Prostaglandin EP3 receptor activation is antinociceptive in sensory neurons via PI3Kγ, AMPK and GRK2.

BACKGROUND AND PURPOSE: Prostaglandin E2 is considered a major mediator of inflammatory pain, by acting on neuronal Gs protein-coupled EP2 and EP4 receptors. However, the neuronal EP3 receptor, colocalized with EP2 and EP4 receptor, is Gi protein-coupled and antagonizes the pronociceptive prostaglandin E2 effect. Here, we investigated the cellular signalling mechanisms by which the EP3 receptor reduces EP2 and EP4 receptor-evoked pronociceptive effects in sensory neurons. EXPERIMENTAL APPROACH: Experiments were performed on isolated and cultured dorsal root ganglion (DRG) neurons from wild type, phosphoinositide 3-kinase γ (PI3Kγ)-/- , and PI3Kγkinase dead (KD)/KD mice. For subtype-specific stimulations, we used specific EP2, EP3, and EP4 receptor agonists from ONO Pharmaceuticals. As a functional readout, we recorded TTX-resistant sodium currents in patch-clamp experiments. Western blots were used to investigate the activation of intracellular signalling pathways. EP4 receptor internalization was measured using immunocytochemistry. KEY RESULTS: Different pathways mediate the inhibition of EP2 and EP4 receptor-dependent pronociceptive effects by EP3 receptor stimulation. Inhibition of EP2 receptor-evoked pronociceptive effect critically depends on the kinase-independent function of the signalling protein PI3Kγ, and adenosine monophosphate activated protein kinase (AMPK) is involved. By contrast, inhibition of EP4 receptor-evoked pronociceptive effect is independent on PI3Kγ and mediated through activation of G protein-coupled receptor kinase 2 (GRK2), which enhances the internalization of the EP4 receptor after ligand binding. CONCLUSION AND IMPLICATIONS: Activation of neuronal PI3Kγ, AMPK, and GRK2 by EP3 receptor activation limits cAMP-dependent pain generation by prostaglandin E2 . These new insights hold the potential for a novel approach in pain therapy.

Spinal interleukin-1ß induces mechanical spinal hyperexcitability in rats: Interactions and redundancies with TNF and IL-6.

Both spinal tumor necrosis factor (TNF) and interleukin-6 (IL-6) contribute to the development of "mechanical" spinal hyperexcitability in inflammatory pain states. Recently, we found that spinal sensitization by TNF was significantly reduced by blockade of spinal IL-6 signaling suggesting that IL-6 signaling is involved in spinal TNF effects. Here, we explored whether spinal interleukin-1ß (IL-1ß), also implicated in inflammatory pain, induces "mechanical" spinal hyperexcitability, and whether spinal IL-1ß effects are related to TNF and IL-6 effects. We recorded the responses of spinal cord neurons to mechanical stimulation of the knee joint in vivo and used cellular approaches on microglial and astroglial cell lines to identify interactions of IL-1ß, TNF, and IL-6. Spinal application of IL-1ß in anesthetized rats modestly enhanced responses of spinal cord neurons to innocuous and noxious mechanical joint stimulation. This effect was blocked by minocycline indicating microglia involvement, and significantly attenuated by interfering with IL-6 signaling. In the BV2 microglial cell line, IL-1ß, like TNF, enhanced the release of soluble IL-6 receptor, necessary for spinal IL-6 actions. Different to TNF, IL-1ß caused SNB-19 astrocytes to release interleukin-11. The generation of "mechanical" spinal hyperexcitability by IL-1ß was more pronounced upon spinal TNF neutralization with etanercept, suggesting that concomitant TNF limits IL-1ß effects. In BV2 cells, TNF stimulated the release of IL-1Ra, an endogenous IL-1ß antagonist. Thus, spinal IL-1ß has the potential to induce spinal hyperexcitability sharing with TNF dependency on IL-6 signaling, but TNF also limited IL-1ß effects explaining the modest effect of IL-1ß.

Oncostatin M induces hyperalgesic priming and amplifies signaling of cAMP to ERK by RapGEF2 and PKA.

