Silvia Capellino

Estrogens and autoimmune diseases

Abstract:  Sex hormones are implicated in the immune response, with estrogens as enhancers at least of the humoral immunity and androgens and progesterone (and glucocorticoids) as natural immune‐suppressors . Several physiological, pathological, and therapeutic conditions may change the serum estrogen milieu and/or peripheral conversion rate, including the menstrual cycle, pregnancy, postpartum period, menopause, being elderly, chronic stress, altered circadian rhythms, inflammatory cytokines, and use of corticosteroids, oral contraceptives, and steroid hormonal replacements, inducing altered androgen/estrogen ratios and related effects. In particular, cortisol and melatonin circadian rhythms are altered, at least in rheumatoid arthritis (RA), and partially involve sex hormone circadian synthesis and levels as well. Abnormal regulation of aromatase activity (i.e., increased activity) by inflammatory cytokine production (i.e., TNF‐alpha, IL‐1, and IL‐6) may partially explain the abnormalities of peripheral estrogen synthesis in RA (i.e., increased availability of 17‐beta estradiol and possible metabolites in synovial fluids) and in systemic lupus erythematosus, as well as the altered serum sex‐hormone levels and ratio (i.e., decreased androgens and DHEAS). In the synovial fluids of RA patients, the increased estrogen concentration is observed in both sexes and is more specifically characterized by the hydroxylated forms, in particular 16alpha‐hydroxyestrone, which is a mitogenic and cell proliferative endogenous hormone. Local effects of sex hormones in autoimmune rheumatic diseases seems to consist mainly in modulation of cell proliferation and cytokine production (i.e., TNF‐alpha, Il‐1, IL‐12). In this respect, it is interesting that male patients with RA seem to profit more from anti‐TNFalpha strategies than do female patients.

Anti-Inflammatory Role of Sympathetic Nerves in Chronic Intestinal Inflammation

Background: Substance P (SP) is a pro-inflammatory neuropeptide in colitis, whereas sympathetic neurotransmitters are anti-inflammatory at high concentrations. Aim and methods: In all layers of the colon, nerve fibre densities of SP+ and sympathetic nerve fibres were investigated (22 Crohn’s disease, six diverticulitis, and 22 controls). In addition, the nerve fibre repellent factor semaphorin 3C (SEMA3C) was studied. The functional role of the sympathetic nervous system was tested in dextran sodium sulfate (DSS) and Il10−/− colitis. Results: In all layers, Crohn’s disease patients demonstrated a loss of sympathetic nerve fibres. Sprouting of SP+ nerve fibres was particularly observed in the mucosa and muscular layer in Crohn’s disease. SEMA3C was detected in epithelial cells, and there was a marked increase of SEMA3C-positive crypts in the mucosa of Crohn’s disease patients compared to controls. In Crohn’s disease, the number of SEMA3C-positive crypts was negatively related to the density of mucosal sympathetic nerve fibres. Sympathectomy reduced acute DSS colitis but increased chronic DSS colitis. Sympathectomy also increased chronic colitis in Il10−/− mice. Conclusions: This study demonstrated a loss of sympathetic and an increase of SP+ nerve fibres in Crohn’s disease. SEMA3C, a sympathetic nerve repellent factor, is highly expressed in the epithelium of Crohn’s disease patients. In chronic experimental colitis, the sympathetic nervous system confers an anti-inflammatory influence. Thus, the loss of sympathetic nerve fibres in the chronic phase of the disease is most probably a pro-inflammatory signal, which might be related to repulsion of these fibres by SEMA3C and other repellents.

Sex hormone modulation of cell growth and apoptosis of the human monocytic/macrophage cell line.

Sex hormones seem to modulate the immune/inflammatory responses by different mechanisms in female and male rheumatoid arthritis patients. The effects of 17β-oestradiol and of testosterone were tested on the cultured human monocytic/macrophage cell line (THP-1) activated with IFN-γ in order to investigate their role in cell proliferation and apoptosis. Activated human THP-1 cells were cultured in the presence of 17β-oestradiol and testosterone (final concentration, 10 nM). The evaluation of markers of cell proliferation included the NF-κB DNA-binding assay, the NF-κB inhibition complex, the proliferating cell nuclear antigen expression and the methyl-tetrazolium salt test. Apoptosis was detected by the annexin V-propidium assay and by the cleaved poly-ADP ribose polymerase expression. Specific methods included flow analysis cytometry scatter analysis, immunocytochemistry and western blot analysis. Cell growth inhibition and increased apoptosis were observed in testosterone-treated THP-1 cells. Increased poly-ADP ribose polymerase-cleaved expression and decreased proliferating cell nuclear antigen expression, as well as an increase of IκB-α and a decrease of the IκB-α phosphorylated form (ser 32), were found in testosterone-treated THP-1 cells. However, the NF-κB DNA binding was found increased in 17β-oestradiol-treated THP-1 cells. The treatment with staurosporine (enhancer of apoptosis) induced decreased NF-κB DNA binding in all conditions, but particularly in testosterone-treated THP-1 cells. Treatment of THP-1 by sex hormones was found to influence cell proliferation and apoptosis. Androgens were found to increase the apoptosis, and oestrogens showed a protective trend on cell death – both acting as modulators of the NF-κB complex.

