Neutralization of Interleukin (IL)-6-signaling by antibodies is considered a promising tool for the treatment of osteoarthritis (OA). To gain further insight into this potential treatment, this study investigated the effects of IL-6-signaling and IL-6 neutralization on chondrocyte metabolism and the release of IL-6-signaling-related mediators by human chondrocytes. Chondrocytes were collected from 49 patients with advanced knee/hip OA or femoral neck fracture. Isolated chondrocytes were stimulated with different mediators to analyze the release of IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130). The effect of IL-6 and IL-6/sIL-6R complex as well as neutralization of IL-6-signaling on the metabolism was analyzed. OA chondrocytes showed high basal IL-6 production and release, which was strongly negatively correlated with the production of cartilage-matrix-proteins. Chondrocytes produced and released sIL-6R and sgp130. The IL-6/sIL-6R complex significantly increased nitric oxide, prostaglandin E₂ and matrix metalloproteinase 1 production, decreased Pro-Collagen Type II and mitochondrial ATP production, and increased glycolysis in OA chondrocytes. Neutralization of IL-6-signaling by antibodies did not significantly affect the metabolism of OA chondrocytes, but blocking of glycoprotein 130 (gp130)-signaling by SC144 significantly reduced the basal IL-6 release. Although IL-6 trans-signaling induced by IL-6/sIL-6R complex negatively affects OA chondrocytes, antibodies against IL-6 or IL-6R did not affect chondrocyte metabolism. Since inhibition of gp130-signaling reduced the enhanced basal release of IL-6, interfering with gp130-signaling may ameliorate OA progression because high cellular release of IL-6 correlates with reduced production of cartilage-matrix-proteins.

Diseases of joints are among the most frequent causes of chronic pain. In the course of joint diseases, the peripheral and the central nociceptive system develop persistent hyperexcitability (peripheral and central sensitization). This review addresses the mechanisms of spinal sensitization evoked by arthritis. Electrophysiological recordings in anesthetized rats from spinal cord neurons with knee input in a model of acute arthritis showed that acute spinal sensitization is dependent on spinal glutamate receptors (AMPA, NMDA, and metabotropic glutamate receptors) and supported by spinal actions of neuropeptides such as neurokinins and CGRP, by prostaglandins, and by proinflammatory cytokines. In several chronic arthritis models (including immune‐mediated arthritis and osteoarthritis) spinal glia activation was observed to be coincident with behavioral mechanical hyperalgesia which was attenuated or prevented by intrathecal application of minocycline, fluorocitrate, and pentoxyfylline. Some studies identified specific pathways of micro‐ and astroglia activation such as the purinoceptor‐ (P₂X₇‐) cathepsin S/CX₃CR1 pathway, the mobility group box‐1 protein (HMGB1), and toll‐like receptor 4 (TLR4) activation, spinal NFκB/p65 activation and others. The spinal cytokines TNF, interleukin‐6, interleukin‐1β, and others form a functional spinal network characterized by an interaction between neurons and glia cells which is required for spinal sensitization. Neutralization of spinal cytokines by intrathecal interventions attenuates mechanical hyperalgesia. This effect may in part result from local suppression of spinal sensitization and in part from efferent effects which attenuate the inflammatory process in the joint. In summary, arthritis evokes significant spinal hyperexcitability which is likely to contribute to the phenotype of arthritis pain in patients. image

Interleukin (IL)-1β is an important pro-inflammatory cytokine in the progression of osteoarthritis (OA), which impairs mitochondrial function and induces the production of nitric oxide (NO) in chondrocytes. The aim was to investigate if blockade of NO production prevents IL-1β-induced mitochondrial dysfunction in chondrocytes and whether cAMP and AMP-activated protein kinase (AMPK) affects NO production and mitochondrial function. Isolated human OA chondrocytes were stimulated with IL-1β in combination with/without forskolin, L-NIL, AMPK activator or inhibitor. The release of NO, IL-6, PGE₂, MMP3, and the expression of iNOS were measured by ELISA or Western blot. Parameters of mitochondrial respiration were measured using a seahorse analyzer. IL-1β significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1β-induced NO release and mitochondrial dysfunction but not IL-1β-induced release of IL-6, PGE₂, and MMP3. Enhancement of cAMP by forskolin reduced IL-1β-induced NO release and prevented IL-1β-induced mitochondrial impairment. Activation of AMPK increased IL-1β-induced NO production and the negative impact of IL-1β on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1β-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1β-induced NO release and mitochondrial dysfunction.

Background and Purpose: Prostaglandin E₂ is considered a major mediator of inflammatory pain, by acting on neuronal G_s protein‐coupled EP2 and EP4 receptors. However, the neuronal EP3 receptor, colocalized with EP2 and EP4 receptor, is G_i 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 PI3Kγ^{kinase 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.

In tumor cells, interleukin-6 (IL-6) signaling can lead to activation of the epidermal growth factor receptor (EGFR), which prolongs Stat3 activation. In the present experiments, we tested the hypothesis that IL-6 signaling activates EGFR signaling in peripheral and spinal nociception and examined whether EGFR localization and activation coincide with pain-related behaviors in arthritis. In vivo in anesthetized rats, spinal application of the EGFR receptor blocker gefitinib reduced the responses of spinal cord neurons to noxious joint stimulation, but only after spinal pretreatment with IL-6 and soluble IL-6 receptor. Using Western blots, we found that IL-6-induced Stat3 activation was reduced by gefitinib in microglial cells of the BV2 cell line, but not in cultured DRG neurons. Immunohistochemistry showed EGFR localization in most DRG neurons from normal rats, but significant downregulation in the acute and most painful arthritis phase. In the spinal cord of mice, EGFR was highly activated mainly in the chronic phase of inflammation, with localization in neurons. These data suggest that spinal IL-6 signaling may activate spinal EGFR signaling. Downregulation of EGFR in DRG neurons in acute arthritis may limit nociception, but pronounced delayed activation of EGFR in the spinal cord may be involved in chronic inflammatory pain.