Hyperalgesic priming is characterized by enhanced nociceptor sensitization by pronociceptive mediators, prototypically PGE2 . Priming has gained interest as a mechanism underlying the transition to chronic pain. Which stimuli induce priming and what cellular mechanisms are employed remains incompletely understood. In adult male rats, we present the cytokine Oncostatin M (OSM), a member of the IL-6 family, as an inducer of priming by a novel mechanism. We used a high content microscopy based approach to quantify the activation of endogenous PKA-II and ERK of thousands sensory neurons in culture. Incubation with OSM increased and prolonged ERK activation by agents that increase cAMP production such as PGE2 , forskolin, and cAMP analogs. These changes were specific to IB4/CaMKIIα positive neurons, required protein translation, and increased cAMP-to-ERK signaling. In both, control and OSM-treated neurons, cAMP/ERK signaling involved RapGEF2 and PKA but not Epac. Similar enhancement of cAMP-to-ERK signaling could be induced by GDNF, which acts mostly on IB4/CaMKIIα-positive neurons, but not by NGF, which acts mostly on IB4/CaMKIIα-negative neurons. In vitro, OSM pretreatment rendered baseline TTX-R currents ERK-dependent and switched forskolin-increased currents from partial to full ERK-dependence in small/medium sized neurons. In summary, priming induced by OSM uses a novel mechanism to enhance and prolong coupling of cAMP/PKA to ERK1/2 signaling without changing the overall pathway structure.

The analgesic potential of cytokine neutralization with biologicals.

Many acute and chronic inflammatory diseases, cancer and neuropathic disorders are accompanied by severe pain states reducing drastically the life quality of the patients. Biologicals which preferentially target cytokines often reduce the disease processes by influencing immune cells, tissue healing, inflammatory aspects and other typical processes of the diseases. Remarkably the effect of biologicals in pain and nociception is often neglected or insufficiently explored. However, because of the dense interaction of the nociceptive system with the surrounding peripheral or central tissue, targeting cytokines has the potential to treat pain at the same time as the other symptoms of the disease. The following review shows mainly results from animal experiments (with some parallels to human studies). It depicts where and how cytokines are involved in nociceptive processing and pain and also indicates possible target strategies. It concentrates on the excitatory cytokines IL-6, TNF-α, IL-1ß, IFN-γ and IL-17.

Involvement of Spinal IL-6 Trans-Signaling in the Induction of Hyperexcitability of Deep Dorsal Horn Neurons by Spinal Tumor Necrosis Factor-Alpha.

UNLABELLED: During peripheral inflammation, both spinal TNF-α and IL-6 are released within the spinal cord and support the generation of inflammation-evoked spinal hyperexcitability. However, whether spinal TNF-α and IL-6 act independently in parallel or in a functionally dependent manner has not been investigated. In extracellular recordings from mechanonociceptive deep dorsal horn neurons of normal rats in vivo, we found that spinal application of TNF-α increased spinal neuronal responses to mechanical stimulation of knee and ankle joints. This effect was significantly attenuated by either sgp130, which blocks IL-6 trans-signaling mediated by IL-6 and its soluble receptor IL-6R (sIL-6R); by an antibody to the IL-6 receptor; or by minocycline, which inhibits the microglia. IL-6 was localized in neurons of the spinal cord and, upon peripheral noxious stimulation in the presence of spinal TNF-α, IL-6 was released spinally. Furthermore, TNF-α recruited microglial cells to provide sIL-6R, which can form complexes with IL-6. Spinal application of IL-6 plus sIL-6R, but not of IL-6 alone, enhanced spinal hyperexcitability similar to TNF-α and the inhibition of TNF-α-induced hyperexcitability by minocycline was overcome by coadministration of sIL-6R, showing that sIL-6R is required. Neither minocycline nor the TNF-α-neutralizing compound etanercept inhibited the induction of hyperexcitability by IL-6 plus sIL-6R. Together, these data show that the induction of hyperexcitability of nociceptive deep dorsal horn neurons by TNF-α largely depends on the formation of IL-6/sIL-6R complexes that are downstream of TNF-α and requires the interactions of neurons and microglia orchestrated by TNF-α. SIGNIFICANCE STATEMENT: Both spinal TNF-α and IL-6 induce a state of spinal hyperexcitability. We present the novel finding that the full effect of TNF-α on the development of spinal hyperexcitability depends on IL-6 trans-signaling acting downstream of TNF-α. IL-6 trans-signaling requires the formation of complexes of IL-6 and soluble IL-6 receptor. Spinal TNF-α furthers the release of IL-6 from neurons in the spinal cord during peripheral noxious stimulation and recruits microglial cells to provide soluble IL-6 receptor, which can form complexes with IL-6. Therefore, a specific interaction between neurons and microglia is required for the full development of TNF-α-induced hyperexcitability of nociceptive deep horsal horn neurons.

Involvement of peripheral and spinal tumor necrosis factor α in spinal cord hyperexcitability during knee joint inflammation in rats.

