Importance of IL-6 trans-signaling and high autocrine IL-6 production in human osteoarthritic chondrocyte metabolism.

OBJECTIVE: 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. DESIGN: 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. RESULTS: 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. CONCLUSION: 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.

Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1ß-Induced Mitochondrial Dysfunction in Human Chondrocytes.

Interleukin (IL)-1ß 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-1ß-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-1ß 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-1ß significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1ß-induced NO release and mitochondrial dysfunction but not IL-1ß-induced release of IL-6, PGE2, and MMP3. Enhancement of cAMP by forskolin reduced IL-1ß-induced NO release and prevented IL-1ß-induced mitochondrial impairment. Activation of AMPK increased IL-1ß-induced NO production and the negative impact of IL-1ß on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1ß-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1ß-induced NO release and mitochondrial dysfunction.

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.

Neuronal prostaglandin E2 receptor subtype EP3 mediates antinociception during inflammation.

The pain mediator prostaglandin E2 (PGE2) sensitizes nociceptive pathways through EP2 and EP4 receptors, which are coupled to Gs proteins and increase cAMP. However, PGE2 also activates EP3 receptors, and the major signaling pathway of the EP3 receptor splice variants uses inhibition of cAMP synthesis via Gi proteins. This opposite effect raises the intriguing question of whether the Gi-protein-coupled EP3 receptor may counteract the EP2 and EP4 receptor-mediated pronociceptive effects of PGE2. We found extensive localization of the EP3 receptor in primary sensory neurons and the spinal cord. The selective activation of the EP3 receptor at these sites did not sensitize nociceptive neurons in healthy animals. In contrast, it produced profound analgesia and reduced responses of peripheral and spinal nociceptive neurons to noxious stimuli but only when the joint was inflamed. In isolated dorsal root ganglion neurons, EP3 receptor activation counteracted the sensitizing effect of PGE2, and stimulation of excitatory EP receptors promoted the expression of membrane-associated inhibitory EP3 receptor. We propose, therefore, that the EP3 receptor provides endogenous pain control and that selective activation of EP3 receptors may be a unique approach to reverse inflammatory pain. Importantly, we identified the EP3 receptor in the joint nerves of patients with painful osteoarthritis.

Tumor necrosis factor reduces the amplitude of rat cortical spreading depression in vivo.

OBJECTIVE: Brain damage and ischemia often trigger cortical spreading depression (CSD), which aggravates brain damage. The proinflammatory cytokine tumor necrosis factor (TNF) is significantly upregulated during brain damage, but it is unknown whether TNF influences spreading depression in cerebral cortex in vivo. This question is important because TNF not only furthers inflammatory reactions but might also be neuroprotective. Here we tested the hypothesis that TNF affects CSD, and we explored the direction in which CSD is modified by TNF. METHODS: CSD, elicited by pressure microinjection of KCl, was recorded in anesthetized rats and mice. TNF was administered locally into a trough, providing local TNF treatment of a cortical area. For further analysis, antibodies to TNF receptor (TNFR) 1 or 2 were applied, or CSD was monitored in TNFR1 and TNFR2 knockout mice. γ-Aminobutyric acid (GABA)A receptors were blocked by bicuculline. Immunohistochemistry localized the cortical expression of TNFR1 and TNFR2. RESULTS: Local application of TNF to the cortex reduced dose-dependently the amplitude of CSD. This effect was prevented by blockade or knockout of TNFR2 but not by blockade or knockout of TNFR1. TNFR2 was localized at cortical neurons including parvalbumin-positive inhibitory interneurons, and blockade of GABAA receptors by bicuculline prevented the reduction of CSD amplitudes by TNF. INTERPRETATION: We identified a functional link between TNF and CSD. TNF activates TNFR2 in cortical inhibitory interneurons. The resulting release of GABA reduces CSD amplitudes. In this manner, TNF might be neuroprotective in pathological conditions.

Galanin diminishes cortical spreading depolarization across rodents - A candidate for treatment?

