Proinflammatory α-Adrenergic Neuronal Regulation of Splenic IFN-γ, IL-6, and TGF-ß of Mice from Day 15 onwards in Arthritis.

INTRODUCTION: In arthritic mice, a sympathetic influence is proinflammatory from the time point of immunization until the onset of disease (days 0-32), but reasons are unknown. Disruption of the major anti-inflammatory pathway through Gαs-coupled receptors probably play a role. For example, noradrenaline cannot operate via anti-inflammatory ß2-adrenoceptors but through proinflammatory α1/2-ad-renoceptors. This might happen, first, through a loss of sympathetic nerve fibers in inflamed tissue with low neurotransmitter levels (noradrenaline only binds to high-affinity α-adrenoceptors) and, second, through an alteration in G-protein receptor coupling with a predominance of α-adrenergic signaling. We hypothesized that both mechanisms play a role in the course of collagen type II-induced arthritis (CIA) in the spleen in mice. METHODS: In CIA mice, nerve fiber density in the spleen was quantified by immunohistochemistry techniques. The functional impact of sympathetic nerve fibers in the spleen was studied by a micro-superfusion technique of spleen slices with a focus on the secretion of IFN-γ and IL-6 (proinflammatory) and TGF-ß (anti-inflammatory). RESULTS: During CIA, sympathetic nerve fibers get increasingly lost from day14 until day 55 after immunization. The influence of electrically released noradrenaline diminishes in the course of arthritis. At all investigated time points (days 14, 32, and 55), only proinflammatory neuronal α-adrenergic effects on cytokine secretion were demonstrated (i.e., stimulation of IFN-γ and IL-6 and inhibition of TGF-ß). CONCLUSION: Sympathetic nerve fibers are rapidly lost in the spleen, and only proinflammatory α-adrenergic neuronal regulation of cytokine secretion takes place throughout the course of arthritis. These results support a predominance of a proinflammatory α-adrenergic sympathetic influence in arthritis.

Increased pain and sensory hyperinnervation of the ligamentum flavum in patients with lumbar spinal stenosis.

Nociceptive sensory nerve fibers have never been investigated in the ligamentum flavum (LF) of patients with LSS. The aim was to analyze nociceptive sensory nerve fibers in the ligamentum flavum (LF) of patients with LSS. A prospective study in patients with lumbar spinal stenosis (LSS) undergoing invasive surgical treatment for lumbar spinal stenosis (LSS) with flavectomy was performed. Patients with LSS were subjected to flavectomy and density of sensory and sympathetic nerve fibers, macrophages, vessels, activated fibroblasts, and cells were investigated by immunostaining techniques. A group of patients with acute disc herniation served as control group. We found a higher density of sensory nerve fibers in LSS patients versus controls. These findings support the role of LF in associated low back pain. Density of sensory nerve fibers in LSS, was positively correlated with typical markers of clinical pain and functional disability, but not with LF density of activated fibroblasts. Inflammation as estimated by macrophage infiltration and higher vascularity does not play a marked role in LF in our LSS patients. In the present study, compared to men with LSS, women with LSS demonstrate more pain and depression, and show a higher density of sensory nerve fibers in LF. This study shed new light on nociceptive nerve fibers, which are increased in LSS compared to controls. The findings speak against a strong inflammatory component in LSS. A higher pain levels in women compared to men can be explained by a higher density of nociceptive nerve fibers. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-7, 2019.

Increased extracellular water measured by bioimpedance and by increased serum levels of atrial natriuretic peptide in RA patients-signs of volume overload.

The aim of the study is to investigate water compartments in patients with rheumatoid arthritis (RA). Acute inflammatory episodes such as infection stimulate water retention, chiefly implemented by the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA) axis. This is an important compensatory mechanism due to expected water loss (sweating etc.). Since SNS and HPA axis are activated in RA, inflammation might be accompanied by water retention. Using bioimpedance analysis, body composition was investigated in 429 controls and 156 treatment-naïve RA patients between January 2008 and December 2014. A group of 34 RA patients was tested before and after 10 days of intensified therapy. Levels of pro-atrial natriuretic peptide (proANP) and expression of atrial natriuretic peptide in synovial tissue were investigated in 15 controls and 14 RA patients. Extracellular water was higher in RA patients than controls (mean ± SEM: 49.5 ± 0.3 vs. 36.7 ± 0.1, % of total body water, p < 0.0001). Plasma levels of proANP were higher in RA than controls. RA patients expressed ANP in synovial tissue, but synovial fluid levels and synovial tissue superfusate levels were much lower than plasma levels indicating systemic origin. Systolic/diastolic blood pressure was higher in RA patients than controls. Extracellular water levels did not change in RA patients despite 10 days of intensified treatment. This study demonstrates signs of intravascular overload in RA patients. Short-term intensification of anti-inflammatory therapy induced no change of a longer-lasting imprinting of water retention indicating the requirement of additional treatment. The study can direct attention to the area of volume overload.

Neuronal α1/2-adrenergic stimulation of IFN-γ, IL-6, and CXCL-1 in murine spleen in late experimental arthritis.

