Anti-inflammatory effects of cell-based therapy with tyrosine hydroxylase-positive catecholaminergic cells in experimental arthritis

Objectives: Studies in rheumatoid arthritis (RA), osteoarthritis (OA) and mice with arthritis demonstrated tyrosine hydroxylase-positive (TH+) cells in arthritic synovium and parallel loss of sympathetic nerve fibres. The exact function of TH+ cells and mode of TH induction are not known. Methods: Synovial cells of RA/OA were isolated and cultured under normoxic/hypoxic conditions with/without stimulating enzyme cofactors of TH and inhibitors of TH. We studied TH expression and release of cytokines/catecholamines. In vivo function was tested by cell therapy with TH+ neuronal precursor cells (TH+ neuronal cells) in DBA/1 mice with collagen type II-induced arthritis (CIA). Results: Compared with normoxic conditions, hypoxia increased TH protein expression and catecholamine synthesis and decreased release of tumour necrosis factor (TNF) in OA/RA synovial cells. This inhibitory effect on TNF was reversed by TH inhibition with α-methyl-para-tyrosine (αMPT), which was particularly evident under hypoxic conditions. Incubation with specific TH cofactors (tetrahydrobiopterin and Fe2+) increased hypoxia-induced inhibition of TNF, which was also reversed by αMPT. To address a possible clinical role of TH+ cells, murine TH+ neuronal cells were generated from mesenchymal stem cells. TH+ neuronal cells exhibited a typical catecholaminergic phenotype. Adoptive transfer of TH+ neuronal cells markedly reduced CIA in mice, and 6-hydroxydopamine, which depletes TH+ cells, reversed this effect. Conclusions: The anti-inflammatory effect of TH+ neuronal cells on experimental arthritis has been presented for the first time. In RA/OA, TH+ synovial cells have TH-dependent anti-inflammatory capacities, which are augmented under hypoxia. Using generated TH+ neuronal cells might open new avenues for cell-based therapy

Association Between Changes in BLyS Levels and the Composition of B and T Cell Compartments in Patients With Refractory Systemic Lupus Erythematosus Treated With Belimumab

Introduction: Belimumab is a monoclonal antibody against soluble BLyS used for treatment of refractory Systemic Lupus Erythematosus (SLE). Although B cells are the main target of this therapy, a BLyS-dependent T cell activation pathway has also been demonstrated. The aim of the study is to analyze B and T cells phenotype modifications in a cohort of SLE patients treated with belimumab in correlation with serum BLyS levels.Materials and Methods: Fourteen SLE patients were enrolled in the study. Lymphocyte immunophenotyping by flow cytometry and determination of serum BLyS levels by high sensitivity ELISA were performed before the first infusion of belimumab, after 6 and 12 months of treatment. Sex and age-matched healthy controls were enrolled for the comparisons.Results: Baseline number of total B cells, especially switched memory B cells, were lower in SLE patients compared to control subjects. After 6 months of treatment, the total number of B cells, particularly, naive and transitional B cells, was significantly reduced in correlation with the reduction of BLyS levels. No significant association was found between baseline counts of B cells and reduction of SLEDAI-2K over time. In terms of response prediction, a significant association between SLEDAI-2K improvement at 12 months and the decrease of total number of B cells within the first 6 months of therapy was observed. Concerning the T cell compartment, the baseline percentage number of CD8+ effector memory was associated with SLEDAI-2K at baseline and with its improvement after 12 months of therapy. Furthermore, T cell lymphopenia and low number of circulating recent thymic emigrants were also observed compared to control subjects measured at baseline.Discussion: The effects of belimumab on B cell subpopulations could be explained by the direct blockage of soluble BLyS, while the mild effects on T cells might be explained indirectly by the reduction of disease activity by means of therapy. B cell immunophenotyping during belimumab might be useful for monitoring the response to treatment

Charakterisierung rekombinant produzierter Virus-ähnlicher Partikel des humanen Parvovirus B19