Galanin (Gal) is a neuropeptide with the potential to ameliorate cortical spreading depolarization (CSD), an electrophysiological phenomenon occurring after brain injury or in migraine aura. Gal is expressed in all cortical neurons both in rat and in mouse cortices. Here we investigated whether the effect of Gal on CSD previously described in the rat is conserved in the mouse cortex. In rats, the topical application of Gal to the cortex for 1 h did not induce any change in CSD amplitudes, propagation velocity, or threshold of elicitation. Rather, topical application of Gal for 3 h was necessary to obtain a significant decrease in these CSD parameters and to develop a remarkable increase in the KCl threshold to elicit a CSD in rat cortex. In contrast, the topical application of Gal on cortical surface for 1 h in mice was sufficient to significantly attenuate CSD amplitudes and increase threshold. A thinner cortex, a faster diffusion or different affinity/expression of receptors for Gal are possible reasons to explain this difference in the time course between rats and mice. Our data are relevant to postulate Gal as a potential target for inhibition of CSD under pathological situations such as stroke or ischemia. SIGNIFICANCE STATEMENT: The neuropeptide Galanin (Gal) is expressed in all neurons throughout the cerebral cortex, both in rats and mice, and is able to reduce or even inhibit Cortical Spreading Depolarization, thus, Gal has the potential to control neuronal excitability that may identify Gal as a target in drug development against CSD.

Neuropathological changes in the TASTPM mouse model of Alzheimer's disease and their relation to hyperexcitability and cortical spreading depolarization.

Although Alzheimer's disease (AD) is characterized by distinct pathological changes, their precise impact on cortical functions are not well understood. Here we used TASTPM mice as an AD model and asked whether the development of neurodegenerative changes has an impact on the extracellular space (ECS) and neuronal excitability, in particular cortical spreading depolarization (CSD) which requires intact neuron and glial functions. We studied wildtype (WT) and TASTPM mice (3, 6, and 12 months old). TASTPM mice showed progressive proliferation of neocortical Amyloid-beta (Aß) plaques between 3 and 12 months (more deposits in females than in males) and Aß accumulation in cortical vessels. As plaques proliferated, neuroinflammatory microglial reaction (CD68, CD39 and Galectin-3) and astrogliosis (GFAP) developed progressively. The cortical ECS volume shrank significantly to about half the size of the WT. CSD in both WT and TASTPM mice showed considerable heterogeneity but did not correlate with the histological changes. However, CSDs were easier to elicit in TASTPM than in WT mice at 3 months, and also compared to older TASTPM mice. Moreover, TASTPM mice showed more hyperexcitability manifested as clonic-tonic behavior after sodium thiopental anesthesia. Thus, AD pathology was associated with abnormal hyperexcitability but did not homogenously alter CSD susceptibility.

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.

Cortical spreading depolarization is a potential target for rat brain excitability modulation by Galanin.

The inhibitory neuropeptide Galanin (Gal) has been shown to mediate anticonvulsion and neuroprotection. Here we investigated whether Gal affects cortical spreading depolarization (CSD). CSD is considered the pathophysiological neuronal mechanism of migraine aura, and a neuronal mechanism aggravating brain damage upon afflictions of the brain. Immunohistochemistry localized Gal and the Gal receptors 1-3 (GalR1-3) in native rat cortex and evaluated microglial morphology after exposure to Gal. In anesthetized rats, Gal was applied alone and together with the GalR antagonists M40, M871, or SNAP 37889 locally to the exposed cortex. The spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection were measured. In rat cortex, Gal was present in all neurons of all cortical layers, but not in astrocytes, microglia and vessels. GalR2 and GalR3 were expressed throughout all neurons, whereas GalR1 was preponderantly located at neurons in layers IV and V, but only in about half of the neurons. In susceptible rats, topical application of Gal on cortex decreased CSD amplitude, slowed CSD propagation velocity, and increased the threshold for KCl to ignite CSD. In some rats, washout of previously applied Gal induced periods of epileptiform patterns in the electrocorticogram. Blockade of GalR2 by M871 robustly prevented all Gal effects on CSD, whereas blockade of GalR1 or GalR3 was less effective. Although microglia did not express GalRs, topical application of Gal changed microglial morphology indicating microglial activation. This effect of Gal on microglia was prevented by blocking neuronal GalR2. In conclusion, Gal has the potential to ameliorate CSD thus reducing pathophysiological neuronal events caused by or associated with CSD.

