Cartilage-binding antibodies induce pain through immune complex-mediated activation of neurons.

Rheumatoid arthritis-associated joint pain is frequently observed independent of disease activity, suggesting unidentified pain mechanisms. We demonstrate that antibodies binding to cartilage, specific for collagen type II (CII) or cartilage oligomeric matrix protein (COMP), elicit mechanical hypersensitivity in mice, uncoupled from visual, histological and molecular indications of inflammation. Cartilage antibody-induced pain-like behavior does not depend on complement activation or joint inflammation, but instead on tissue antigen recognition and local immune complex (IC) formation. smFISH and IHC suggest that neuronal Fcgr1 and Fcgr2b mRNA are transported to peripheral ends of primary afferents. CII-ICs directly activate cultured WT but not FcRγ chain-deficient DRG neurons. In line with this observation, CII-IC does not induce mechanical hypersensitivity in FcRγ chain-deficient mice. Furthermore, injection of CII antibodies does not generate pain-like behavior in FcRγ chain-deficient mice or mice lacking activating FcγRs in neurons. In summary, this study defines functional coupling between autoantibodies and pain transmission that may facilitate the development of new disease-relevant pain therapeutics.

Increased levels of IL-6 in the cerebrospinal fluid of patients with chronic schizophrenia--significance for activation of the kynurenine pathway.

BACKGROUND: Accumulating evidence indicates that schizophrenia is associated with brain immune activation. While a number of reports suggest increased cytokine levels in patients with schizophrenia, many of these studies have been limited by their focus on peripheral cytokines or confounded by various antipsychotic treatments. Here, well-characterized patients with schizophrenia, all receiving olanzapine treatment, and healthy volunteers were analyzed with regard to cerebrospinal fluid (CSF) levels of cytokines. We correlated the CSF cytokine levels to previously analyzed metabolites of the kynurenine (KYN) pathway. METHODS: We analyzed the CSF from patients and controls using electrochemiluminescence detection with regard to cytokines. Cell culture media from human cortical astrocytes were analyzed for KYN and kynurenic acid (KYNA) using high-pressure liquid chromatography or liquid chromatography/mass spectrometry. RESULTS: We included 23 patients and 37 controls in our study. Patients with schizophrenia had increased CSF levels of interleukin (IL)-6 compared with healthy volunteers. In patients, we also observed a positive correlation between IL-6 and the tryptophan:KYNA ratio, indicating that IL-6 activates the KYN pathway. In line with this, application of IL-6 to cultured human astrocytes increased cell medium concentration of KYNA. LIMITATIONS: The CSF samples had been frozen and thawed twice before analysis of cytokines. Median age differed between patients and controls. When appropriate, all present analyses were adjusted for age. CONCLUSION: We have shown that IL-6, KYN and KYNA are elevated in patients with chronic schizophrenia, strengthening the idea of brain immune activation in patients with this disease. Our concurrent cell culture and clinical findings suggest that IL-6 induces the KYN pathway, leading to increased production of the N-methyl-D-aspartate receptor antagonist KYNA in patients with schizophrenia.

Influence of model and matrix on cytokine profile in rat and human.

OBJECTIVE: Emerging evidence indicates that low-grade inflammation is part of the clinical picture of OA and that there is a need to identify soluble biomarkers of ongoing inflammation in the joint from a translational aspect. The aim of this study was to compare levels of pro-inflammatory biomarkers in SF, serum and/or EDTA plasma. METHODS: SF and blood from rats subjected to Freund's complete adjuvant (FCA; n = 48) or monoiodoacetate (MIA; n = 88) monoarthritis and from control rats were collected over time. SF, EDTA plasma and serum were obtained from six individuals with OA of the knee and healthy controls. Levels of IL-6, KC/GRO, IL-8, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 3α (MIP-3α), IL-1ß, TNF and l(+)-lactate were assessed either by immune assay or by a colorimetric method. RESULTS: Elevated levels of biomarkers were shown in monoarthritic animals in SF compared with the control groups, although with considerably lower magnitude in the MIA groups, which also indicated a biphasic pattern. Levels of KC/GRO and MIP-3α in serum from the FCA model and IL-6 in the MIA model followed the pattern of SF. In serum samples from OA individuals, MIP-3α correlated significantly with levels in SF. CONCLUSION: While we found increased levels of markers in joint fluid and blood, no single systemic biochemical biomarkers that were a common denominator between the animal models and the patient material could be identified. Our data indicate that it is critical to delineate the temporal profile of multiple local and systemic factors in order to pinpoint soluble biomarkers for OA.

Spinal HMGB1 induces TLR4-mediated long-lasting hypersensitivity and glial activation and regulates pain-like behavior in experimental arthritis.