Das Kapsid des ältesten bekannten humanpathogenen Vertreters innerhalb der Parvoviridae, Parvovirus B19, besteht aus zwei Strukturproteinen VP1 und VP2. Das VP2-Protein stellt im viralen Partikel einen Anteil von 95 %. Es spielt eine wichtige Rolle bei der T-Zell-Antwort und VP2-spezifische konformationelle Epitope induzieren eine lang anhaltende Immunantwort. Rekombinant hergestellte VP2-Kapside im Baculovirus-Expressionssystem sind für viele Tests zum Nachweis von Parvovirus-B19-spezifischen Antikörpern ein wichtiges Antigen. Ziel vorliegender Arbeit war es, ein alternatives Expressionssystem zu etablieren, das VP2- und VP1/VP2-Mischkapside in hoher Qualität und Ausbeute produzieren kann. Der Grund für die Etablierung eines alternativen Expressionssystems sind die hohen Kosten und die umständliche Handhabung die das Baculovirussystem auszeichnen. Die rekombinant produzierten Virus-ähnlichen Partikel sollten molekularbiologisch, histologisch sowie immunologisch charakterisiert und mit Kapsiden aus dem Baculovirus-Expressionssystem verglichen werden. Als eukaryontische Expressionssysteme standen die Bäckerhefe, Saccharomyces cerevisiae, Drosophila-Schneider-2-Zellen sowie die trypanosomenartigen Leishmania-tarentolae-Zellen zur Verfügung. Einzig in der Bäckerhefe war es möglich, VP2-Kapside zu produzieren. Als nächstes wurde die Kapsidsynthese optimiert. Dabei wurden verschiedene Anzuchtsbedingungen, Nährmediumzusammensetzungen und Hefestämme getestet. Der Protease-defiziente Saccharomyces-Stamm HT393 produzierte im Vergleich zu dem kommerziell erhältlichen Stamm INVSc2 deutlich mehr VP2-Protein und zeigte weniger Proteindegradation. Desweiteren wurden Methoden zur Reinigung der rekombinanten VP2-Kapside entwickelt und anschließend optimiert. Der Aufschluss der Bäckerhefe gelang über Hochdruck, der über 95 % der Zellen zerstörte. Die Aufreinigung der VP2-Kapside wurde über mehrere Ultrazentrifugationsschritte erreicht. Gewählt wurde eine Kombination aus Differential- und isopyknischer Zentrifugation. Diese beiden Ultrazentrifugationstechniken lieferten den besten Kompromiss aus Ausbeute und Reinheit. Im Elektronenmikroskop konnten keine Unterschiede zwischen Partikeln aus der Bäckerhefe, Baculovirus infizierten Insektenzellen oder nativen Viren festgestellt werden. Ähnlich waren auch die Ergebnisse aus vergleichenden ELISA-Tests und dem T-Zell-Proliferationsassay. Demnach haben in Bäckerhefe produzierte VP2-Kapside dieselbe Antigenizität und Immunogenität wie Partikel aus dem Baculovirus-Expressionssystem. Schließlich wurde überprüft, ob mittels eines dualen Vektorsystems beide Strukturproteine VP1 und VP2 in Saccharomyces cerevisiae produziert werden können. Die Ergebnisse zeigten eine Co-Synthese des VP1- und VP2-Proteins in selben Mengenverhältnissen. Zur Generierung von Mischkapsiden muß allerdings die Menge an VP1-Protein auf maximal 40 % der Gesamtmenge an Strukturprotein beschränkt werden

The synthetic cannabinoid WIN55,212-2 mesylate decreases the production of inflammatory mediators in rheumatoid arthritis synovial fibroblasts by activating CB2, TRPV1, TRPA1 and yet unidentified receptor targets

Background: In rheumatoid arthritis (RA), synovial fibroblasts (SF) secrete large amounts of IL-6, IL-8 and matrix metalloproteinases (MMPs) which are crucial for cartilage destruction. RASFs are sensitive to the action of cannabinoids and they not only express cannabinoid receptors type I and II (CB1 and CB2) but also transient receptor potential channels type vanilloid (TRPV1) and ankyrin (TRPA1). The synthetic cannabinoid WIN55,212-2 mesylate (WIN) demonstrated strong anti-inflammatory effects in monocytes and synovial fibroblasts only in high concentrations in a non-cannabinoid receptor dependent manner. In this study we assessed the ability of WIN to modulate cytokine and MMP-3 production in SFs over a wide concentration range and identified specific receptor targets that mediate the effects of this synthetic cannabinoid. Methods: MMP-3, IL-6 and IL-8 were determined by ELISA. Adhesion was measured by the XCELLigence system. Proliferation was assessed by cell titer blue assays. Results: WIN significantly reduced TNF-induced IL-6, IL-8 and MMP-3 production in concentrations below 2 mu M, while higher concentrations completely inhibited production of IL-6 and IL-8 but increased extracellular MMP-3 levels. The inhibitory effect at low concentrations (<2 mu M)was independent on activation of either CB1 or CB2 but was attenuated by TRPV1 or TRPA1 inhibition in OASFs and RASFs. The effects of high concentrations of WIN on cytokine and MMP-3 production were decreased by the calcium chelating agent BAPTA, the AMPK activator metformin, the TRPA1 antagonist A967079 and the CB2 antagonist COR170. Furthermore, fetal calf serum content in culture media strongly influenced the efficacy of WIN at high concentrations. In addition, high concentrations of WIN also diminished SF adhesion and proliferation without altering cell viability whereas low concentrations promoted SF adhesion without any influence on proliferation. Conclusion: The synthetic cannabinoid WIN in low concentrations exhibits anti-inflammatory effects in synovial fibroblasts independent of CB1 and CB2 while CB2 and yet unidentified receptor targets are responsible for WIN effects in micromolar concentrations. Our results indicate a TRPV1/TRPA1 dependent mechanism of SF regulation that might be coupled to cellular energy status and calcium content