Molecular effects of interleukin-1ß on dorsal root ganglion neurons: prevention of ligand-induced internalization of the bradykinin 2 receptor and downregulation of G protein-coupled receptor kinase 2.

In dorsal root ganglion sections numerous small-to medium-sized neurons were found to exhibit extensive colocalization of the bradykinin receptor 2, the interleukin-1 receptor 1 and G protein-coupled receptor kinase 2. Application of bradykinin to cultured DRG neurons caused substantial internalization of the bradykinin 2 receptor which significantly reduced the responsiveness of DRG neurons to a second application of bradykinin. Such an internalization was not observed in DRG neurons which were exposed to long-term pretreatment with interleukin-1ß. The long-term incubation with interleukin-1ß on its own did neither change the proportion of neurons which expressed the bradykinin 2 receptor in the cytoplasma nor the proportion of neurons expressing the bradykinin 2 receptor in the membrane but it reduced the proportion of neurons expressing G protein-coupled receptor kinase 2, an enzyme which facilitates the internalization of G protein-coupled receptors. These results show that interleukin-1ß maintains the responsiveness of DRG neurons to bradykinin in the long-term range, and they suggest that the downregulation of G protein-coupled receptor kinase 2 could be a cellular mechanism involved in this interleukin-1ß effect.

Osteoarthritis-Induced Metabolic Alterations of Human Hip Chondrocytes.

Osteoarthritis (OA) alters chondrocyte metabolism and mitochondrial biology. We explored whether OA and non-OA chondrocytes show persistent differences in metabolism and mitochondrial function and different responsiveness to cytokines and cAMP modulators. Hip chondrocytes from patients with OA or femoral neck fracture (non-OA) were stimulated with IL-1ß, TNF, forskolin and opioid peptides. Mediators released from chondrocytes were measured, and mitochondrial functions and glycolysis were determined (Seahorse Analyzer). Unstimulated OA chondrocytes exhibited significantly higher release of IL-6, PGE2 and MMP1 and lower production of glycosaminoglycan than non-OA chondrocytes. Oxygen consumption rates (OCR) and mitochondrial ATP production were comparable in unstimulated non-OA and OA chondrocytes, although the non-mitochondrial OCR was higher in OA chondrocytes. Compared to OA chondrocytes, non-OA chondrocytes showed stronger responses to IL-1ß/TNF stimulation, consisting of a larger decrease in mitochondrial ATP production and larger increases in non-mitochondrial OCR and NO production. Enhancement of cAMP by forskolin prevented IL-1ß-induced mitochondrial dysfunction in OA chondrocytes but not in non-OA chondrocytes. Endogenous opioids, present in OA joints, influenced neither cytokine-induced mitochondrial dysfunction nor NO upregulation. Glycolysis was not different in non-OA and OA chondrocytes, independent of stimulation. OA induces persistent metabolic alterations, but the results suggest upregulation of cellular mechanisms protecting mitochondrial function in OA.

From spreading depolarization to epilepsy with neuroinflammation: The role of CGRP in cortex.

CGRP release plays a major role in migraine pain by activating the trigeminal pain pathways. Here we explored putative additional effects of CGRP on cortical circuits and investigated whether CGRP affects cortical excitability, cortical spreading depolarization (CSD), a phenomenon associated with migraine aura, blood-brain-barrier (BBB) and microglial morphology. We used immunohistochemistry to localize CGRP and the CGRP receptor (CGRP-R) in native cortex and evaluated morphology of microglia and integrity of the BBB after exposure to CGRP. In anesthetized rats we applied CGRP and the CGRP-R antagonist BIBN4096BS locally to the exposed cortex and monitored the spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection. In mouse brain slices CGRP effects on neuronal activity were explored by multielectrode array. CGRP immunoreactivity was detectable in intracortical vessels, and all cortical neurons showed CGRP-R immunoreactivity. In rat cortex in vivo, topical CGRP induced periods of epileptiform discharges, however, also dose-dependently reduced CSD amplitudes and propagation velocity. BIBN4096BS prevented these effects. CGRP evoked synchronized bursting activity in mouse cortical but not in cerebellar slices. Topical application of CGRP to rat cortex induced plasma extravasation and this was associated with reduced ramification of microglial cells. From these findings we conclude that CGRP induces a pathophysiological state in the cortex, consisting in neuronal hyperexcitability and neuroinflammation. Thus, CGRP may have a pronounced impact on brain functions during migraine episodes supporting the benefit of CGRP antagonists for clinical use. However, increased cortical CGRP may end the CSD-induced aura phase of migraine.

