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.

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.

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.

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.

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.

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.

Selective killing of proinflammatory synovial fibroblasts via activation of transient receptor potential ankyrin (TRPA1).

BACKGROUND: Studies in rheumatoid arthritis synovial fibroblasts (RASF) demonstrated the expression of several transient receptor potential channels (TRP) such as TRPV1, TRPV2, TRPV4, TRPA1 and TRPM8. Upon ligation, these receptors increase intracellular calcium but they have also been linked to modulation of inflammation in several cell types. TNF was shown to increase the expression of TRPA1, the receptor for mustard oil and environmental poisons in SF, but the functional consequences have not been investigated yet. METHODS: TRPA1 was detected by immunocytochemistry, western blot and cell-based ELISA. Calcium measurements were conducted in a multimode reader. Cell viability was assessed by quantification of lactate dehydrogenase (LDH) in culture supernatants and "RealTime-Glo" luminescent assays. IL-6 and IL-8 production by SF was quantified by ELISA. Proliferation was determined by cell titer blue incorporation. RESULTS: After 72 h, mimicking proinflammatory conditions by the innate cytokine TNF up-regulated TRPA1 protein levels in RASF which was accompanied by increased sensitivity to TRPA1 agonists AITC and polygodial. Under unstimulated conditions, polygodial elicited calcium flux only in the highest concentrations used (50 µM and 25 µM). TNF preincubation substantially lowered the activation threshold for polygodial (from 25 µM to 1 µM). In the absence of TNF pre-stimulation, only polygodial in high concentrations was able to reduce viability of synovial fibroblasts as determined by a real-time viability assay. However, following TNF preincubation, stimulation of TRPA1 led to a fast (<30 min) viability loss by necrosis of synovial fibroblasts. TRPA1 activation was also associated with decreased proliferation of RASFs, an effect that was also substantially enhanced by TNF preincubation. On the functional level, IL-6 and IL-8 production was attenuated by the TRPA1 antagonist A967079 but also polygodial, although the latter mediated this effect by reducing cell viability. CONCLUSION: Simulating inflamed conditions by preincubation of synovial fibroblasts with TNF up-regulates and sensitizes TRPA1. Subsequent activation of TRPA1 increases calcium flux and substantially reduces cell viability by inducing necrosis. Since TRPA1 agonists in the lower concentration range only show effects in TNF-stimulated RASF, this cation channel might be an attractive therapeutic target in chronic inflammation to selectively reduce the activity of proinflammatory SF in the joint.

α-MSH modulates cell adhesion and inflammatory responses of synovial fibroblasts from osteoarthritis patients.

INTRODUCTION: The synovium is a target for neuropeptides. Melanocortins have attained particular attention as they elicit antiinflammatory effects. Although synovial fluid from patients with rheumatic diseases contains α-melanocyte-stimulating hormone (α-MSH) it is unknown whether synovial fibroblasts generate α-MSH and respond to melanocortins. METHODS: Synovial tissue was obtained from osteoarthritis (OA) patients. Cells were isolated and prepared either as primary mixed synoviocytes or propagated as synovial fibroblasts (OASFs). Melanocortin receptor (MC) and proopiomelanocortin (POMC) expression were investigated by endpoint RT-PCR, immunofluorescence and Western immunoblotting. Functional coupling of MC1 was assessed by cAMP and Ca(2+) assays. Cell adhesion was monitored by the xCELLigence system. Secretion of α-MSH, tumour necrosis factor (TNF), interleukin (IL)-6 and IL-8 was determined by ELISA. RESULTS: OASFs in vitro expressed MC1. MC1 transcripts were present in synovial tissue and appropriate immunoreactivity was detected in synovial fibroblasts in situ. OASFs contained truncated POMC transcripts but neither full-length POMC mRNA, POMC protein nor α-MSH were detectable. In accordance with this only truncated POMC transcripts were present in synovial tissue. α-MSH increased cAMP dose-dependently but did not alter calcium in OASFs. α-MSH also enhanced adhesion of OASFs to fibronectin and reduced TNF, IL-6 and IL-8 secretion in primary mixed synoviocyte cultures. In OASFs, α-MSH modulated basal and TNF/IL-1ß-mediated secretion of IL-6 and IL-8. CONCLUSION: Synovial fibroblasts express MC1in vitro and in situ. α-MSH elicits biological effects in these cells suggesting an endogenous immunomodulatory role of melanocortins within the synovium. Our results encourage in vivo studies with melanocortins in OA models.

