Induction of chronic destructive arthritis in SCID mice by arthritogenic fibroblast-like synoviocytes derived from mice with antigen-induced arthritis.

BACKGROUND: Fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) are autonomously activated to maintain inflammation and joint destruction in co-transplantation models. To elucidate inducing mechanisms involved in this altered behavior, the arthritogenic potential of FLSs from murine antigen-induced arthritis (AIA) were investigated in a transfer model. METHODS: FLSs were isolated, expanded in vitro, and transferred into knee joint cavities of severe combined immunodeficient (SCID) mice. Their arthritogenic capacity was assessed by monitoring joint swelling and evaluation of histological parameters 70 to 100 days after transfer. RESULTS: FLSs from AIA mice were able to transfer arthritis into recipient SCID mice. FLS transfer induced a chronic arthritis with recruitment of inflammatory cells and marked cartilage destruction. Long-lasting inflammation was not required for imprinting of arthritogenicity in FLSs since cells isolated from acute arthritic joints were fully competent to transfer arthritis. We also observed arthritogenic potential in FLSs isolated from contralateral non-arthritic joints in our monoarticular arthritis model. CONCLUSIONS: We show that the transformation of FLSs into arthritogenic cells occurs early in arthritis development. This challenges current hypotheses on the role of these cells in arthritis pathogenesis and opens up the way for further mechanistic studies.

In situ foamable, degradable polyurethane as biomaterial for soft tissue repair.

Degradable foams which can be inserted endoscopically as liquid or pasty mixtures into soft tissue defects possess a promising potential for the surgical treatment of such defects. The defects can be sealed under in situ foaming and simultaneous material expansion. We developed an in situ foamable (l-lactide-co-ε-caprolactone)-based, star-shaped prepolymer by ring opening polymerization of l-lactide and ε-caprolactone in the presence of meso-erythritol as starter. By conversion of the terminal hydroxyl groups of the formed oligoester with lysine diisocyanate ethyl ester (LDI) an isocyanate-endcapped, reactive prepolymer has been received. Foaming can be initiated by addition of 1,4-diazabicyclo[2,2,2]octane (DABCO), water, LDI and DMSO. By varying the composition of these additives, the foaming and curing time could be varied within a clinically acceptable range. A porosity of approximately 90%, and an average tensile strength of 0.3MPa with elongations of 90% were determined for the foams. In vitro cytotoxicity on cured foams was assayed on 3T3 fibroblasts and demonstrated an excellent cytocompatibility. This was also confirmed in an in vivo study using an established rat model, where prefabricated foams and in situ hardening material were inserted into subdermal skin incisions in parallel. The feature of chronic inflammation was only weakly developed in both groups and slightly more pronounced and persisted for longer time in the group of in situ foamed material. In both groups the foreign materials were vascularized, degraded and substituted by connective tissue. The results encourage to proceed with trials where the materials are used to fill more heavily loaded defects.

Pseudo-HE images derived from CARS/TPEF/SHG multimodal imaging in combination with Raman-spectroscopy as a pathological screening tool.

BACKGROUND: Due to the steadily increasing number of cancer patients worldwide the early diagnosis and treatment of cancer is a major field of research. The diagnosis of cancer is mostly performed by an experienced pathologist via the visual inspection of histo-pathological stained tissue sections. To save valuable time, low quality cryosections are frequently analyzed with diagnostic accuracies that are below those of high quality embedded tissue sections. Thus, alternative means have to be found that enable for fast and accurate diagnosis as the basis of following clinical decision making. METHODS: In this contribution we will show that the combination of the three label-free non-linear imaging modalities CARS (coherent anti-Stokes Raman-scattering), TPEF (two-photon excited autofluorescence) and SHG (second harmonic generation) yields information that can be translated into computational hematoxylin and eosin (HE) images by multivariate statistics. Thereby, a computational HE stain is generated resulting in pseudo-HE overview images that allow for identification of suspicious regions. The latter are analyzed further by Raman-spectroscopy retrieving the tissue's molecular fingerprint. RESULTS: The results suggest that the combination of non-linear multimodal imaging and Raman-spectroscopy possesses the potential as a precise and fast tool in routine histopathology. CONCLUSIONS: As the key advantage, both optical methods are non-invasive enabling for further pathological investigations of the same tissue section, e.g. a direct comparison with the current pathological gold-standard.

