Endogenously produced catecholamines improve the regulatory function of TLR9-activated B cells.

The sympathetic nervous system (SNS) contributes to immune balance by promoting anti-inflammatory B cells. However, whether B cells possess a self-regulating mechanism by which they modulate regulatory B cell (Breg) function is not well understood. In this study, we investigated the ability of B cells to synthesize their own catecholamines upon stimulation with different B cell activators and found that expression of the enzyme tyrosine hydroxylase (TH), required to generate catecholamines, is up-regulated by Toll-like receptor (TLR)9. This TLR9-dependent expression of TH correlated with up-regulation of adrenergic receptors (ADRs), enhanced interleukin (IL)-10 production, and overexpression of the co-inhibitory ligands programmed death ligand 1 (PD-L1) and Fas ligand (FasL). Moreover, concomitant stimulation of ß1-3-ADRs together with a B cell receptor (BCR)/TLR9 stimulus clearly enhances the anti-inflammatory potential of Bregs to suppress CD4 T cells, a crucial population in the pathogenesis of autoimmune diseases, like rheumatoid arthritis (RA). Furthermore, TH up-regulation was also demonstrated in B cells during the course of collagen-induced arthritis (CIA), a mouse model for the investigation of RA. In conclusion, our data show that B cells possess an autonomous mechanism to modulate their regulatory function in an autocrine and/or paracrine manner. These findings help to better understand the function of B cells in the regulation of autoimmune diseases and the interplay of SNS.

Anti-Inflammatory Effects of Cannabigerol in Rheumatoid Arthritis Synovial Fibroblasts and Peripheral Blood Mononuclear Cell Cultures Are Partly Mediated by TRPA1.

Since its medical legalization, cannabis preparations containing the major phytocannabinoids (cannabidiol (CBD) and δ9-tetrahydrocannabinol (THC)) have been used by patients with rheumatoid arthritis (RA) to alleviate pain and inflammation. However, minor cannabinoids such as cannabigerol (CBG) also demonstrated anti-inflammatory properties, but due to the lack of studies, they are not widely used. CBG binds several cellular target proteins such as cannabinoid and α2-adrenergic receptors, but it also ligates several members of the transient potential receptor (TRP) family with TRPA1 being the main target. TRPA1 is not only involved in nnociception, but it also protects cells from apoptosis under oxidative stress conditions. Therefore, modulation of TRPA1 signaling by CBG might be used to modulate disease activity in RA as this autoimmune disease is accompanied by oxidative stress and subsequent activation of pro-inflammatory pathways. Rheumatoid synovial fibroblasts (RASF) were stimulated or not with tumor necrosis factor (TNF) for 72 h to induce TRPA1 protein. CBG increased intracellular calcium levels in TNF-stimulated RASF but not unstimulated RASF in a TRPA1-dependent manner. In addition, PoPo3 uptake, a surrogate marker for drug uptake, was enhanced by CBG. RASF cell viability, IL-6 and IL-8 production were decreased by CBG. In peripheral blood mononuclear cell cultures (PBMC) alone or together with RASF, CBG-modulated interleukin (IL)-6, IL-10, TNF and immunoglobulin M and G production which was dependent on activation stimulus (T cell-dependent or independent). However, effects on PBMCs were only partially mediated by TRPA1 as the antagonist A967079 did inhibit some but not all effects of CBG on cytokine production. In contrast, TRPA1 antagonism even enhanced the inhibitory effects of CBG on immunoglobulin production. CBG showed broad anti-inflammatory effects in isolated RASF, PBMC and PBMC/RASF co-cultures. As CBG is non-psychotropic, it might be used as add-on therapy in RA to reduce IL-6 and autoantibody levels.

Joints for joints: cannabinoids in the treatment of rheumatoid arthritis.

