selectively mediates heat and mechanical hypersensitivity associated with nerve injury. Our data clarifies the role of the Trpv1-Cre population and the dependence of VGLUT2-mediated glutamatergic transmission in nerve injury-induced hyperalgesia.

Multimodal use of calcitonin gene-related peptide and substance P in itch and acute pain uncovered by the elimination of vesicular glutamate transporter 2 from transient receptor potential cation channel subfamily V member 1 neurons.

Primary afferents are known to use glutamate as their principal fast neurotransmitter. However, it has become increasingly clear that peptides have an influential role in both mediating and modulating sensory transmission. Here we describe the transmission accounting for different acute pain states and itch transmitted via the transient receptor potential cation channel subfamily V member 1 (TRPV1) population by either ablating Trpv1-Cre-expressing neurons or inducing vesicular glutamate transporter 2 (VGLUT2) deficiency in Trpv1-Cre-expressing neurons. Furthermore, by pharmacological inhibition of substance P or calcitonin gene-related peptide (CGRP) signaling in Vglut2-deficient mice, we evaluated the contribution of substance P or CGRP to these sensory modulations, with or without the presence of VGLUT2-mediated glutamatergic transmission in Trpv1-Cre neurons. This examination, together with c-Fos analyses, showed that glutamate via VGLUT2 in the Trpv1-Cre population together with substance P mediate acute cold pain, whereas glutamate together with CGRP mediate noxious heat. Moreover, we demonstrate that glutamate together with both substance P and CGRP mediate tissue-injury associated pain. We further show that itch, regulated by the VGLUT2-mediated transmission via the Trpv1-Cre population, depends on CGRP and gastrin-releasing peptide receptor (GRPR) transmission because pharmacological blockade of the CGRP or GRPR pathway, or genetic ablation of Grpr, led to a drastically attenuated itch. Our study reveals how different neurotransmitters combined can cooperate with each other to transmit or regulate various acute sensations, including itch.

Glutamate, substance P, and calcitonin gene-related peptide cooperate in inflammation-induced heat hyperalgesia.

The transient receptor potential cation channel subfamily V member 1 (TRPV1) is known as a thermosensor and integrator of inflammation-induced hyperalgesia. TRPV1 is expressed in a subpopulation of primary afferent neurons that express several different neurotransmitters. The role of the TRPV1 channel in the development of hyperalgesia is established, but the role of the neurotransmitter glutamate, used partially by the same neuronal population and thus probably mediating the response, is still under investigation. We have used a Trpv1-Cre mouse line in which we either ablated Trpv1-Cre expressing neurons or induced vesicular glutamate transporter 2 (Vglut2) deficiency in Trpv1-Cre expressing neurons and investigated specific states of hyperalgesia after persistent inflammation. Furthermore, by pharmacologic inhibition of substance P (SP) or calcitonin gene-related peptide (CGRP) signaling in Vglut2-deficient mice, we also evaluated the contribution of SP or CGRP to inflammation-induced hyperalgesia, with or without the presence of vesicular glutamate transporter 2 (VGLUT2)-mediated glutamatergic transmission in Trpv1-Cre neurons. This examination, together with c-Fos analyses, showed that VGLUT2-mediated glutamatergic transmission in Trpv1-Cre afferents together with SP or CGRP is essential for the development of the heat hyperalgesia associated with persistent inflammation. Additionally, SP-, CGRP-, and VGLUT2-mediated transmission together were found to play a role in the development of mechanical hyperalgesia after persistent inflammation.

VGLUT2-dependent glutamatergic transmission in primary afferents is required for intact nociception in both acute and persistent pain modalities.

Glutamate is an essential transmitter in pain pathways. However, its broad usage in the central and peripheral nervous system prevents us from designing efficient glutamate-based pain therapies without causing harmful side effects. The discovery of vesicular glutamate transporters (VGLUT1-3) has been a crucial step in describing specific glutamatergic neuronal subpopulations and glutamate-dependent pain pathways. To assess the role of VGLUT2-mediated glutamatergic contribution to pain transmission from the entire primary sensory population, we crossed our Vglut2(f/f) line with the Ht-Pa-Cre line. Such Vglut2-deficient mice showed significantly decreased, but not completely absent, acute nociceptive responses. The animals were less prone to develop an inflammatory-related state of pain and were, in the partial sciatic nerve ligation chronic pain model, much less hypersensitive to mechanical stimuli and did not develop cold allodynia or heat hyperalgesia. To take advantage of this neuropathic pain-resistant model, we analyzed Vglut2-dependent transcriptional changes in the dorsal spinal cord after nerve injury, which revealed several novel candidate target genes potentially relevant for the development of neuropathic pain therapeutics. Taken together, we conclude that VGLUT2 is a major mediator of nociception in primary afferents, implying that glutamate is the key somatosensory neurotransmitter.