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.

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.

Synovial fibroblasts integrate inflammatory and neuroendocrine stimuli to drive rheumatoid arthritis.

Rheumatoid arthritis is a chronic inflammatory disease that is characterized by pannus tissue consisting of synovial fibroblasts (SF), macrophages and lymphocytes. The inflammatory milieu in the joint activates resident SF and transforms them in a tumor-like phenotype. These changes manifest in resistance to Fas-induced apoptosis and production of cytokines, chemokines and matrix metalloproteinases. By alterations in DNA methylation, SF retain their transformed phenotype even in the absence of pro-inflammatory stimuli and are able to spread arthritis to unaffected joints. Furthermore, SF integrate neuroendocrine input to modulate inflammation since they possess receptors for several neurotransmitters (e.g., dopamine, norepinephrine and glutamate). Until now, no specific therapy targeting SF is available; however, reprogramming them to a regulatory phenotype might limit joint destruction and cartilage degradation.

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.

Increased expression of dopamine receptors in synovial fibroblasts from patients with rheumatoid arthritis: inhibitory effects of dopamine on interleukin-8 and interleukin-6.

OBJECTIVE: Observations in both animal models of arthritis and patients with rheumatoid arthritis (RA) suggest a role for dopamine and its receptors in RA. Because synovial fibroblasts (SFs) contribute to inflammation and joint destruction in RA, the aim of this study was to investigate dopaminergic pathways in SFs obtained from patients with RA and, for comparison, in SFs from patients with osteoarthritis (OA) undergoing knee joint replacement surgery. METHODS: The expression of all dopamine receptors (D1 -D5 ) and dopamine transporter was assessed by immunofluorescence and immunohistochemical staining. The levels of dopamine receptor and tyrosine hydroxylase messenger RNA were measured by real-time polymerase chain reaction. The intracellular content of dopamine, its precursor, and its main metabolites was assayed by high-performance liquid chromatography. The influence of dopamine on proinflammatory interleukin-6 (IL-6) and IL-8, matrix metalloproteinase 3, and tissue inhibitor of metalloproteinases 1 (TIMP-1) and TIMP-2 was studied in SFs. RESULTS: SFs possess an intrinsic dopaminergic system, including dopamine receptors, dopamine transporter, and tyrosine hydroxylase, and contain dopamine, its precursor, and its main metabolites. SFs from patients with RA, in comparison with those from patients with OA, showed increased expression of dopamine receptors D1 and D5 , and exogenous dopamine strongly inhibited the production of IL-8 in patients with RA. CONCLUSION: SFs from patients with RA and patients with OA show a dopaminergic phenotype. The expression of D1-like dopamine receptors was higher in RASFs, and this increased expression may lead to antiinflammatory effects, as demonstrated by the expression of IL-8. Studies in animal models and patients with RA are needed to assess the therapeutic potential of endogenous, local production of dopamine in synoviocytes.

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.

Integrins and their ligands in rheumatoid arthritis.

Integrins play an important role in cell adhesion to the extracellular matrix and other cells. Upon ligand binding, signaling is initiated and several intracellular pathways are activated. This leads to a wide variety of effects, depending on cell type. Integrin activation has been linked to proliferation, secretion of matrix-degrading enzymes, cytokine production, migration, and invasion. Dysregulated integrin expression is often found in malignant disease. Tumors use integrins to evade apoptosis or metastasize, indicating that integrin signaling has to be tightly controlled. During the course of rheumatoid arthritis, the synovial tissue is infiltrated by immune cells that secrete large amounts of cytokines. This pro-inflammatory milieu leads to an upregulation of integrin receptors and their ligands in the synovial tissue. As a consequence, integrin signaling is enhanced, leading to enhanced production of matrix-degrading enzymes and cytokines. Furthermore, in analogy to invading tumors, synovial fibroblasts start invading and degrading cartilage, thereby generating extracellular matrix debris that can further activate integrins.