Current knowledge on PB1-F2 of influenza A viruses.

Almost 10 years ago, an eleventh protein of influenza A viruses was discovered in a search for CD8+ T-cell epitopes. This protein was named PB1-F2 since it is encoded in the +1 reading frame of the PB1 gene segment. Various studies have shown that PB1-F2 has a pleiotropic effect: (1) The protein can induce apoptosis in a cell type-dependent manner, (2) PB1-F2 is able to promote inflammation, and (3) finally it up-regulates viral polymerase activity by its interaction with the PB1 subunit. These properties could contribute to an enhanced pathogenicity. However, the underlying mechanism is not fully understood yet. New data suggest that some effects of PB1-F2 are strain-specific and host-specific.

Impact of Diabetes Mellitus on Knee Osteoarthritis Pain and Physical and Mental Status: Data From the Osteoarthritis Initiative.

OBJECTIVE: Diabetes mellitus (DM) appears to increase osteoarthritic knee pain, which may be related to greater adiposity and more advanced disease status often observed in individuals with osteoarthritis (OA) and DM. We aimed to assess whether OA knee pain and health status are worse in individuals with OA and DM, independent of these potential confounders. METHODS: We included 202 OA participants with DM and 2,279 without DM from the Osteoarthritis Initiative. Knee pain was evaluated using the Knee Injury and Osteoarthritis Outcome Score (KOOS) and a numeric rating scale (NRS). Physical and mental status were assessed by the Medical Outcomes Study Short Form 12 (SF-12) questionnaire, physical component summary (PCS) score and mental component summary (MCS) score, and by the Center for Epidemiologic Studies Depression Scale (CES-D). Linear regression models assessed the influence of DM, adjusted for age, sex, body mass index (BMI), and radiographic severity. RESULTS: OA participants with DM reported worse knee pain and greater physical and mental issues compared with participants without DM. Individuals with DM had worse KOOS pain (ß = -4.72 [95% confidence interval (95% CI) -7.22, -2.23]) and worse NRS pain (ß = 0.42 [95% CI 0.04, 0.80]) independent of BMI, OA severity, age, and sex. The negative influence of DM was also apparent for SF-12 PCS (ß = -3.49 [95% CI -4.73, -2.25]), SF-12 MCS (ß = -1.42 [95% CI -2.57, -0.26]), and CES-D (ß = 1.08 [95% CI 0.08, 2.08]). CONCLUSION: Individuals with knee OA experience on average higher pain intensity and a worse physical and mental health status if they have DM. Linear regression models show that DM is a risk factor for higher pain, in addition to and independent of greater BMI and radiographic OA severity.

Substance P and Alpha-Calcitonin Gene-Related Peptide Differentially Affect Human Osteoarthritic and Healthy Chondrocytes.

Osteoarthritis (OA) is a degenerative joint disease that not only causes cartilage loss but also structural damage in all joint tissues. Joints are innervated by alpha-calcitonin gene-related peptide (αCGRP) and substance P (SP)-positive sensory nerve fibers. Alteration of sensory joint innervation could be partly responsible for degenerative changes in joints that contribute to the development of OA. Therefore, our aim was to analyze and compare the molecular effects of SP and αCGRP on the metabolism of articular chondrocytes from OA patients and non-OA cartilage donors. We treated the cells with SP or αCGRP and analysed the influence of these neuropeptides on chondrocyte metabolism and modulation of signaling pathways. In chondrocytes from healthy cartilage, SP had minimal effects compared with its effects on OA chondrocytes, where it induced inflammatory mediators, inhibited chondrogenic markers and promoted apoptosis and senescence. Treatment with αCGRP also increased apoptosis and senescence and reduced chondrogenic marker expression in OA chondrocytes, but stimulated an anabolic and protective response in healthy chondrocytes. The catabolic influence of SP and αCGRP might be due to activation of ERK signaling that could be counteracted by an increased cAMP response. We suggest that a switch between the G-subunits of the corresponding receptors after binding their ligands SP or αCGRP plays a central role in mediating the observed effects of sensory neuropeptides on chondrocytes.