PURPOSE OF REVIEW: An increasing number of patients with rheumatoid arthritis (RA) are using cannabis to treat their symptoms, although systematic studies regarding efficacy in RA are lacking. Within this review we will give an overview on the overall effects of cannabinoids in inflammation and why they might be useful in the treatment of RA. RECENT FINDINGS: Peripherally, cannabinoids show anti-inflammatory effects by activating cannabinoid type 2 receptors (CB2) which decrease cytokine production and immune cell mobilization. In contrast, cannabinoid type 1 receptor (CB1) activation on immune cells is proinflammatory while CB1 antagonism provides anti-inflammatory effects by increasing ß2-adrenergic signaling in the joint and secondary lymphoid organs. In addition, the nonpsychotropic cannabinoid, cannabidiol (CBD) demonstrated antiarthritic effects independent of cannabinoid receptors. In addition to controlling inflammation, cannabinoids reduce pain by activating central and peripheral CB1, peripheral CB2 receptors and CBD-sensitive noncannabinoid receptor targets. SUMMARY: Cannabinoids might be a suitable treatment for RA, but it is important to target the right receptors in the right place. For clinical studies, we propose a combination of a CB2 agonist to decrease cytokine production, a peripheral CB1 antagonist to prevent detrimental CB1 signaling and to support anti-inflammatory effects of CB2 via activation of ß2-adrenergic receptors and CBD to induce cannabinoid-receptor-independent anti-inflammatory effects.

Tryptophan metabolism in rheumatoid arthritis is associated with rheumatoid factor and predicts joint pathology evaluated by the Rheumatoid Arthritis MRI Score (RAMRIS).

OBJECTIVES: Tryptophan and its metabolites have been suggested to play a role in inflammatory processes. However, studies in rheumatoid arthritis (RA) are scarce, which prompted us to investigate two cohorts of RA patients to better understand the importance of tryptophan metabolism in this disease. METHODS: Tryptophan and its metabolites were characterised by ELISA in a cross-sectional cohort 1 (81 RA, 55 OA) and a longitudinal cohort 2 (25 RA, 3 visits over 6 months) to investigate discriminatory power between diseases and predicitive value for radiologic outcome, respectively. Radiologic outcome was monitored by RA MRI Score (RAMRIS), including grading of synovitis, bone oedema and erosion, as well as delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) index assessing cartilage quality of the MCP II joint. RESULTS: RA patients showed higher levels of serum serotonin (RA: 206.8 ng/ml ± 156.7; OA: 81.2 ng/ml ± 63.6) and estimated indoleamine (2,3)-dioxygenase (IDO) activity (kynurenine / tryptophan ratio; RA: 0.065±0.067; OA: 0.021±0.010). IDO activity showed similar, or better discriminatory power between RA and OA (AUC 0.914) than anti-CCP antibody level (AUC 0.922) and rheumatoid factor (RF, AUC 0.783), respectively. In cohort 2, regression analysis revealed a predictive value of baseline serotonin levels and IDO activity for changes in RAMRIS score and erosions at month six, respectively. CONCLUSIONS: This study supports the hypothesis that tryptophan and its metabolites can be used as biomarkers predicting radiologic outcome and discriminate between RA and OA patients. Overall, our results strengthen the notion that tryptophan metabolism is closely linked to RA disease mechanisms.

Peripheral elimination of the sympathetic nervous system stimulates immunocyte retention in lymph nodes and ameliorates collagen type II arthritis.

OBJECTIVES: In collagen type II-induced arthritis (CIA), early activation of the sympathetic nervous system (SNS) is proinflammatory. Here, we wanted to find new target organs contributing to proinflammatory SNS effects. In addition, we wanted to clarify the importance of SNS-modulated immunocyte migration. METHODS: A new technique termed spatial energy expenditure configuration (SEEC) was developed to demonstrate bodily areas of high energy demand (to find new targets). We studied homing of labeled cells in vivo, lymphocyte expression of CCR7, supernatant concentration of CCL21, and serum levels of sphingosine-1-phosphate (S1P) in sympathectomized control/arthritic animals. RESULTS: During the course of arthritis, SEEC identified an early marked increase of energy expenditure in draining lymph nodes and spleen (nowhere else!). Although early sympathectomy ameliorated later disease, early sympathectomy increased energy consumption, organ weight, and cell numbers in arthritic secondary lymphoid organs, possibly a sign of lymphocyte retention (also in controls). Elimination of the SNS retained lymph node cells, elevated expression of CCR7 on lymph node cells, and increased CCL21. Serum levels of S1P, an important factor for lymphocyte egress, were higher in arthritic than control animals. Sympathectomy decreased S1P levels in arthritic animals to control levels. Transfer of retained immune cells from draining lymph nodes of sympathectomized donors to sympathectomized recipients markedly increased arthritis severity over weeks. CONCLUSIONS: By using the SEEC technique, we identified draining lymph nodes and spleen as major target organs of the SNS. The data show that the SNS increases egress of lymphocytes from draining lymph nodes to stimulate arthritic inflammation.