Extracellular high mobility group box-1 protein (HMGB1) plays important roles in the pathogenesis of nerve injury- and cancer-induced pain. However, the involvement of spinal HMGB1 in arthritis-induced pain has not been examined previously and is the focus of this study. Immunohistochemistry showed that HMGB1 is expressed in neurons and glial cells in the spinal cord. Subsequent to induction of collagen antibody-induced arthritis (CAIA), Hmgb1 mRNA and extranuclear protein levels were significantly increased in the lumbar spinal cord. Intrathecal (i.t.) injection of a neutralizing anti-HMGB1 monoclonal antibody or recombinant HMGB1 box A peptide (Abox), which each prevent extracellular HMGB1 activities, reversed CAIA-induced mechanical hypersensitivity. This occurred during ongoing joint inflammation as well as during the postinflammatory phase, indicating that spinal HMGB1 has an important function in nociception persisting beyond episodes of joint inflammation. Importantly, only HMGB1 in its partially oxidized isoform (disulfide HMGB1), which activates toll-like receptor 4 (TLR4), but not in its fully reduced or fully oxidized isoforms, evoked mechanical hypersensitivity upon i.t. injection. Interestingly, although both male and female mice developed mechanical hypersensitivity in response to i.t. HMGB1, female mice recovered faster. Furthermore, the pro-nociceptive effect of i.t. injection of HMGB1 persisted in Tlr2- and Rage-, but was absent in Tlr4-deficient mice. The same pattern was observed for HMGB1-induced spinal microglia and astrocyte activation and cytokine induction. These results demonstrate that spinal HMGB1 contributes to nociceptive signal transmission via activation of TLR4 and point to disulfide HMGB1 inhibition as a potential therapeutic strategy in treatment of chronic inflammatory pain.

Spinal actions of lipoxin A4 and 17(R)-resolvin D1 attenuate inflammation-induced mechanical hypersensitivity and spinal TNF release.

Lipoxins and resolvins have anti-inflammatory and pro-resolving actions and accumulating evidence indicates that these lipid mediators also attenuate pain-like behavior in a number of experimental models of inflammation and tissue injury-induced pain. The present study was undertaken to assess if spinal administration of lipoxin A4 (LXA4) or 17 (R)-resolvin D1 (17(R)-RvD1) attenuates mechanical hypersensitivity in the carrageenan model of peripheral inflammation in the rat. Given the emerging role of spinal cytokines in the generation and maintenance of inflammatory pain we measured cytokine levels in the cerebrospinal fluid (CSF) after LXA4 or 17(R)-RvD1 administration, and the ability of these lipid metabolites to prevent stimuli-induced release of cytokines from cultured primary spinal astrocytes. We found that intrathecal bolus injection of LXA4 and17(R)-RvD1 attenuated inflammation-induced mechanical hypersensitivity without reducing the local inflammation. Furthermore, both LXA4 and 17(R)-RvD1 reduced carrageenan-induced tumor necrosis factor (TNF) release in the CSF, while only 17(R)-RvD1attenuated LPS and IFN-γ-induced TNF release in astrocyte cell culture. In conclusion, this study demonstrates that lipoxins and resolvins potently suppress inflammation-induced mechanical hypersensitivity, possibly by attenuating cytokine release from spinal astrocytes. The inhibitory effect of lipoxins and resolvins on spinal nociceptive processing puts them in an intriguing position in the search for novel pain therapeutics.

Pentoxifylline and propentofylline prevent proliferation and activation of the mammalian target of rapamycin and mitogen activated protein kinase in cultured spinal astrocytes.

Astrocyte activation is an important feature in many disorders of the central nervous system, including chronic pain conditions. Activation of astrocytes is characterized by a change in morphology, including hypertrophy and increased size of processes, proliferation, and an increased production of proinflammatory mediators. The xanthine derivatives pentoxifylline and propentofylline are commonly used experimentally as glial inhibitors. These compounds are generally believed to attenuate glial activity by raising cyclic AMP (cAMP) levels and inhibiting glial tumor necrosis factor (TNF) production. In the present study, we show that these substances inhibit TNF and serum-induced astrocyte proliferation and signaling through the mammalian target of rapamycin (mTOR) pathway, demonstrated by decreased levels of phosphorylated S6 kinase (S6K), commonly used as a marker of mTOR complex (mTORC) activation. Furthermore, we show that pentoxifylline and propentofylline also inhibit JNK and p38, but not ERK, activation induced by TNF. In addition, the JNK antagonist SP600125, but not the p38 inhibitor SB203580, prevents TNF-induced activation of S6 kinase, suggesting that pentoxifylline and propentofylline may regulate mTORC activity in spinal astrocytes partially through inhibition of the JNK pathway. Our results suggest that pentoxifylline and propentofylline inhibit astrocyte activity in a broad fashion by attenuating flux through specific pathways.