Impact of &Delta;9-Tetrahydrocannabinol on Rheumatoid Arthritis Synovial Fibroblasts Alone and in Co-Culture with Peripheral Blood Mononuclear Cells

&delta;9-Tetrahydrocannabinol (THC) has demonstrated anti-inflammatory effects in animal models of arthritis, but its mechanism of action and cellular targets are still unclear. The purpose of this study is to elucidate the effects of THC (0.1&ndash;25 &micro;M) on synovial fibroblasts from patients with rheumatoid arthritis (RASF) and peripheral blood mononuclear cells (PBMC) from healthy donors in respect to proliferation, calcium mobilization, drug uptake, cytokine and immunoglobulin production. Intracellular calcium and drug uptake were determined by fluorescent dyes Cal-520 and PoPo3, respectively. Cytokine and immunoglobulin production were evaluated by ELISA. Cannabinoid receptors 1 and 2 (CB1 and CB2) were detected by flow cytometry. RASF express CB1 and CB2 and the latter was increased by tumor necrosis factor (TNF). In RASF, THC (&ge;5 &micro;M) increased intracellular calcium levels/PoPo3 uptake in a TRPA1-dependent manner and reduced interleukin-8 (IL-8) and matrix metalloprotease 3 (MMP-3) production at high concentrations (25 &micro;M). Proliferation was slightly enhanced at intermediate THC concentrations (1&ndash;10 &micro;M) but was completely abrogated at 25 &micro;M. In PBMC alone, THC decreased interleukin-10 (IL-10) production and increased immunoglobulin G (IgG). In PBMC/RASF co-culture, THC decreased TNF production when cells were stimulated with interferon-&gamma; (IFN-&gamma;) or CpG. THC provides pro- and anti-inflammatory effects in RASF and PBMC. This is dependent on the activating stimulus and concentration of THC. Therefore, THC might be used to treat inflammation in RA but it might need titrating to determine the effective concentration

Anti-inflammatory effects of N-acylethanolamines in rheumatoid arthritis synovial cells are mediated by TRPV1 and TRPA1 in a COX-2 dependent manner

Introduction The endocannabinoid system modulates function of immune cells and mesenchymal cells such as fibroblasts, which contribute to cartilage destruction in rheumatoid arthritis (RA). The aim of the study was to determine the influence of N-acylethanolamines anandamide (AEA), palmitoylethanolamine (PEA) and oleylethanolamine (OEA) on several features of arthritic inflammation in vitro (human material) and in vivo (a mouse model). Methods Immunofluorescence and western blotting were used to detect cannabinoid receptors and related enzymes. Cytokines and MMP-3 were measured by ELISA. Intracellular signaling proteins were detected by proteome profiling. Proliferation was quantified by CTB reagent. Adhesion was assessed by the xCELLigence system. After onset of collagen type II arthritis, mice were treated daily with the FAAH inhibitor JNJ1661010 (20 mg/kg) or vehicle. Results IL-6, IL-8 and MMP-3 (determined only in synovial fibroblasts (SFs)) were downregulated in primary synoviocytes and SFs of RA and OA after AEA, PEA and OEA treatment. In SFs, this was due to activation of TRPV1 and TRPA1 in a COX-2-dependent fashion. FAAH inhibition increased the efficacy of AEA in primary synoviocytes but not in SFs. The effects of OEA and PEA on SFs were diminished by FAAH inhibition. Adhesion to fibronectin was increased in a CB 1 -dependent manner by AEA in OASFs. Furthermore, elevation of endocannabinoids ameliorated collagen-induced arthritis in mice. Conclusions N-acylethanolamines exert anti-inflammatory effects in SFs. A dual FAAH/COX-2 inhibitor, increasing N-acylethanolamine levels with concomitant TRP channel desensitization, might be a good candidate to inhibit the production of proinflammatory mediators of synovial cells and to reduce erosions