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 Fe(2+)) 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.

Proinflammatory receptor switch from Gαs to Gαi signaling by ß-arrestin-mediated PDE4 recruitment in mixed RA synovial cells.

OBJECTIVE: In chronic inflammation, prevention of cAMP degradation by phosphodiesterase-4 (PDE4) inhibition can be anti-inflammatory therapy. However, PDE4 inhibition was uneffective in rheumatoid arthritis (RA). Recent studies demonstrated that PDE4/ß-arrestin interaction at ß-adrenoceptors resulted in switching from Gαs to Gαi signaling and ERK1/2 activation. Such a switch in signaling might elicit proinflammatory effects. We aimed to investigate this possible Gαs to Gαi signaling switch in RA and osteoarthritis (OA) mixed synoviocytes. METHODS: Synoviocytes were treated alone or with combinations of adrenergic, dopaminergic, and adenosinergic drugs, rolipram (PDE4 inhibitor), inhibitors of Gαi signaling (pertussis toxin), and blockers of protein kinase A (PKA). Under normoxic or hypoxic conditions, proinflammatory TNF was the readout-parameter. We investigated co-expression and interaction of PDE4 and ß-arrestin by imaging techniques. Expression of pERK1/2 was analyzed by western blotting. RESULTS: Mixed synoviocytes in RA and OA possessed all major Gαs-coupled neurotransmitter receptors. Under hypoxia, particularly in RA cells, Gαs-coupled receptor agonists unexpectedly increased TNF and respective antagonists decreased TNF. Under hypoxia, rolipram alone or rolipram plus Gαs agonists increased TNF, which was reversed by pertussis toxin or PKA inhibition. Co-localization and interaction of PDE4 and ß-arrestin in synovial tissue and cells was demonstrated. Gαs agonists or rolipram plus Gαs agonists increased pERK1/2 expression. CONCLUSIONS: This study in human arthritic synovial tissue presents an unexpected proinflammatory switch from Gαs to Gαi signaling, which depends on PDE4/ß-arrestin interaction. This phenomenon is most probably responsible for reduced efficacy of PDE4 inhibitors and Gαs agonists in RA.

A sustained high fat diet for two years decreases IgM and IL-1 beta in ageing Wistar rats.

BACKGROUND: The immune system undergoes several alterations of innate and adaptive immunity during ageing. The main features of the aged immune system are a reduced diversity of T cell receptors and a reduced activity of innate immune cells with subsequent changes in adaptive immunity resulting in a less effective, less specific, and dys-regulated immune response and in an increased susceptibility towards infection, malignancy, and autoimmunity. The process is referred to as immunosenescence and is also modulated by environmental modifiers, such as dietary factors. High fat diet (HFD), via direct modulation of immune cell function by fatty acids and/or increased body fat mass, influences immune function. However, it is not clear whether HFD is beneficial or detrimental for the functioning of the ageing immune system. METHODS: Male Wistar rats fed with either a high fat diet (HFD 43 en% of fat) or control diet (SD, 25 en% of fat) over up to 24 month and were analyzed for plasma IL-1ß, IL-6, TNF, IgM, IgG1, IgA, IgG2a, IgG2b, IgG2c, light chains lambda and kappa, testosterone, prolactin and percentage of splenic B cells and apoptosis rate, respectively. RESULTS: In general, all analyzed immunoglobuline isotypes increased with age, except for IgA. This increase was attenuated by HFD. In HFD and SD rats the percentage of B cells in the spleen and also their apoptotic rate was lower in aged as compared to young animals with no additional diet-induced effect. Testosterone and prolactin levels were lower in old animals, as expected. There was a statistical trend towards an increased prolactin/testosterone ratio in middle aged (6-12 monthsnth) HFD rats as compared to SD. IL-6 was neither affected by HFD nor age. On the other hand, HFD rats showed a decrease in IL-1ß as compared to SD, which correlated with the above-mentioned suppressive effect on immunoglobulin isotypes, especially IgM. CONCLUSION: In Wistar rats, HFD reveals an immunosuppressive effect in ageing animals by decreasing immunoglobulins, especially IgM, and IL-1ß when compared to SD.