Systematic evaluation of the biological variance within the Raman based colorectal tissue diagnostics.

Being among the most common cancers worldwide screening and early diagnosis of colorectal cancer is of high interest for the health system, the patients and for research. Raman microspectroscopy as a label-free, non-invasive and non-destructive technique is a promising tool for an early diagnosis. However, to ensure a reliable diagnosis specially designed statistical analysis workflows are required. Several statistical approaches have been introduced leading to varying results in the overall accuracy, sensitivity and specificity. In this study a systematic evaluation of different statistical analysis approaches has been performed using a colon cancer mouse model with genotypic identical individuals. Based on the inter-individual Raman spectral variances a measure for the biological variance can be estimated. By applying a leave-one-individual-out cross-validation a clinically relevant discrimination of healthy tissue versus adenoma and carcinoma with an accuracy of 95% is shown. Furthermore, the transfer of a model from tissue to biopsy specimen is demonstrated.

Interleukin-6-dependent influence of nociceptive sensory neurons on antigen-induced arthritis.

INTRODUCTION: Interleukin-6 (IL-6) is an important mediator of inflammation. In addition to cells involved in inflammation, sensory nociceptive neurons express the IL-6 signal-transducer glycoprotein 130 (gp130). These neurons are not only involved in pain generation but also produce neurogenic inflammation by release of neuropeptides such as calcitonin gene-related peptide (CGRP). Whether IL-6 activation of sensory neurons contributes to the induction of inflammation is unknown. This study explored whether the action of IL-6 on sensory neurons plays a role in the generation of neurogenic inflammation and arthritis induction. METHODS: In SNS-gp130(-/-) mice lacking gp130 selectively in sensory neurons and appropriate control littermates (SNS-gp130(flox/flox)), we induced antigen-induced arthritis (AIA), and assessed swelling, histopathological arthritis scores, pain scores, expression of CGRP in sensory neurons, serum concentrations of CGRP and cytokines, and the cytokine release from single cell suspensions from lymph nodes and spleens. In wild-type mice CGRP release was determined during development of AIA and, in cultured sensory neurons, upon IL-6 stimulation. RESULTS: Compared to SNS-gp130(flox/flox) mice SNS-gp130(-/-) mice showed significantly weaker initial swelling, reduced serum concentrations of CGRP, IL-6, and IL-2, no inflammation-evoked upregulation of CGRP in sensory neurons, but similar histopathological arthritis scores during AIA. During the initial swelling phase of AIA, CGRP was significantly increased in the serum, knee and spleen. In vitro, IL-6 augmented the release of CGRP from cultured sensory neurons. Upon antigen-specific restimulation lymphocytes from SNS-gp130(-/-) mice released more interleukin-17 and interferon-γ than lymphocytes from SNS-gp130(flox/flox) mice. In naive lymphocytes from SNS-gp130(flox/flox) and SNS-gp130(-/-) mice CGRP reduced the release of IL-2 (a cytokine which inhibits the release of interleukin-17 and interferon-γ). CONCLUSIONS: IL-6 signaling in sensory neurons plays a role in the expression of arthritis. Selective deletion of gp130 signaling in sensory neurons reduces the swelling of the joint (most likely by reducing neurogenic inflammation) but increases some proinflammatory systemic cellular responses such as the release of interleukin-17 and interferon-γ from lymphocytes upon antigen-specific restimulation. Thus IL-6 signaling in sensory neurons is not only involved in pain generation but also in the coordination of the inflammatory response.

1,25-Dihydroxyvitamin D3 prevents bone loss of the secondary spongiosa in arthritic rats by an increase of bone formation and mineralization and inhibition of bone resorption.