Catecholamine-producing cells in the synovial tissue during arthritis: modulation of sympathetic neurotransmitters as new therapeutic target

Background The proinflammatory and anti-inflammatory role of the sympathetic nervous system in early and late inflammation is an unresolved paradox. A drastic loss of sympathetic nerve fibres in the synovial tissue of patients with rheumatoid arthritis (RA) has previously been demonstrated. The presence of tyrosine hydroxylase (TH)-positive cells in RA and osteoarthritis (OA) has been determined, but the role of these cells in inflammation is still unclear. Objective To characterise TH-positive cells in inflamed RA and OA synovial tissue and to study their role in inflammation. Methods Synovial samples were obtained from 32 patients with OA and 19 patients with RA and from 10 control patients. Synovial tissue samples were used for immunofluorescence staining. Synovial cells were isolated by tissue digestion and immediately used for cell culture. For in vivo experiments, collagen type-II arthritis in DBA/1J mice was induced. Results TH+ cells were present only in inflamed tissue and not in controls. Catecholamine-storing vesicles and vesicular monoamine transporter 2 (VMAT2) were identified in the synovial tissue. Experimental increase of cytoplasmic catecholamines by VMAT2 blockade strongly reduced tumour necrosis factor (TNF) independently of canonical extracellular β-adrenergic signalling. In addition, VMAT2 blockade increased cyclic AMP (cAMP) and cAMP responsive element binding protein, responsible for TNF inhibition. In vivo, appearance of VMAT2 positive cells was confirmed. VMAT2 blockade ameliorated inflammation also in vivo. Conclusions This study demonstrates that local catecholamine-producing cells start to replace sympathetic nerve fibres around the onset of disease, and modulation of locally produced catecholamines has strong anti-inflammatory effects in vivo and in vitro.

Serum cytokines and steroidal hormones in polymyalgia rheumatica and elderly-onset rheumatoid arthritis

Background: Polymyalgia rheumatica (PMR) may create some difficulties in the differential diagnosis of elderly-onset rheumatoid arthritis (EORA) and of EORA with PMR-like onset (EORA/PMR). Aim: To investigate possible differences between three groups of patients, with regard to serum levels of inflammatory cytokines and steroidal hormones at baseline and after 1 month of treatment with glucocorticoids (prednisone 7.5–12.5 mg/day). Patients and methods: 14 patients with PMR, 15 with EORA and 14 with EORA/PMR, as well as 15 healthy, matched controls were analysed. Tumour necrosis factor α (TNFα), interleukin (IL)6, IL1 receptor antagonist (IL1Ra), cortisol, dehydroepiandrosterone sulphate (DHEAS) and 17-hydroxy-progesterone (PRG) were evaluated. Results: Serum levels of both TNFα and IL6 were significantly higher in all three groups of patients than in controls (p<0.01). Serum IL6 levels were significantly higher in patients with both PMR and EORA/PMR than in patients with EORA (p<0.05). IL1Ra serum levels were significantly higher in patients with EORA than in controls (p<0.001) and in patients with PMR and EORA/PMR (p<0.05). DHEAS was significantly lower in patients with EORA/PMR than in those with EORA (p<0.05). PRG was significantly higher in all patient groups (p<0.05). After glucocorticoid treatment, serum TNFα and IL6 levels significantly decreased in all patient groups; IL1Ra significantly increased in patients with PMR and in those with EORA/PMR; cortisol, DHEAS, and PRG significantly decreased in patients with PMR and in those with EORA/PMR (p<0.05). Conclusions: Different cytokine and steroidal hormone patterns suggest that patients with PMR and those with EORA/PMR seem to be have a more intensive inflammatory reaction and are more efficient responders to glucocorticoid treatment than patients with EORA.