Cortisol-mediated adhesion of synovial fibroblasts is dependent on the degradation of anandamide and activation of the endocannabinoid system.

OBJECTIVE: In rheumatoid arthritis (RA) synovial fluid, levels of the endocannabinoids anandamide (AEA) and 2-arachidonylglycerol are elevated. Since synovial fibroblasts (SFs) possess all of the enzymes necessary for endocannabinoid synthesis, it is likely that these cells contribute significantly to elevated endocannabinoid levels. While glucocorticoids initiate endocannabinoid synthesis in neurons, this study was undertaken to test whether cortisol also regulates endocannabinoid levels in mesenchymal cells such as SFs, and whether this interferes with integrin-mediated adhesion. METHODS: Adhesion was determined in 1-minute intervals over 60 minutes using an xCELLigence system. Slopes from individual treatment groups were averaged and compared to the control. Fatty acid amide hydrolase (FAAH) and cyclooxygenase 2 (COX-2) were detected by immunocytochemistry, and AEA was detected by mass spectrometry. RESULTS: Cortisol increased the adhesion of RASFs and osteoarthritis SFs with a maximum of 200% at both 10(-7) M and 10(-8) M. When cortisol was administered together with either cannabinoid receptor 1 (CB(1) ) antagonist (rimonabant; 100 nM), CB(2) antagonist (JTE907; 100 nM), transient receptor potential vanilloid channel 1 (TRPV-1) antagonist (capsazepine; 1 µM), FAAH inhibitor, or COX-2 inhibitor, adhesion was reduced below the level in controls. Concomitant inhibition of FAAH and COX-2 reversed these effects. Mass spectrometry revealed the presence of AEA in SFs. CONCLUSION: Our findings indicate that glucocorticoid-induced adhesion is dependent on CB(1) /CB(2) /TRPV-1 activation. Since AEA is produced in SFs, this endocannabinoid is the most likely candidate to mediate these effects. Since AEA levels are regulated by COX-2 and FAAH, inhibition of both enzymes along with low-dose glucocorticoids may provide a therapeutic option to maximally boost the endocannabinoid system in RA, with possible beneficial effects.

Relationship between placenta growth factor 1 and vascularization, dehydroepiandrosterone sulfate to dehydroepiandrosterone conversion, or aromatase expression in patients with rheumatoid arthritis and patients with osteoarthritis.

OBJECTIVE: Proliferating pannus is in many aspects similar to placental tissue. Both fibroblast-rich tissues have high vascularity, and tissue from patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA) demonstrates conversion of androgenic prehormones to downstream estrogens. We undertook this study to investigate similarities between proliferating pannus and placental tissue by focusing on angiogenic placenta growth factor 1 (PlGF-1) in patients with OA and patients with RA. METHODS: We used immunohistochemistry to study the presence of PlGF-1, its synovial distribution, and the PlGF-1-expressing synovial cell type. The relationship between PlGF-1 and conversion of the biologically inactive placental prehormone dehydroepiandrosterone sulfate (DHEAS) to the biologically active dehydroepiandrosterone (DHEA) was investigated in mixed synovial cells. The effects of DHEA on PlGF-1 expression were studied by intracellular fluorescence-activated cell sorting analysis. RESULTS: PlGF-1-positive cells were detected in the lining and sublining areas in patients with RA and patients with OA, and cellular density was similar. Double staining revealed that PlGF-1-positive cells were macrophages. In RA and OA, the density of PlGF-1-positive cells correlated positively with the density of macrophages and the density of type IV collagen-positive vessels. The supernatant concentration of (3) H-DHEA after conversion from (3) H-DHEAS and the density of aromatase-positive cells were positively correlated with the density of PlGF-1-positive cells only in OA. Low DHEA concentrations (≤10(-9) M) had stimulatory effects on PlGF-1 when compared to serum concentrations (10(-8) M to 10(-7) M) in the monocytic cell line THP-1 and in primary mixed synovial cells. CONCLUSION: PlGF-1 functions similarly in inflamed synovium and in the placenta. It is related to vessel formation and, in OA patients, to androgen/estrogen conversion. Evolutionarily conserved functions of PlGF-1 for placental phenomena are obviously also present in synovial inflammation.