BACKGROUND: Active vitamin D metabolites have been shown to have protective effects in experimental arthritis especially when used as preventive treatment. However, because the direct effects of 1,25-dihydroxyvitamin D3 (1,25(OH) 2D3) on bone formation and resorption are very complex, the net effect of 1,25(OH)2D3 on histomorphometric parameters of bone turnover and mineralisation should be investigated. Therefore, we examined the influence of 1,25(OH)2D3 therapy on arthritis-induced alterations of periarticular and axial bone as well as disease activity, inflammation and joint destruction in antigen-induced arthritis (AIA) of the rat. METHODS: AIA was induced in 20 eight-week-old female Wistar rats. 10 rats without arthritis were used as healthy controls. AIA rats received 1,25(OH)2D3 (0.2 µg/kg/day, i.p., n = 10) or vehicle (n = 10) at regular intervals for 28 consecutive days beginning 3 days before arthritis induction. Bone structure of the secondary spongiosa of the periarticular and axial bone was analyzed using histomorphometry. Parameters of mineralization were investigated using tetracycline labelling. Clinical disease activity, inflammation and joint destruction were measured by joint swelling and histological investigation, respectively. RESULTS: AIA led to significant periarticular bone loss. 1,25(OH)2D3 treatment resulted in a highly significant increase in trabecular bone volume and bone formation rate in comparison to both vehicle-treated AIA and healthy controls at periarticular (p < 0.01 and p < 0.001, respectively) and axial bone (p < 0.001 and p < 0.001, respectively). In addition, bone resorption was reduced by 1,25(OH)2D3 at the axial bone (p < 0.05 vs. vehicle-treated AIA). Joint swelling as well as histological signs of inflammation and joint destruction were not influenced by 1,25(OH)2D3. CONCLUSIONS: The results of the study indicate a marked osteoanabolic effect of 1,25(OH)2D3 presumably due to a substantial increase in mineralization. Thus, 1,25(OH)2D3 may be an effective osteoanabolic treatment principle to antagonize the inflammation-associated suppression of bone formation in rheumatoid arthritis.

Collagenase-3 (MMP-13) deficiency protects C57BL/6 mice from antibody-induced arthritis.

INTRODUCTION: Matrix metalloproteinases (MMPs) are important in tissue remodelling. Here we investigate the role of collagenase-3 (MMP-13) in antibody-induced arthritis. METHODS: For this study we employed the K/BxN serum-induced arthritis model. Arthritis was induced in C57BL/6 wild type (WT) and MMP-13-deficient (MMP-13­/­) mice by intraperitoneal injection of 200 µl of K/BxN serum. Arthritis was assessed by measuring the ankle swelling. During the course of the experiments, mice were sacrificed every second day for histological examination of the ankle joints. Ankle sections were evaluated histologically for infiltration of inflammatory cells, pannus tissue formation and bone/cartilage destruction. Semi-quantitative PCR was used to determine MMP-13 expression levels in ankle joints of untreated and K/BxN serum-injected mice. RESULTS: This study shows that MMP-13 is a regulator of inflammation. We observed increased expression of MMP-13 in ankle joints of WT mice during K/BxN serum-induced arthritis and both K/BxN serum-treated WT and MMP-13­/­ mice developed progressive arthritis with a similar onset. However, MMP-13­/­ mice showed significantly reduced disease over the whole arthritic period. Ankle joints of WT mice showed severe joint destruction with extensive inflammation and erosion of cartilage and bone. In contrast, MMP-13­/­ mice displayed significantly decreased severity of arthritis (50% to 60%) as analyzed by clinical and histological scoring methods. CONCLUSIONS: MMP-13 deficiency acts to suppress the local inflammatory responses. Therefore, MMP-13 has a role in the pathogenesis of arthritis, suggesting MMP-13 is a potential therapeutic target.

Assessment of rat antigen-induced arthritis and its suppression during glucocorticoid therapy by use of hemicyanine dye probes with different molecular weight in near-infrared fluorescence optical imaging.