Effects of Testosterone, 17β-Estradiol, and Downstream Estrogens on Cytokine Secretion from Human Leukocytes in the Presence and Absence of Cortisol

Abstract:  Estrogens at physiological concentrations are thought to play an immune‐stimulating role, whereas androgens have an anti‐inflammatory impact. However, their role on cytokine secretion in the presence or absence of cortisol has not been investigated. Furthermore, the role of hydroxylated estrogens downstream of 17β‐estradiol (E2) on secretion of tumor necrosis factor (TNF) is not known. In this study on peripheral blood leukocytes of healthy male subjects, we scrutinized the influence of prior sex hormones (for 24 h) with and without later addition of cortisol (for another 24 h) on stimulated secretion of TNF, IL‐2, IL‐4, IL‐6, IL‐10, and interferon‐γ (IFN‐γ). E2 stabilized or increased immune stimuli–induced secretion of TNF, IL‐2, IL‐4, IL‐6, IL‐10, and IFNγ in relation to testosterone. Testosterone, in contrast, inhibited (IL‐2, IL‐4, IL‐10) or tended to inhibit stimulated secretion of these cytokines (TNF, IFNγ). This effect of E2 was pronounced at a concentration of 10−10 M (testosterone: 10−7 M) in the presence of cortisol. E2 (10−8 M, 10−10 M) and testosterone (10−7 M) did not change glucocorticoid receptor expression. The downstream estrogens 2OH‐estradiol(one), 4OH‐estradiol(one), and 16OH‐estradiol(one) did not stimulate TNF secretion at 10−10 M, but even inhibited its secretion at 10−11 M. However, the combination of 16OH‐estradiol(one) on one side and 2OH‐estradiol(one) or 4OH‐estradiol(one) on the other side markedly stimulated TNF secretion that was only observable in the presence of cortisol. In conclusion, at physiological concentrations, E2 and a combination of downstream estrogens stabilized or increased immune stimuli–induced TNF secretion. These effects are dependent on the presence of physiological concentrations of cortisol. This study underlines the proinflammatory role of E2, which is probably dependent on conversion to a proinflammatory cocktail of downstream estrogens and the presence of cortisol.

The immunomodulatory effects of estrogens: clinical relevance in immune-mediated rheumatic diseases.

Immunological, epidemiological, and clinical evidence suggest that female sex hormones play an important role in the etiology and pathophysiology of chronic immune/inflammatory diseases. Several significant factors generate confusion and opposite conclusions in evaluating the role of estrogens in these diseases, including relatively superficial translational studies from animals to the human condition, the different effects of estrogens on their different receptors or on different target cells, the different estrogen concentrations employed, and opposite effects (especially on cell proliferation) exerted by different peripheral estrogen metabolites. A preponderance of 16α‐hydroxylated estrogens, as observed in rheumatoid arthritis synovial fluids, is an unfavorable sign in synovial inflammation. Since 17β‐estradiol administered during hormone replacement therapy will rapidly increase estrone sulfate after conversion in adipose tissue by aromatases, hormone replacement therapy can have proinflammatory effects by providing estrone sulfate to the inflamed synovial tissue. In addition, it appears that the use of combined oral contraceptives is associated with an increased risk of at least systemic lupus erythematosus. In conclusion, estrogens are generally considered as enhancers of cell proliferation and humoral immune response.

Estrone/17β-estradiol conversion to, and tumor necrosis factor inhibition by, estrogen metabolites in synovial cells of patients with rheumatoid arthritis and patients with osteoarthritis

OBJECTIVE The role of estrogens in rheumatoid arthritis (RA) is debated since both proinflammatory and antiinflammatory effects have been reported. Important evidence of the dual role of estrogens is conversion to various proinflammatory or antiinflammatory metabolites. This study was undertaken to examine the downstream conversion of estrogens in synovial cells from patients with RA or osteoarthritis (OA). METHODS We studied serum levels of estrone, estrone sulfate, and estrone sulfate membrane transporters, intracellular interconversion of estrone and 17beta-estradiol, and conversion of estrone/17beta-estradiol to various estrogen metabolites in RA and OA synovial cells. The effect of estrogen metabolites on tumor necrosis factor (TNF) secretion was also studied in RA and OA synovial cells. RESULTS Serum levels of estrone sulfate were similar in healthy controls and RA patients. Estrone sulfate transporters were present in synovial tissue. Interconversion of estrone and 17beta-estradiol and the expression of converting enzymes of the cytochrome P450 family were similar in RA and OA cells. Using estrone and 17beta-estradiol as substrates, RA and OA synovial cells produced 16alpha-, 4-, and 2-hydroxylated estrogens and their 4- and 2-methylation products. The levels of 16alpha-hydroxylated estrone/17beta-estradiol (16alphaOH-estrone/16alphaOH-17beta-estradiol) were higher than the levels of all other estrogen metabolites. RA synovial cells produced more 16alphaOH-estrone than did OA synovial cells. Importantly, the 16alphaOH estrogens did not inhibit TNF secretion, whereas all other estrogen metabolites had marked inhibitory effects. CONCLUSION Our findings indicate that precursor estrogens are converted to proinflammatory metabolites, particularly in RA synovial cells. RA synovial cells mainly produce the proproliferative 16alphaOH-estrone, which, in addition to 16alphaOH-17beta-estradiol, is one of the only 2 estrogens studied that does not inhibit TNF secretion. A preponderance of 16alpha-hydroxylated estrogens is an unfavorable sign in synovial inflammation.