Components of the sympathetic nervous system as targets to modulate inflammation - rheumatoid arthritis synovial fibroblasts as neuron-like cells?

BACKGROUND: Catecholamines are major neurotransmitters of the sympathetic nervous system (SNS) and they are of pivotal importance in regulating numerous physiological and pathological processes. Rheumatoid arthritis (RA) is influenced by the activity of the SNS and its neurotransmitters norepinephrine (NE) and dopamine (DA) and early sympathectomy alleviates experimental arthritis in mice. In contrast, late sympathectomy aggravates RA, since this procedure eliminates anti-inflammatory, tyrosine hydroxylase (TH) positive cells that appear in the course of RA. While it has been shown that B cells can take up, degrade and synthesize catecholamines it is still unclear whether this also applies to synovial fibroblasts, a mesenchymal cell that is actively engaged in propagating inflammation and cartilage destruction in RA. Therefore, this study aims to present a detailed description of the catecholamine pathway and its influence on human RA synovial fibroblasts (RASFs). RESULTS: RASFs express all catecholamine-related targets including the synthesizing enzymes TH, DOPA decarboxylase, dopamine beta-hydroxylase, and phenylethanolamine N-methyltransferase. Furthermore, vesicular monoamine transporters 1/2 (VMAT1/2), dopamine transporter (DAT) and norepinephrine transporter (NET) were detected. RASFs are also able to degrade catecholamines as they express monoaminoxidase A and B (MAO-A/MAO-B) and catechol-O-methyltransferase (COMT). TNF upregulated VMAT2, MAO-B and NET levels in RASFs. DA, NE and epinephrine (EPI) were produced by RASFs and extracellular levels were augmented by either MAO, COMT, VMAT or DAT/NET inhibition but also by tumor necrosis factor (TNF) stimulation. While exogenous DA decreased interleukin-6 (IL-6) production and cell viability at the highest concentration (100 µM), NE above 1 µM increased IL-6 levels with a concomitant decrease in cell viability. MAO-A and MAO-B inhibition had differential effects on unstimulated and TNF treated RASFs. The MAO-A inhibitor clorgyline fostered IL-6 production in unstimulated but not TNF stimulated RASFs (10 nM-1 µM) while reducing IL-6 at 100 µM with a dose-dependent decrease in cell viability in both groups. The MAO-B inhibitor lazabemide hydrochloride did only modestly decrease cell viability at 100 µM while enhancing IL-6 production in unstimulated RASFs and decreasing IL-6 in TNF stimulated cells. CONCLUSIONS: RASFs possess a complete and functional catecholamine machinery whose function is altered under inflammatory conditions. Results from this study shed further light on the involvement of sympathetic neurotransmitters in RA pathology and might open therapeutic avenues to counteract inflammation with the MAO enzymes being key candidates.

Cannabidiol: Influence on B Cells, Peripheral Blood Mononuclear Cells, and Peripheral Blood Mononuclear Cell/Rheumatoid Arthritis Synovial Fibroblast Cocultures.