Pain-related behaviors associated with persistence of mechanical hyperalgesia after antigen-induced arthritis in rats.

Upon transient musculoskeletal diseases, some patients develop persistent pain while others recover from pain. Here, we studied whether such heterogeneity also occurs in rats after recovery from unilateral antigen-induced arthritis (AIA) in the knee joint, and which pain phenotype may predict the course of pain. Typically, inflammatory swelling lasts about 3 weeks. Pain-related behaviors were monitored for 84 days after AIA induction. Unbiased cluster analysis of intragroup differences at day 84 of AIA revealed that about one-third of the rats (cluster 1) showed persistent mechanical hyperalgesia at the injected knee joint, whereas the other rats (cluster 2) had recovered from pain. Retrograde analysis of pain-related behaviors revealed that cluster 1 rats exhibited more severe mechanical hyperalgesia at the injected knee from day 3 of AIA and mechanical hyperalgesia at the contralateral knee. Cluster 1 and 2 rats did not show different inflammatory swelling, secondary mechanical and thermal hyperalgesia at the ipsilateral hindpaw, guarding score, and asymmetry of weight bearing during AIA. Thus, in particular, early severe mechanical hyperalgesia in the inflamed joint and segmental contralateral mechanical hyperalgesia seem to be a risk factor for the development of persistent mechanical hyperalgesia pointing to the importance of spinal mechanisms. However, none of the rats showed an expression of ATF3 in dorsal root ganglion neurons, nor morphological spinal microglia activation thus not suggesting development of neuropathic pain. Both clusters showed a persistent upregulation of pCREB in dorsal root ganglion neurons, inversely correlated with mechanical hyperalgesia at the knee. The role of pCREB needs to be further explored.

Met receptor tyrosine kinase transactivation is involved in proteinase-activated receptor-2-mediated hepatocellular carcinoma cell invasion.

The expression of proteinase-activated receptor (PAR)(2) in human hepatocellular carcinoma (HCC) was established by reverse transcription-polymerase chain reaction, confocal immunofluorescence and electron microscopy in permanent cell lines, primary HCC cell cultures and HCC tumor tissue. Stimulation of HCC cells with trypsin and the PAR(2)-selective activating peptide, 2-furoyl-LIGRLO-NH(2), increased cell invasion across Matrigel. Both effects were blocked by a PAR(2)-selective pepducin antagonist peptide (pal-PAR(2)) and by PAR(2) silencing with specific small interfering RNA (siRNA). PAR(2)-initiated HCC cell invasion was also blocked by inhibiting the hepatocyte growth factor receptor (Met receptor tyrosine kinase) with the receptor-targeted kinase inhibitors, SU 11274 and PHA 665752, or by downregulation of Met with specific siRNA. The involvement of Met in PAR(2)-mediated HCC invasive signaling was further supported by the finding that treatment of HCC cells with trypsin or the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated Met activation-phosphorylation. In addition, Met-dependent stimulation of p42/p44 mitogen-activated protein Kinases was found to be critical for the PAR(2)-Met receptor tyrosine kinase-invasive signaling axis in HCC cells. Our study establishes an important link between the PAR(2) and Met receptor tyrosine kinase signaling in promoting HCC cell invasion.

Host cell cytokines induced by Chlamydia pneumoniae decrease the expression of interstitial collagens and fibronectin in fibroblasts.