Glucocorticoids increase alpha5 integrin expression and adhesion of synovial fibroblasts but inhibit ERK signaling, migration, and cartilage invasion.

OBJECTIVE: In rheumatoid arthritis (RA), integrins mediate cell adhesion, migration, and invasion, and their expression is regulated by cytokines and growth factors. The aim of this study was to investigate whether hormones such as cortisol or other steroids can influence integrin expression and function in the synovial cells of patients with RA. METHODS: We performed immunofluorescence and fluorescence-activated cell sorting analyses to quantify surface integrin levels. Adhesion and migration assays were performed to study the function of synovial fibroblasts (SFs). ERK activation was measured by cellular activation of a signaling enzyme-linked immunosorbent assay. Invasion of SFs into cartilage was determined in the SCID mouse coimplantation model of RA in vivo. RESULTS: In RA, expression of integrin subunits alpha5, alphav, and beta1 was higher at the site of invasion compared with the sublining zone. Testosterone and 17beta-estradiol had no influence on integrin levels, but cortisol up-regulated expression of the alpha5 subunit in a time-dependent and dose-dependent manner. In addition, cortisol increased the adhesion of SFs to fibronectin and inhibited ERK signaling upon integrin activation or upon stimulation with tumor necrosis factor. Small interfering RNA or a neutralizing antibody to alpha5 integrin increased SF migration, indicating that up-regulated alpha5 integrin is responsible for an immobile phenotype. In addition, in the SCID mouse model, SF invasion into cartilage was attenuated by glucocorticoid treatment in vivo. CONCLUSION: Glucocorticoids increase integrin expression and the adhesion of cells to fibronectin, inhibit ERK signaling, and down-regulate the invasiveness of SFs in vivo. This study demonstrates that an important antiinflammatory aspect of glucocorticoids is regulating the expression and function of alpha5 integrin.

Neuronally released sympathetic neurotransmitters stimulate splenic interferon-gamma secretion from T cells in early type II collagen-induced arthritis.

OBJECTIVE: The sympathetic nervous system confers a proinflammatory effect during the early phase of type II collagen-induced arthritis (CIA). These effects might be mediated by up-regulation of cytokines such as interferon-gamma (IFNgamma) or chemokines such as CXCL1 (cytokine-induced neutrophil chemoattractant, or KC). This study aimed to identify the role of sympathetic neurotransmitters in splenic secretion of IFNgamma and KC shortly after the onset of CIA. METHODS: At different time points during CIA, we determined the density of sympathetic nerve fibers in the spleens of mice. Spleens were removed when the mouse joints were assessed an arthritis score of 3 (at approximately day 32). Spleen slices (0.35 mm thick) were transferred to superfusion microchambers to allow observation of the effects of physiologically released sympathetic neurotransmitters on secretion of IFNgamma and KC. RESULTS: Compared with control mice, mice with CIA demonstrated a decrease in sympathetic nerve fiber density in the spleens, which reached a minimum density shortly after the start of symptomatic arthritis (day 32). T cell depletion markedly reduced splenic secretion of IFNgamma and KC. Electrical-field stimulation of the spleen slices reduced the secretion of IFNgamma, which was attenuated by an alpha1-adrenergic antagonist. In addition, splenic IFNgamma secretion was stimulated by norepinephrine, via beta-adrenergic receptors, and adenosine, via A1 adenosine receptors. Similarly, splenic KC secretion was stimulated by norepinephrine, via beta-adrenergic receptors. CONCLUSION: The results of this study demonstrate a reduction of sympathetic nerve fibers in the spleens of arthritic animals. Nevertheless, sympathetic nerves help to increase secretion of IFNgamma and KC, which, at the early stages shortly after the onset of CIA, can contribute to the proinflammatory effect of the sympathetic nervous system.