PURPOSE: Arthritic joints are ideal disease entities to be assessed via optical imaging. Here, we investigated the selective accumulation behavior of two differently sized hemicyanine optical probes in arthritic joints and its modification during glucocorticoid therapy in the course of inflammation. MATERIALS AND METHODS: The well-standardized preclinical antigen-induced arthritis (AIA) model in rats was used. The animals were divided into 3 groups: arthritic, arthritic and dexamethasone-treated, and immunized only. After intravenous coinjection of DY-752 (size, 800 Da) and DY-682-(rat) IgG (size, 150 kDa) probes, spectrally unmixed near-infrared fluorescence images were acquired and analyzed semiquantitatively. Probe organ distribution, joint swelling, blood cell counts, joint vessel density, and histological scoring of arthritis were determined. RESULTS: The local joint accumulation kinetics of the DY-752 probe differed from the DY-682-IgG one. In the course of AIA, probe signaling in arthritic joints was similar between each other. Joint swelling and histological scoring were in accordance with signaling. Dexamethasone treatment of rats with AIA significantly reduced both the near-infrared fluorescence signals and severity of arthritis but did not change the joint vascular density or the uptake of the probes by phagocytes. A differential biodistribution of both probes was encountered, but such an accumulation was prevented by dexamethasone treatment. CONCLUSIONS: Near-infrared fluorescence signaling in the course of AIA closely reflects the pathophysiological events of the arthritic joint and the effects of therapy independently of the molecular size of the probe. The results show the suitability of our hemicyanine probes for imaging of arthritis.

Histamine 4 receptor plays an important role in auto-antibody-induced arthritis.

Rheumatoid arthritis is a widespread autoimmune disease. In the murine K/B×N arthritis model, anti-GPI (anti-glucose 6-phosphate isomerase) antibodies lead to the formation of immune complexes. In the course of pathogenesis, these complexes activate the immune system and induce degranulation of mast cells, which are essential in this model of rheumatoid arthritis. A major mediator in mast cell granules is histamine, which is proven to be indispensable for joint inflammation in K/B×N mice. Histamine is known to bind to four different receptors (HR1-4), which have different expression profiles and exert a variety of different functions, including activation of the immune system. To analyze the contribution of the different histamine receptors, we employed histamine receptor antagonists (cetirizine, ranitidine, thioperamide and clozapine) blocking the receptors in C57BL/6 mice. Arthritis was induced via K/B×N serum injection. The results demonstrated that mice treated with all four histamine receptor antagonists simultaneously showed no arthritic symptoms, while positive control mice injected with K/B×N serum and vehicle suffered from severe symptoms. When antagonists specific for HR1-4 were applied individually, only the HR4 antagonist clozapine could protect mice from arthritis, reflecting its expression and functionality in the immune system.

Macrophage-derived, macrophage migration inhibitory factor (MIF) is necessary to induce disease in the K/BxN serum-induced model of arthritis.

Rheumatoid arthritis (RA) is characterized by the interaction of multiple mediators, among the most important of which are cytokines. In recent years, extensive studies demonstrate a pivotal role for one cytokine, macrophage migration inhibitory factor (MIF), in fundamental events in innate and adaptive immunity. MIF has now been demonstrated to be involved in the pathogenesis of many diseases, but in the case of RA the evidence for a role of MIF is very strong. MIF is abundantly expressed in the sera of RA patients and in RA synovial tissue correlating with disease activity. MIF-deficient mice were used to induce arthritis by serum transfer from K/BxN mice. K/BxN serum transfer arthritis was markedly attenuated in MIF(-) mice, with reduction in clinical index and histological severity as well as decrease in synovial cytokines. Macrophage transfers were done to investigate the specific role of macrophage-derived MIF. We show that adoptive transfer of wild-type macrophages into MIF(-) mice restores the sensitivity of MIF(-) mice to arthritis development, and this affect was associated with a restoration in serum IL-1ß and IL-6 production. These results indicate that MIF plays a critical role in inflammation and joint destruction in K/BxN serum-induced arthritis and that the systemic expression of MIF by a subpopulation of macrophages is necessary and sufficient for the full development of arthritis.

Neuronal IL-17 receptor upregulates TRPV4 but not TRPV1 receptors in DRG neurons and mediates mechanical but not thermal hyperalgesia.