OBJECTIVE: Functional cross-talk exists between sympathetic nerve fibers and cytokine-producing splenic cells in early collagen type II-induced arthritis (CIA) (day 32). These earlier experiments demonstrated exclusively neuronal sympathetic regulation of IFN-γ, CXCL1, IL-6, and TGF-ß. However, in late arthritis, the sympathetic influence might change due to loss of sympathetic nerve fibers and appearance of neurotransmitter-producing cells. We aimed to investigate neurotransmitter-dependent regulation of IFN-γ, CXCL1, IL-6, and TGF-ß in murine spleen in late CIA. METHODS: Spleens were removed when animals reached day 58 (46-68) after immunization to generate 0.35 mm-thick spleen slices, which were transferred to superfusion microchambers to electrically induce release of neurotransmitters. Using respective neurotransmitter antagonists, effects of released neurotransmitters on cytokine secretion were investigated. RESULTS: There was electrically induced inhibition of IFN-γ, CXCL1, and IL-6, and stimulation of TGF-ß, which was much less pronounced than in early CIA. There existed ß adrenergic inhibition of IFN-γ, IL-6, and TGF-ß (and stimulation of CXCL1) independent of electrical stimulation (interpreted as non-neuronal). However, there was a neuronal α1/2 adrenergic stimulation of IFN-γ, CXCL1, and IL-6 and, we observed neuronal A1-adenosinergic stimulation of TGF-ß. CONCLUSIONS: In the late phase of CIA, non-neuronal modulation of cytokine secretion increases while neuronal regulation strikingly decreases. Particularly, ß-adrenergic effects are non-neuronal while α1/2-adrenergic effects are clearly neuronal. We suggest that alterations in sympathetic innervation of the spleen fundamentally change the functional neuroimmune interplay in the spleen of arthritic mice.

Intra-articular glucocorticoid injections decrease the number of steroid hormone receptor positive cells in synovial tissue of patients with persistent knee arthritis.

OBJECTIVES: To explore changes in the number of steroid hormone receptor positive cells in synovial tissue (ST) after intra-articular glucocorticoid injection, to correlate these changes with changes in clinical variables, and to evaluate whether the number of steroid hormone receptor positive cells predicted the clinical response to glucocorticoid injection. METHODS: Fourteen patients with persistent knee arthritis despite at least two previous injections in an outpatient setting received an intra-articular injection with glucocorticoids, followed by 3 days of admission with bed rest. Clinical efficacy was assessed at 6 and 12 weeks. ST biopsies were performed 2 weeks before and 12 weeks after the injection. The presence of different cell types (T cells, macrophages, fibroblast-like synoviocytes) and numbers of glucocorticoid, androgen and oestrogen α and ß receptor positive cells were evaluated by histochemistry. RESULTS: Patients showed, despite previous failures, good clinical response to glucocorticoid injection, with significant improvement in erythrocyte sedimentation rate, visual analogue scale (VAS) for pain, and joint disability score. The number of steroid hormone receptor positive cells decreased markedly (p<0.05 for all four receptors). The decrease in oestrogen receptor α positive cells correlated significantly with the improvement in VAS for pain and joint disability score. The number of glucocorticoid, androgen and oestrogen α and ß receptor positive cells before injection did not predict the effect of treatment. CONCLUSIONS: Intra-articular glucocorticoid injections followed by bed rest for persistent arthritis are clinically effective and significantly decrease the number of steroid hormone receptor positive cells in ST. The relevance of the latter needs further study.

Sympathetic inhibition of IL-6, IFN-γ, and KC/CXCL1 and sympathetic stimulation of TGF-ß in spleen of early arthritic mice.

OBJECTIVES: The connection between sympathetic nerve fibers and immune cells in the spleen is known. In the context of arthritis, the functional meaning of the neuroimmune contact remains unclear. From immunization until disease outbreak, the sympathetic nervous system (SNS) has a proinflammatory influence which is converted into an anti-inflammatory influence after disease outbreak. This study investigated the influence of neuronally released neurotransmitters on IFN-γ, KC (CXCL1), IL-6, and TGF-ß in spleen of mice shortly after outbreak of collagen type II-induced arthritis. METHODS: Spleens were removed when animals reached an arthritis score of 3 on a scale of 1-16 (approx. on day 32) in order to generate 0.35 mm-thick spleen slices. Spleen slices were transferred to superfusion microchambers in order to electrically induce release of sympathetic neurotransmitters. By means of this technique, the effect of physiologically released neurotransmitters was investigated on secretion of IFN-γ, KC, IL-6, and TGF-ß. RESULTS: High amounts of IFN-γ, KC, IL-6, and TGF-ß were released from superfused spleen, and electrical stimulation markedly inhibited IFN-γ, KC, and IL-6 release but pronouncedly stimulated TGF-ß. The adrenergic influence via ß-adrenoceptors stimulated release of IL-6 and, particularly, TGF-ß. However, catecholamines inhibit release of IL-6 via α1-adrenergic pathways but without any effect on TGF-ß. The co-transmitter adenosine stimulated IL-6 release via A1-adenosine receptors but no influence was recognized on TGF-ß. CONCLUSION: At disease outbreak, electrically released endogenous neurotransmitters of the SNS inhibit IFN-γ, KC, and IL-6 but ß-adrenergically stimulate TGF-ß. This creates an anti-inflammatory milieu that might be responsible for the observed dual influence of the SNS on arthritis.

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.

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.