A sensory subpopulation depends on vesicular glutamate transporter 2 for mechanical pain, and together with substance P, inflammatory pain.

Ablating or functionally compromising sets of sensory neurons has provided important insights into peripheral modality-specific wiring in the somatosensory system. Inflammatory hyperalgesia, cold pain, and noxious mechanosensation have all been shown to depend upon Na(v)1.8-positive sensory neurons. The release of fast-acting neurotransmitters, such as glutamate, and more slowly released neuropeptides, such as substance P (SP), contribute to the diversified responses to external stimuli. Here we show that deleting Vglut2 in Na(v)1.8(Cre)-positive neurons compromised mechanical pain and NGF-induced thermal hyperalgesia, whereas tactile-evoked sensation, thermal, formalin-evoked, and chronic neuropathic pain were normal. However, when Vglut2(f/f);Na(v)1.8(Cre) mice were injected with a SP antagonist before the formalin test, the second phase pain response was nearly completely abolished, whereas in control mice, the pain response was unaffected. Our results suggest that VGLUT2-dependent signaling originating from Na(v)1.8-positive neurons is a principal sensing mechanism for mechanical pain and, together with SP, inflammatory pain. These data define sets of primary afferents associated with specific modalities and provide useful genetic tools with which to analyze the pathways that are activated by functionally distinct neuronal populations and transmitters.

VGLUT2-dependent sensory neurons in the TRPV1 population regulate pain and itch.

The natural response to itch sensation is to scratch, which relieves the itch through an unknown mechanism. Interaction between pain and itch has been frequently demonstrated, and the selectivity hypothesis of itch, based on data from electrophysiological and behavioral experiments, postulates the existence of primary pain afferents capable of repressing itch. Here, we demonstrate that deletion of vesicular glutamate transporter (VGLUT) 2 in a subpopulation of neurons partly overlapping with the vanilloid receptor (TRPV1) primary afferents resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. The increased itch behavior was reduced by administration of antihistaminergic drugs and by genetic deletion of the gastrin-releasing peptide receptor, demonstrating a dependence on VGLUT2 to maintain normal levels of both histaminergic and nonhistaminergic itch. This study establishes that VGLUT2 is a major player in TRPV1 thermal nociception and also serves to regulate a normal itch response.

Effect of co-administration of fluoxetine and amantadine on immunoendocrine parameters in rats subjected to a forced swimming test.

Considerable attention has been paid to a possible role of immunological dysregulation in the pathogenesis of depression. It has been reported that combined administration of antidepressant drugs and the non-competitive NMDA receptor antagonist amantadine reduces immobility time in the forced swimming test (FST). Moreover, preliminary clinical data show that such a combination of drugs has a beneficial effect on treatment-resistant depressed patients. Since immune activation and a pro-inflammatory response are clearly evident in treatment-resistant depression, the aim of the present study was to examine the effect of a combination of the antidepressant fluoxetine and amantadine on immunoendocrine parameters in rats subjected to the forced swimming test. The obtained results revealed synergistic antidepressant effects of the combined administration of fluoxetine (10 mg/kg) and amantadine (10 mg/kg) - drugs otherwise ineffective when given separately in the above doses. Antidepressant activity was accompanied with a significant decrease in the capacity of splenocytes to proliferate in response to concanavalin A. Moerover, fluoxetine and the combination of amantadine and fluoxetine reduced relative spleen weight in rats subjected to the FST, compared to rats treated with the vehicle. The combination of amantadine and fluoxetine enhanced the production of the negative immunoregulator interleukin-10 (but not interferon-gamma) in rats subjected to the FST. The exposure to the FST produced an increase in plasma corticosterone levels, which was significantly attenuated by pretreatment with fluoxetine and amantadine. In summary, the antidepressive efficacy of a combination of fluoxetine and amantadine given in suboptimal doses may be related to the negative immunoendocrine effects of these drugs.