In addition to the proinflammatory cytokines tumor necrosis factor-α, interleukin-6 and interleukin-1ß, the cytokine interleukin-17 (IL-17) is considered an important mediator of autoimmune diseases such as rheumatoid arthritis. Because tumor necrosis factor-α and interleukin-1ß have the potential to influence the expression of transduction molecules such as transient receptor potential vanilloid 1 (TRPV1) in dorsal root ganglion (DRG) neurons and thus to contribute to pain we explored in the present study whether IL-17A activates DRG neurons and influences the expression of TRPV1. The IL-17A receptor was visualized in most neurons in dorsal root ganglion (DRG) sections as well as in cultured DRG neurons. Upon long-term exposure to IL-17A, isolated and cultured rat DRG neurons showed a significant upregulation of extracellular-regulated kinase (ERK) and nuclear factor κB (NFκB). Long-term exposure of neurons to IL-17A did not upregulate the expression of TRPV1. However, we found a pronounced upregulation of transient receptor potential vanilloid 4 (TRPV4) which is considered a candidate transduction molecule for mechanical hyperalgesia. Upon the injection of zymosan into the paw, IL-17A-deficient mice showed less mechanical hyperalgesia than wild type mice but thermal hyperalgesia was not attenuated in IL-17A-deficient mice. These data show, therefore, a particular role of IL-17 in mechanical hyperalgesia, and they suggest that this effect is linked to an activation and upregulation of TRPV4.

Interleukin-17 sensitizes joint nociceptors to mechanical stimuli and contributes to arthritic pain through neuronal interleukin-17 receptors in rodents.

OBJECTIVE: Interleukin-17 (IL-17) is considered a proinflammatory cytokine, but whether neuronal IL-17 receptors contribute to the generation of arthritic pain is unknown. This study was undertaken to explore whether IL-17A acts on neurons, whether it sensitizes joint nociceptors, and whether neutralization of IL-17 is antinociceptive. METHODS: We recorded action potentials from rat joint nociceptors after intraarticular injection of IL-17A. We studied the expression of the IL-17A receptor in the rat dorsal root ganglia (DRG), explored the effect of IL-17A on signaling pathways in cultured rat DRG neurons, and using patch clamp recordings, monitored changes of excitability by IL-17A. We tested whether an antibody to IL-17 influences pain behaviors in mice with antigen-induced arthritis (AIA). RESULTS: A single injection of IL-17A into the rat knee joint elicited a slowly developing and long-lasting sensitization of nociceptive C fibers of the joint to mechanical stimuli, which was not attenuated by neutralizing tumor necrosis factor α or IL-6. The IL-17A receptor was visualized in most rat DRG neurons, the cell bodies of primary sensory neurons. In isolated and cultured rat DRG neurons, IL-17A caused rapid phosphorylation of protein kinase B and ERK, and it rapidly enhanced excitability. In mice with unilateral AIA in the knee, an antibody against IL-17 improved the guarding score and reduced secondary mechanical hyperalgesia at the ipsilateral paw. CONCLUSION: Our findings indicate that IL-17A has the potential to act as a pain mediator by targeting IL-17 receptors in nociceptive neurons, and these receptors are particularly involved in inflammation-evoked mechanical hyperalgesia.

The role of interleukin-1ß in arthritic pain: main involvement in thermal, but not mechanical, hyperalgesia in rat antigen-induced arthritis.

OBJECTIVE: Interleukin-1ß (IL-1ß) is considered a pronociceptive cytokine, but its role in the generation of arthritic pain is unknown. The aim of this study was to investigate the role of IL-1ß in arthritic pain and to explore the antinociceptive potential of the IL-1 receptor type I (IL-1RI) antagonist anakinra. METHODS: Antigen-induced arthritis (AIA) was induced in rats. Expression of IL-1RI in the dorsal root ganglia (DRGs) was determined, and the effects of anakinra on inflammation, pain-related behavior, and receptor expression were assessed. In cultured DRG neurons, the effect of IL-1ß on the expression of the transient receptor potential vanilloid 1 (TRPV-1) ion channel was examined. Recordings of action potentials from joint nociceptors were made after intraarticular injection of IL-1ß into the rat knee joints. RESULTS: AIA generated pronounced and persistent mechanical and thermal hyperalgesia, and IL-1RI expression in the lumbar DRGs was significantly up-regulated. Treatment with anakinra did not significantly reduce the severity of arthritis or mechanical hyperalgesia, but did result in a pronounced reduction in thermal hyperalgesia. In cultured DRG neurons, IL-1ß up-regulated the expression of TRPV-1, a major transduction molecule involved in thermal hyperalgesia. During AIA, anakinra treatment down-regulated the expression of TRPV-1, consistent with the pronounced reduction in thermal hyperalgesia. IL-1ß increased the mechanosensitivity of C-fibers of the joint, but reduced the mechanosensitivity of Aδ-fibers, thus having opposite effects on these mechanonociceptive nerve fibers. CONCLUSION: In the context of arthritic knee pain, IL-1ß and IL-1 receptors appear to be involved in thermal, rather than mechanical, hyperalgesia. Therefore, neutralization of IL-1ß may be mainly antinociceptive in disease states characterized by thermal hyperalgesia, but not in disease states mainly characterized by mechanical hyperalgesia.