Background: Cannabidiol (CBD), one major nonintoxicating phytocannabinoid from Cannabis sativa demonstrated anti-inflammatory effects in animal models of several inflammatory conditions, including arthritis. However, it is still unknown which cell types mediate these anti-inflammatory effects of CBD, and, since CBD binds to a plethora of receptors and enzymes, it is complicated to pinpoint its mechanism of action. In this study, we elucidate the effects of CBD on B cells and peripheral blood mononuclear cells (PBMCs) in respect to survival, calcium mobilization, drug uptake, and cytokine (IL-6, IL-10, and TNF) and immunoglobulin production. Methods: Modulation of intracellular calcium and drug uptake in B cells was determined by using the fluorescent dyes Cal-520 and PoPo3, respectively. Cytokine and immunoglobulin production was evaluated by enzyme-linked immunosorbent assay. PBMC composition and B cell survival after CBD treatment was assessed by flow cytometry. Results: B cells express two major target receptors for CBD, TRPV2 (transient receptor potential vanilloid 2) and TRPA1 (transient receptor potential ankyrin 1), which are not regulated by B cell activation. CBD increased intracellular calcium levels in mouse and human B cells, which was accompanied by enhanced uptake of PoPo3. These effects were not dependent on transient receptor potential channel activation. CBD increased the number of early apoptotic B cells at the expense of viable cells and diminished interleukin (IL)-10 and tumor necrosis factor (TNF) production when activated T cell independently. In PBMCs, CBD increased IL-10 production when B cells were activated T cell dependent, while decreasing TNF levels when activated T cell independently. In PBMC/rheumatoid synovial fibroblast cocultures, CBD reduced IL-10 production when B cells were activated T cell independently. Immunoglobulin M production was augmented by CBD when B cells were activated with CpG. Conclusion: CBD is able to provide pro- and anti-inflammatory effects in isolated B cells and PBMCs. This is dependent on the activating stimulus (T cell dependent or independent) and concentration of CBD. Therefore, CBD might be used to dampen B cell activity in autoimmune conditions such as rheumatoid arthritis, in which B cells are activated by specific autoantigens.

Increased density of sympathetic nerve fibers in metabolically activated fat tissue surrounding human synovium and mouse lymph nodes in arthritis.

OBJECTIVE: To investigate the density of sympathetic nerve fibers in and the metabolic activation of fat tissue surrounding human synovium in rheumatoid arthritis (RA)/osteoarthritis (OA) and in the draining lymph nodes of arthritic and normal mice. METHODS: Using immunofluorescence and immunohistochemistry, the density of sympathetic nerve fibers and the presence of nerve repellent factors were investigated. The metabolic activation of fat tissue was estimated by the occurrence of small-vacuole adipocytes, expression of ß3-adrenoceptors, and adipose tissue weight. RESULTS: The density of sympathetic nerve fibers was markedly increased in fat tissue surrounding RA synovium compared with that in fat tissue surrounding OA synovium. In adipose tissue adjacent to draining lymph nodes, the density of sympathetic nerve fibers was higher in arthritic mice compared with normal mice. In human synovium and mouse draining lymph nodes, the 2 sympathetic nerve repellent factors, semaphorin 3C and semaphorin 3F, were highly expressed. In arthritic compared with normal mice, the fat tissue around lymph nodes was markedly lighter, adipocytes had more fragmented lipid droplets, and fat tissue demonstrated high expression of ß3-adrenoceptors. CONCLUSION: This study demonstrated an increased density of sympathetic nerve fibers in metabolically activated fat tissue surrounding human RA synovium and the draining lymph nodes of arthritic mice. Because sympathetic neurotransmitters stimulate lipolysis, the repulsion of sympathetic nerve fibers from inflamed regions and their increased occurrence in fat tissue probably represent an adaptive program to support the proinflammatory process by releasing energy-rich substrates.

Impact of Δ9-Tetrahydrocannabinol on Rheumatoid Arthritis Synovial Fibroblasts Alone and in Co-Culture with Peripheral Blood Mononuclear Cells.

δ9-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 (0.1-25 µM) 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 (≥5 µM) 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 µM). Proliferation was slightly enhanced at intermediate THC concentrations (1-10 µM) but was completely abrogated at 25 µM. 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-γ (IFN-γ) 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.

Characterization of B- and T-Cell Compartment and B-Cell Related Factors Belonging to the TNF/TNFR Superfamily in Patients With Clinically Active Systemic Lupus Erythematosus: Baseline BAFF Serum Levels Are the Strongest Predictor of Response to Belimumab after Twelve Months of Therapy.