First appearance and location of catecholaminergic cells during experimental arthritis and elimination by chemical sympathectomy.

OBJECTIVE The sympathetic nervous system is proinflammatory in early collagen-induced arthritis (CIA) and antiinflammatory in late disease. In late arthritis, sympathetic innervation of synovial and lymphoid tissue is markedly reduced. Thus, its suggested antiinflammatory role is difficult to explain. We hypothesized that newly discovered catecholamine-producing (catecholaminergic) cells are targets of chemical sympathectomy. However, in CIA, the time point of appearance, the location, and the possible chemical elimination of catecholaminergic cells have not been studied. The purpose of this study was to investigate the emergence and location of catecholamine-producing and -storing cells in different organs and joints of mice after induction of CIA and to determine whether catecholamine-producing cells can be depleted by 6-hydroxydopamine (6-OHDA) during the early and late phases of CIA in vivo. METHODS The presence of cells positive for tyrosine hydroxylase (TH) and vesicular monoamine transporter 2 (VMAT-2) was evaluated immunohistologically in the lymph nodes, thymus, bone marrow, spleen, and joints of control and arthritic mice. Density was evaluated at different time points after early and late chemical sympathectomy. (131)I-metaiodobenzylguanidine ((131)I-MIBG) scintigraphy demonstrated functional activity of these cells in joint inflammation. RESULTS The density of TH+ and VMAT-2+ cells was highest after arthritis onset (from day 28 onward) and was observed to occur in the following sequence: lymph nodes, thymus, joints, bone marrow, and spleen. Even before arthritis onset (days 5-21), these cells were already more numerous, particularly in the draining lymph nodes, thymus, and joints. (131)I-MIBG scintigraphy demonstrated catecholamine-storing cells in inflammatory hot spots in the paw. Chemical sympathectomy strongly reduced the density of catecholaminergic cells in vitro and in vivo. CONCLUSION After disease onset, catecholaminergic cells are particularly present in primary and secondary lymphoid organs and joints. Since catecholaminergic cells have been reported to have antiinflammatory properties in arthritis, the proinflammatory role played by chemical sympathectomy in late arthritis, as we previously determined, is probably dependent on catecholaminergic cell elimination.

Low density of sympathetic nerve fibers relative to substance P-positive nerve fibers in lesional skin of chronic pruritus and prurigo nodularis

BACKGROUND In prurigo nodularis (PN), an increase in the density of dermal substance P-positive (SP+) sensory nerve fibers has been demonstrated. In addition, the density of sympathetic nerve fibers is unchanged. OBJECTIVE We aimed to investigate the density and balance of sensory and sympathetic dermal nerves in pruritus on normally appearing skin in comparison to PN. METHODS In a parallel investigation in lesional and non-lesional skin routine histological and immunofluorescence staining against SP and tyrosine hydroxylase (TH) were performed. RESULTS We found an increased density of dermal SP+ nerve fibers in PN and also in pruritus relative to sympathetic nerve fibers in affected areas compared to the unaffected site. The density of SP+ and TH+ nerves did not correlate with clinical parameters such as itch quality, duration or intensity. Sparse lymphocytic infiltration as found in affected pruritus skin may be a source of nerve growth factor and explain the hyperinnervation. CONCLUSION Similar to the situation in PN, chronic pruritus lesions also demonstrate a preponderance of SP+ sensory nerve fibers relative to sympathetic nerve fibers, which probably acts as a causal pro-inflammatory signal in development of pruritus. These findings suggest new therapeutic approaches in patients with chronic pruritus.