Decreased arthritis severity in cathepsin L-deficient mice is attributed to an impaired T helper cell compartment.

OBJECTIVE: Cathepsin L (CL) is potentially involved in joint destruction and in antigen presentation in rheumatoid arthritis. In order to define the roles of this protease in arthritis development we analysed the antigen-induced arthritis (AIA) in CL-deficient (CL(-/-)) mice. METHODS: Antigen-induced arthritis was induced in CL(-/-) and wild-type mice. Complete CL deficiency resulted in an impaired positive selection of conventional CD4(+) T helper (Th) cells and finally in a reduced number of Th cells. Thus, we addressed the effect of this phenotype by rescuing CD4(+) Th cell numbers by transgenic expression of the human CL-like protease cathepsin V (hCV) in thymic epithelium of CL(-/-) mice [Tg(K14-hCV);CL(-/-)]. The arthritis development was monitored by measuring joint swelling. Joint inflammation and destruction were assessed histopathologically. RESULTS: The severity of AIA was decreased in CL(-/-) mice characterized by reduced swelling, decreased inflammation and destruction, and diminished cellular and humoral immune responsiveness. AIA in Tg(K14-hCV);CL(-/-) mice was associated with a reconstitution of all parameters by normalization of the ratio of regulatory to conventional T cells. CONCLUSIONS: Cathepsin L has a significant impact on AIA severity by influencing the selection of Th cell populations in the thymus, but seems not play any significant role in the direct joint destruction.

The anti-inflammatory effects of sympathectomy in murine antigen-induced arthritis are associated with a reduction of Th1 and Th17 responses.

BACKGROUND: Both facilitatory and inhibitory effects of the sympathetic nervous system (SNS) on experimental arthritis have been reported. It is unknown whether such bidirectional effects are inherent to all experimental arthritis models and/or whether critical time windows exist for influences of the SNS on inflammation. OBJECTIVES: To assess the effect of sympathectomy at different time points on the course and severity of murine antigen-induced arthritis (AIA). METHODS: AIA was induced in mice. Chemical sympathectomy with 6-hydroxydopamine was carried out either neonatally, in the immunisation phase, or immediately before AIA elicitation, or during the chronic phase. In sympathectomised and non-sympathectomised AIA mice the inflammatory process (joint swelling, histopathology of inflammation and joint destruction), pain-related behaviour and cellular and humoral immune responses were analysed. RESULTS: Sympathectomy during AIA induction or neonatal sympathectomy significantly reduced the severity of acute AIA. Neither sympathectomy in the immunisation phase nor in the chronic phase influenced AIA. Flare-up reactions were reduced by sympathectomy just before flare-up or during the initial acute AIA stage. Sympathectomised AIA mice showed less hyperalgesia. Sympathectomy significantly reduced interleukin (IL) 2, IL-17 and transforming growth factor ß in supernatants from lymph nodes and/or spleen cells and antigen-specific Th1-associated IgG2a in serum; IgG1 titres were unaffected. The ß blocker, propranolol, and the norepinephrine reuptake inhibitor bupropion produced similar anti-inflammatory effects, whereas the ß-adrenergic agonist isoproterenol increased AIA severity in neonatally sympathectomised mice. CONCLUSIONS: Sympathetic activity mainly increases the severity of acute episodes of immune-mediated arthritis. Therapeutic reduction of sympathetic activity at acute stages attenuates inflammation, hyperalgesia and proinflammatory immune parameters.

Glucocorticoid therapy of antigen-induced arthritis depends on the dimerized glucocorticoid receptor in T cells.