Background: Patients with systemic lupus erythematosus (SLE) show increased serum levels of tumor necrosis factor (TNF)/TNF receptor (R) superfamily member, e.g. BAFF (B lymphocyte stimulator). Belimumab, a monoclonal antibody against soluble BAFF, is used for treatment of SLE. Although B cells are the main target, a BAFF-dependent T-cell activation pathway also plays a role. High levels of anti-DNA antibodies and low complement at baseline are known predictors of response to Belimumab. Objectives: To explore the association of circulating lymphocytes and serum levels of B- cell related TNF/TNFR superfamily members with response to Belimumab in SLE patients. Methods: Twenty-one SLE patients received Belimumab. Clinical evaluation and laboratory tests were performed at baseline, at 6 and 12 months. TNF super-family members (BAFF, APRIL, sBCMA, sCD40L, sTACI, TWEAK) were tested by high-sensitivity ELISA in all patients, and lymphocyte immunophenotyping was performed by flow cytometry in ten subjects. SLE-disease activity was assessed by SLEDAI-2K score. Linear regression modeling was used to investigate parameters influencing SLEDAI-2K and anti-dsDNA antibody titers over time and for predictive models. Results: Clinical improvement was observed in all patients. A global reduction of circulating B cells, especially naïve, was detected, without variation in the T-cell compartment. All TNF family members decreased, whereas APRIL remained constant. The increase in serum levels of C3 (p = 0.0004) and sTACI (p = 0.0285) was associated with a decrease of SLEDAI-2K. The increase of C4 (p = 0.027) and sBCMA (p = 0.0015) and the increase of CD8+ T cells (p = 0.0160) were associated with a decrease, whereas an increase of sCD40L in serum (p = 0.0018) and increased number of CD4+ T cells (p = 0.0029) were associated with an increase, in anti-dsDNA antibody titers, respectively. Using stepwise forward inclusion, the minimal model to predict SLEDAI-2K response at 12 months included BAFF (p = 3.0e - 07) and SLEDAI-2K (p = 7.0e - 04) at baseline. Baseline APRIL levels also showed an association, although the overall model fit was weaker. Conclusion: In our real-life cohort, baseline serum levels of BAFF were the best predictor of response to Belimumab, confirming post-hoc results of the BLISS study and suggesting the utility of this particular biomarker for the identification of patients who are more likely to respond.

Cannabidiol (CBD): a killer for inflammatory rheumatoid arthritis synovial fibroblasts.

Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated anti-inflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF). These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin (TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A, also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltage-dependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses anti-arthritic activity and might ameliorate arthritis via targeting synovial fibroblasts under inflammatory conditions.

Positive and negative cooperativity of TNF and Interferon-γ in regulating synovial fibroblast function and B cell survival in fibroblast/B cell co-cultures.

Synovial fibroblasts (SF) were reported to produce B cell activating factor (BAFF) in response to stimulation with interferon-γ (IFN-γ) or tumor necrosis factor (TNF). However, the influence of these pro-inflammatory cytokines on other receptors/ligands of the TNF superfamily or associated cytokine receptors in SF has not been investigated yet. Here we show the differential regulation of BAFF (CD257), Fn14 (CD266), TACI (CD267), BAFF-R (CD268), BCMA (CD269), CD40 ligand (CD40L, CD154), IFN-γR (CD119), Leptin receptor (ObR, CD295), VCAM-1 (CD106) and membrane TGF-ß in isolated SF and the impact of IFN-γ/TNF co-incubation on proliferation, IL-6 and IL-8 production. In addition, the impact of differentially stimulated SF on B cell survival in co-cultures was assessed. Surface cytokines and cytokine receptors were detected by flow cytometry. Soluble cytokine receptors and cytokines were quantified by ELISA. Proliferation was assessed by cell titer blue. Murine B cell survival in fibroblast/ B cell co-cultures was determined by annexin V/propidium iodide staining and flow cytometry. IFN-γ together with TNF synergistically and significantly increased the cell surface levels of BAFF, Fn14, TACI, BAFF-R, BCMA, CD40L, ObR and IFN-γR in rheumatoid arthritis SF after 72 h incubation. Soluble BAFF was only induced by IFN-γ and inhibited by TNF. Addition of TWEAK had no influence on proliferation or IL-8 production but decreased TNF-induced IL-6 production, whereas APRIL, BAFF and leptin did not modulate TNF or TNF/IFN-γ-induced proliferation or cytokine production. Proliferation was increased by TNF and further enhanced by the addition of IFN-γ. In co-culture experiments, SF stimulated with TNF/IFN but not TNF or IFN-γ alone increased shedding of VCAM-1 and expression of membrane TGFß, which was associated with reduced survival of murine B cells. IFN-γ and TNF regulate the expression of TNF family member cytokines and associated receptors. Ligation of IFN-γR and Fn14 under pro-inflammatory conditions modulated IL-6/IL-8 production and proliferation. In B cell/SF co-cultures, the combination of TNF/IFN reduced B cell survival possibly via enhanced VCAM-1 shedding and/or increased TGF-ß production. IFN-γ is necessary for the observed effects on B cell survival and SF cytokine production and emphasizes its anti-inflammatory role in rheumatoid arthritis.