Chlamydia pneumoniae infection has been associated with chronic obstructive airway disease (COPD), asthma, and atherosclerosis. Inflammation and airway remodeling in asthma and COPD result in subepithelial fibrosis that is characterized by the deposition of interstitial collagens and fibronectin. The progression of atherosclerosis is also accompanied by an increased production of interstitial collagens in the intima. As shown by reverse transcription-PCR and immunoblotting, infection of human fibroblasts and smooth muscle cells by C. pneumoniae TW-183 downregulated the expression of type I and III collagen and fibronectin, whereas the level of type IV collagen remained unchanged. Conditioned medium from infected fibroblasts as well as epithelial WISH cells also reduced the expression of interstitial collagens and fibronectin in uninfected cells. In experiments using blocking antibodies, beta interferon was found to contribute to the inhibitory effects of conditioned medium collected from infected fibroblasts. In contrast, downregulation of matrix protein expression by conditioned medium from epithelial cells was caused by interleukin-1alpha, which was not secreted from fibroblasts following chlamydial infection. C. pneumoniae-mediated inhibition of collagen and fibronectin expression was diminished following transfection of fibroblasts with specific small interfering RNA targeting the transcription factor CCAAT/enhancer-binding protein beta. The downregulation of interstitial collagens and fibronectin by the Chlamydia-induced host cell cytokine response may modulate tissue remodeling processes in airway diseases. In atherosclerosis the inhibition of collagen synthesis by C. pneumoniae infection may promote plaque vulnerability, thereby increasing the risk of plaque rupture.

Contribution of Inflammation and Bone Destruction to Pain in Arthritis: A Study in Murine Glucose-6-Phosphate Isomerase-Induced Arthritis.

OBJECTIVE: Arthritis is often characterized by inflammation and bone destruction. This study was undertaken to investigate the contribution of inflammation and bone destruction to pain. METHODS: Inflammation, bone resorption, pain-related behaviors, and molecular markers (activating transcription factor 3 [ATF-3], p-CREB, and transient receptor potential vanilloid channel 1) in sensory neurons were measured in murine glucose-6-phosphate isomerase (G6PI)-induced arthritis, a model of rheumatoid arthritis. Depletion of Treg cells before immunization changed self-limiting arthritis into nonremitting arthritis with pronounced bone destruction. Zoledronic acid (ZA) was administered to reduce bone resorption. RESULTS: Compared to nondepleted mice, Treg cell-depleted mice exhibited arthritis with more severe bone destruction and higher guarding scores (P < 0.05; n = 10 mice per group) as well as more persistent thermal hyperalgesia (P < 0.05), but displayed similar mechanical hyperalgesia at the hindpaws (n = 18-26 mice per group). These pain-related behaviors, as well as an up-regulation of the neuronal injury marker ATF-3 in sensory neurons (studied in 39 mice), appeared before the clinical score (inflammation) became positive and persisted in Treg cell-depleted and nondepleted mice. In the late stage of arthritis, Treg cell-depleted mice treated with ZA showed less bone resorption (<50%; P < 0.01) and less thermal hyperalgesia (P < 0.01) than Treg cell-depleted mice without ZA treatment (n = 15 mice per group), but ZA treatment did not reduce the clinical score and local mechanical hyperalgesia. CONCLUSION: Pain-related behaviors precede and outlast self-limiting arthritis. In nonremitting arthritis with enhanced bone destruction, mainly local thermal, but not local mechanical, hyperalgesia was aggravated. The up-regulation of ATF-3 indicates an early and persisting affection of sensory neurons by G6PI-induced arthritis.

The potential of substance P to initiate and perpetuate cortical spreading depression (CSD) in rat in vivo.

The tachykinin substance P (SP) increases neuronal excitability, participates in homeostatic control, but induces brain oedema after stroke or trauma. We asked whether SP is able to induce cortical spreading depression (CSD) which often aggravates stroke-induced pathology. In anesthetized rats we applied SP (10-5, 10-6, 10-7, or 10-8 mol/L) to a restricted cortical area and recorded CSDs there and in remote non-treated areas using microelectrodes. SP was either applied in artificial cerebrospinal fluid (ACSF), or in aqua to perform a preconditioning. Plasma extravasation in cortical grey matter was assessed with Evans Blue. Only SP dissolved in aqua induced self-regenerating CSDs. SP dissolved in ACSF did not ignite CSDs even when excitability was increased by acetate-preconditioning. Aqua alone elicited as few CSDs as the lowest concentration of SP. Local pretreatment with 250 nmol/L of a neurokinin 1 receptor antagonist prevented the SP-induced plasma extravasation, the initiation of CSDs by 10-5 mol/L SP diluted in aqua, and the initiation of CSDs by aqua alone, but did not suppress KCl-induced CSD. Thus neurokinin 1 receptor antagonists may be used to explore the involvement of SP in CSDs in clinical studies.