Despite several side effects, glucocorticoids (GCs) have been widely used for 60 y to treat rheumatoid arthritis on the basis of their antiinflammatory effects. However, the cells targeted by GCs and the transcriptional mechanisms underlying their actions through the glucocorticoid receptor (GR) in steroid therapy remain poorly defined. Using cell type-specific GR-deficient mice subjected to antigen-induced arthritis (AIA) as a model of human rheumatoid arthritis, we show that GC action on T cells but not myeloid cells is critical for therapeutic intervention in AIA. Furthermore, the resistance of mice expressing a DNA binding-defective GR (GR(dim)) to GC treatment reveals that dimerization of the GR is indispensable for the antiinflammatory effects. In these mice, the GC-induced suppression of T(H)1 and T(H)17 cell-derived proinflammatory cytokines is impaired. Our finding that IL-17A(-/-) mice are resistant to GC therapy, whereas IFN-γ(-/-) mice respond as efficiently as WT mice implies that IL-17-producing T cells and not IFN-γ-producing T cells are the most important targets for an efficient GC therapy. The present study's identification of the critical cell type and the mode of GR action in steroid therapy of AIA significantly advances our understanding of steroid therapy and should lead to therapies with greater efficiency and fewer side effects.

Potent anti-inflammatory and antinociceptive activity of the endothelin receptor antagonist bosentan in monoarthritic mice.

INTRODUCTION: Endothelins are involved in tissue inflammation, pain, edema and cell migration. Our genome-wide microarray analysis revealed that endothelin-1 (ET-1) and endothelin-2 (ET-2) showed a marked up-regulation in dorsal root ganglia during the acute phase of arthritis. We therefore examined the effects of endothelin receptor antagonists on the development of arthritis and inflammatory pain in monoarthritic mice. METHODS: Gene expression was examined in lumbar dorsal root ganglia two days after induction of antigen-induced arthritis (AIA) using mRNA microarray analysis. Effects of drug treatment were determined by repeated assessment of joint swelling, pain-related behavior, and histopathological manifestations during AIA. RESULTS: Daily oral administration of the mixed ET(A) and ET(B) endothelin receptor antagonist bosentan significantly attenuated knee joint swelling and inflammation to an extent that was comparable to dexamethasone. In addition, bosentan reduced inflammatory mechanical hyperalgesia. Chronic bosentan administration also inhibited joint swelling and protected against inflammation and joint destruction during AIA flare-up reactions. In contrast, the ET(A)-selective antagonist ambrisentan failed to promote any detectable antiinflammatory or antinociceptive activity. CONCLUSIONS: Thus, the present study reveals a pivotal role for the endothelin system in the development of arthritis and arthritic pain. We show that endothelin receptor antagonists can effectively control inflammation, pain and joint destruction during the course of arthritis. Our findings suggest that the antiinflammatory and antinociceptive effects of bosentan are predominantly mediated via the ET(B) receptor.

Differential antiinflammatory and antinociceptive effects of the somatostatin analogs octreotide and pasireotide in a mouse model of immune-mediated arthritis.

OBJECTIVE: Clinical and preclinical evidence suggests that somatostatin exhibits potent antiinflammatory and antinociceptive properties. However, it is not known which of the 5 somatostatin receptor subtypes (SSTRs 1-5) is involved in these actions. The purpose of this study was to assess the effects of the stable somatostatin analogs octreotide and pasireotide (SOM230) in a mouse model of antigen-induced arthritis (AIA). METHODS: Studies were performed in SSTR2-deficient mice (SSTR2(-/-)) and their wild-type littermates (SSTR2(+/+)). The expression of SSTR1, SSTR2A, SSTR3, and SSTR5 in dorsal root ganglia was examined by immunohistochemistry. RESULTS: Untreated SSTR2(-/-) mice with AIA displayed joint swelling and mechanical hyperalgesia similar to that seen in SSTR2(+/+) mice. In wild-type mice, both octreotide and pasireotide significantly attenuated knee joint swelling and histopathologic manifestations of arthritis to an extent comparable to that of dexamethasone. In SSTR2(-/-) mice, the antiinflammatory effects of both octreotide and pasireotide were completely abrogated. Prolonged administration of pasireotide also inhibited joint swelling and protected against joint destruction during AIA flare reactions. In addition, both octreotide and pasireotide reduced inflammatory hyperalgesia. The antinociceptive actions of octreotide were abolished in SSTR2(-/-) mice, but those of pasireotide were retained. In dorsal root ganglia of naive wild-type mice, only SSTR1 and SSTR2A, but not SSTR3 or SSTR5, were detected in a subset of small- and medium-diameter neurons. CONCLUSION: Our findings indicate that the antinociceptive and antiinflammatory actions of octreotide and pasireotide are largely mediated via the SSTR2 receptor. In addition, we identified the SSTR1 receptor as a novel pharmacologic target for somatostatin-mediated peripheral analgesia in inflammatory pain.