A thyroid hormone network exists in synovial fibroblasts of rheumatoid arthritis and osteoarthritis patients.

While patients with rheumatoid arthritis (RA) sometimes demonstrate thyroidal illness, the role of thyroid hormones in inflamed synovial tissue is unknown. This is relevant because thyroid hormones stimulate immunity, and local cells can regulate thyroid hormone levels by deiodinases (DIO). The study followed the hypothesis that elements of a thyroid hormone network exist in synovial tissue. In 12 patients with RA and 32 with osteoarthritis (OA), we used serum, synovial fluid, synovial tissue, and synovial fibroblasts (SF) in order to characterize the local thyroid hormone network using ELISAs, immunohistochemistry, imaging methods, tissue superfusion studies, cell-based ELISAs, flow cytometry, and whole genome expression profiling. Serum/synovial fluid thyroid hormone levels were similar in RA and OA (inclusion criteria: no thyroidal illness). The degradation product termed reverse triiodothyronine (reverse T3) was much lower in serum compared to synovial fluid indicating biodegradation of thyroid hormones in the synovial environment. Superfusion experiments with synovial tissue also demonstrated biodegradation, particularly in RA. Cellular membrane transporters of thyroid hormones, DIOs, and thyroid hormone receptors were present in tissue and SF. Density of cells positive for degrading DIOs were higher in RA than OA. TNF increased protein expression of degrading DIOs in RASF and OASF. Gene expression studies of RASF revealed insignificant gene regulation by bioactive T3. RA and OA synovial tissue/SF show a local thyroid hormone network. Thyroid hormones undergo strong biodegradation in synovium. While bioactive T3 does not influence SF gene expression, SF seem to have a relay function for thyroid hormones.

Comprehensive Longitudinal Surveillance of the IgG Autoantibody Repertoire in Established Systemic Lupus Erythematosus.

OBJECTIVE: To investigate the role of epitope spreading in established systemic lupus erythematosus (SLE). METHODS: IgG autoantibody reactivity with 398 distinct recombinant proteins was measured over a period of 6 years in 69 SLE patients and compared to that in 45 controls. Changes in mean fluorescence intensity (MFI), number of autoantibodies to distinct antigens, and reactivity with distinct clones of established antigenic targets (e.g., U1 RNP, Sm, and ribosomal P) representing epitope fine mapping were assessed. Linear mixed modeling, adjusted with Bonferroni correction for age and sex, was applied. RESULTS: The total number of autoantibodies, mean MFI, and number of autoantibodies in epitope fine mapping were higher in SLE patients compared to controls (P < 0.0001). The total number of antibodies to distinct autoantigens remained stable over time, while the mean MFI decreased over time in SLE (P < 0.021). SLE patients showed variable recognition of epitopes in fine mapping over time. In particular, in SLE patients, more clones of the U1 RNP complex were recognized at the time of new organ involvement (+0.65) (P = 0.007). Mean MFI was higher in patients with lupus nephritis (P = 0.047). The time-averaged MFIs of 22 individual autoantibodies (including double-stranded DNA [dsDNA]) were higher, after Bonferroni correction, in SLE (P < 0.0001). The MFIs of dsDNA and histone cluster 2 H3c were associated with scores on the Systemic Lupus Activity Measure (P < 0.0001). CONCLUSION: Longitudinal surveillance of the IgG autoantibody repertoire in established SLE reveals evidence of sustained breadth of autoantibody repertoire without significant expansion. Associations of disease activity with dsDNA and with histone H3 autoantibodies were confirmed.

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.