OBJECTIVE: Tumor necrosis factor α (TNFα) is produced not only in peripheral tissues, but also in the spinal cord. The purpose of this study was to address the potential of peripheral and spinal TNFα to induce and maintain spinal hyperexcitability, which is a hallmark of pain states in the joints during rheumatoid arthritis and osteoarthritis. METHODS: In vivo recordings of the responses of spinal cord neurons to nociceptive knee input under normal conditions and in the presence of experimental knee joint inflammation were obtained in anesthetized rats. TNFα, etanercept, or antibodies to TNF receptors were applied to either the knee joint or the spinal cord surface. RESULTS: Injection of TNFα into the knee joint cavity increased the responses of spinal cord neurons to mechanical joint stimulation, and injection of etanercept into the knee joint reduced the inflammation-evoked spinal activity. These spinal effects closely mirrored the induction and reduction of peripheral sensitization. Responses to joint stimulation were also enhanced by spinal application of TNFα, and spinal application of either etanercept or anti-TNF receptor type I significantly attenuated the generation of inflammation-evoked spinal hyperexcitability, which is characterized by widespread pain sensitization beyond the inflamed joint. Spinally applied etanercept did not reduce established hyperexcitability in the acute kaolin/carrageenan model. In antigen-induced arthritis, etanercept decreased spinal responses on day 1, but not on day 3. CONCLUSION: While peripheral TNFα increases spinal responses to joint stimulation, spinal TNFα supports the generation of the full pattern of spinal hyperexcitability. However, established spinal hyperexcitability may be maintained by downstream mechanisms that are independent of spinal TNFα.

Spinal interleukin-6 is an amplifier of arthritic pain in the rat.

OBJECTIVE: Significant joint pain is usually widespread beyond the affected joint, which results from the sensitization of nociceptive neurons in the central nervous system (central sensitization). This study was undertaken to explore whether the proinflammatory cytokine interleukin-6 (IL-6) in the joint induces central sensitization, whether joint inflammation causes the release of IL-6 from the spinal cord, and whether spinal IL-6 contributes to central sensitization. METHODS: In anesthetized rats, electrophysiologic recordings of spinal cord neurons with sensory input from the knee joint were made. Neuronal responses to mechanical stimulation of the rat knee and leg were monitored. IL-6 and soluble IL-6 receptor (sIL-6R) were applied to the knee joint or the spinal cord. Spinal release of IL-6 was measured by enzyme-linked immunosorbent assay. Soluble gp130, which neutralizes IL-6/sIL-6R, was spinally applied during the development of joint inflammation or during established inflammation. RESULTS: A single injection of IL-6/sIL-6R into the rat knee joint as well as application of IL-6/sIL-6R to the rat spinal cord significantly increased the responses of spinal neurons to mechanical stimulation of the knee and ankle joint, i.e., induced central sensitization. Application of soluble gp130 to the rat spinal cord attenuated this effect of IL-6. The development of knee inflammation in the rat caused spinal release of IL-6. Spinal application of soluble gp130 attenuated the development of inflammation-evoked central sensitization but did not reverse it. CONCLUSION: Our findings indicate that the generation of joint pain in the rat involves not only IL-6 in the joint but also IL-6 released from the spinal cord. Spinal IL-6 contributes to central sensitization and thus promotes the widespread hyperalgesia observed in the course of joint disease.

Nociceptive neurons in the rat caudal trigeminal nucleus respond to blood plasma perfusion of the subarachnoid space: the involvement of complement.

The meninges of the brain are innervated by afferent nerve fibres containing SP and CGRP, two typical peptides found in sensory neurons. These fibres project to the trigeminal nuclear complex and the cervical dorsal horn. Discharge of the afferents may provide a physiological basis for some types of headaches. Considerable speculation surrounds the possible causes of meningeal afferent activation. Blood-borne substances released during subarachnoid haemorrhage are one possibility and there is a possibility that these also play a role in migraine. In the case of migraine, blood components, e.g. from platelets, cannot be excluded. To investigate the possible effects of platelets and plasma factors, the subarachnoid space of the rat was continuously perfused with artificial cerebrospinal fluid during extracellular recordings from single units of the caudal trigeminal nucleus. Washed and concentrated suspensions of adenosindiphosphate (ADP)--activated platelets and plasma, from which platelets had been removed--were introduced as a bolus into the continuous flow. Neurons in the caudal nucleus of the trigeminal complex receiving input from the meninges were stimulated. They did not respond to the activated platelet suspensions but showed intense responses to plasma. Plasma completely lost its ability to excite trigeminal neurons after heat inactivation (30 min, 56 degrees C). It is concluded that the complement system may be involved in the excitatory nociceptive effect of platelet-poor plasma.