In vivo molecular imaging of experimental joint inflammation by combined (18)F-FDG positron emission tomography and computed tomography.

INTRODUCTION: The purpose of this work was to establish and validate combined small animal positron emission tomography - computed tomography (PET/CT) as a new in vivo imaging method for visualisation and quantification of joint inflammation. METHODS: Signalling of radioisotope ¹8F labelled Fluorodeoxyglucose (¹8F-FDG) injected in mice with glucose-6-phosphate isomerase (G6PI)-induced arthritis was analysed by PET/CT. Accumulation of ¹8F-FDG in tissue was quantified by PET measurement, whereas high definition CT delivered anatomical information. The fusion of both images revealed in detail spatial and temporal distribution and metabolism of ¹8F-FDG. RESULTS: A distinct ¹8F-FDG signal could be measured by PET in carpal and tarsal joints, from mice with early or established arthritis. In contrast, no accumulation of ¹8F-FDG was detectable before arthritis onset. Comparison of ¹8F-FDG joint uptake with histopathological evaluation revealed a significant correlation of both methods. CONCLUSIONS: Small animal PET/CT using ¹8F-FDG is a feasible method for monitoring and, more importantly, quantitative assessment of inflammation in G6PI-arthritis. Since it is possible to perform repeated non-invasive measurements in vivo, not only numbers of animals in preclinical studies can markedly be reduced by this method, but also longitudinal studies come into reach, e. g. for individual flare-up reactions or monitoring therapy response in progressive arthritis.

Differential effects of locally and systemically administered soluble glycoprotein 130 on pain and inflammation in experimental arthritis.

INTRODUCTION: Interleukin-6 (IL-6) is a key player in systemic arthritis, involved in inflammation and joint destruction. IL-6 signalling has also been revealed in nerve cells. Recently, IL-6 and in particular IL-6 together with its soluble IL-6 receptor (sIL-6R) were shown to induce a long-lasting robust sensitization of joint nociceptors for mechanical stimuli which was difficult to reverse, suggesting that IL-6 signalling plays a significant role in the generation and maintenance of arthritic pain. Here we tested in a preclinical model of arthritis, antigen-induced arthritis (AIA) in the rat, whether systemic or local neutralization of IL-6/sIL-6R complexes with soluble glycoprotein 130 (sgp130) alters arthritic pain and how sgp130 influences the inflammatory process in AIA. METHODS: Rats with AIA were either treated with sgp130 or saline intra-peritoneally or intra-articularly (each group n = 9). Then, pain-related and locomotor behaviour, as well as joint swelling, were measured during an observation period of 21 days, followed by histopathological end-point analysis for inflammatory and destructive changes. RESULTS: A single intra-articular application of sgp130 at the time of AIA induction barely reduced the development of AIA, but significantly attenuated pain-related behaviour, that is, primary mechanical hyperalgesia in the acute phase of AIA. By contrast, repeated systemic application of sgp130 after onset of AIA only slightly attenuated pain at a late stage of AIA. None of the treatments reduced secondary hyperalgesia. Furthermore, in the present study joint destruction at 21 days was significantly attenuated after intra-articular sgp130 treatment, but not after systemic sgp130. CONCLUSIONS: In addition to its role in chronic inflammation, IL-6 in the joint plays a significant role in the generation and maintenance of arthritic joint pain at acute and chronic stages of AIA. The particular effectiveness of intra-articular injection of sgp130 indicates, first, that IL-6/sIL-6R in the inflamed joint, rather than circulating IL-6/sIL-6R, is responsible for the generation of hyperalgesia, and, second, that early neutralization of IL-6/sIL-6R is particularly successful in producing antinociception. Furthermore, neutralization of IL-6/sIL-6R (and possibly other cytokines which use the transmembrane signal-transducing subunit gp130) directly at the site of joint inflammation seems to be effective in the prevention of joint destruction.