A circulating cell population showing both M1 and M2 monocyte/macrophage surface markers characterizes systemic sclerosis patients with lung involvement.

BACKGROUND: Systemic sclerosis (SSc) is a disorder characterized by immune system alterations, vasculopathy and fibrosis. SSc-related interstitial lung disease (ILD) represents a common and early complication, being the leading cause of mortality. Monocytes/macrophages seem to have a key role in SSc-related ILD. Interestingly, the classically (M1) and alternatively (M2) activated monocyte/macrophage phenotype categorization is currently under revision. Our aim was to evaluate if circulating monocyte/macrophage phenotype could be used as biomarker for lung involvement in SSc. To this purpose we developed a wide phenotype characterization of circulating monocyte/macrophage subsets in SSc patients and we evaluated possible relations with lung involvement parameter values. METHODS: A single centre cross-sectional study was performed in fifty-five consecutive SSc patients, during the year 2017. All clinical and instrumental tests requested for SSc follow up and in particular, lung computed tomography (CT) scan, pulmonary function tests (PFTs), Doppler echocardiography with systolic pulmonary artery pressure (sPAP) measurement, blood pro-hormone of brain natriuretic peptide (pro-BNP) evaluation, were performed in each patient in a maximum one-month period. Flow cytometry characterization of circulating cells belonging to the monocyte/macrophage lineage was performed using specific M1 (CD80, CD86, TLR2 and TLR4) and M2 surface markers (CD204, CD163 and CD206). Non-parametric tests were used for statistical analysis. RESULTS: A higher percentage of circulating CD204+CD163+CD206+TLR4+CD80+CD86+ and CD14+CD206+CD163+CD204+TLR4+CD80+CD86+ mixed M1/M2 monocyte/macrophage subsets, was identified to characterize patients affected by SSc-related ILD and higher systolic pulmonary artery pressure. Mixed M1/M2 monocyte/macrophage subset showed higher percentages in patients positive for anti-topoisomerase antibody, a known lung involvement predictor. CONCLUSIONS: The present study shows for the first time, through a wide flow cytometry surface marker analysis, that higher circulating mixed M1/M2 monocyte/macrophage cell percentages are associated with ILD, sPAP and anti-topoisomerase antibody positivity in SSc, opening the path for research on their possible role as pathogenic or biomarker elements for SSc lung involvement.

Dual endothelin receptor antagonists contrast the effects induced by endothelin-1 on cultured human microvascular endothelial cells.

OBJECTIVES: To evaluate the ability of dual endothelin (ET) receptor antagonists (ETA/ETB -ETA/BRAs) to contrast the ET-1-induced effects on cultured human microvascular endothelial cells (HMVECs). METHODS: Some cultured HMVECs were untreated, or treated with ET-1 (100nM) or transforming growth factor ß1 (TGFß1, 10ng/mL) alone for 6 days, in order to induce the endothelial-to-mesenchymal transition (EndoMT). Other cultured HMVECs were pre-treated for 1hr with ETA/BRAs bosentan (10µM) or macitentan (1µM, 10µM) before the stimulation with ET-1 for 6 days. At the end of treatments, a mechanical injury was induced to cultured HMVECs (by scratching the cell monolayer with a sterile tip), and then the cell ability to re-fill the damaged area was determined after 24hrs. EndoMT phenotype markers and monocyte chemoattractant protein-1 (MCP-1) were evaluated by qRT-PCR and Western blotting. Statistical analysis was performed using Mann-Whitney-U non-parametric test. RESULTS: Both ET-1 and TGFß1 induced EndoMT and the MCP-1 over-expression in cultured HMVECs, as well as reduced the process of endothelial cell damage repair. Pre-treatment with ETA/BRAs let cultured HMVECs to significantly restore the in vitro damage of the cell monolayer and antagonised the EndoMT process as well as the MCP-1 over-expression (range p<0.05 - p<0.001). Conversely, untreated or TGFß1-treated HMVECs were found unaffected by the ETA/BRAs treatments. CONCLUSIONS: The treatment with dual ETA/BRAs seems to partially restore the altered cell function induced by ET-1 in cultured endothelial cells, and might justify their therapeutic efficiency in clinical conditions characterised by increased concentrations of ET-1.

Effects of combined treatments with CTLA4-IG (abatacept), dexamethasone and methotrexate on cultured human macrophages.

OBJECTIVES: To evaluate the anti-inflammatory effect of CTLA4-Ig (abatacept) and dexamethasone (DEX) monotreatment versus their combination and adding methotrexate (MTX) on cultured human macrophages. METHODS: THP-1 cells, activated into macrophages (PMA 0.05 µg/ml), were cultured for 3 and 24 hrs with CTLA4-Ig (500 µg/ml), DEX (10-8 M), MTX (0.05 µg/ml), and CTLA4-Ig combined with DEX or CTLA4-Ig combined with DEX plus MTX. CTLA4-Ig/CD86 interaction was evaluated by FACS analysis. Quantitative real time-PCR (qRT-PCR), immunocytochemistry (ICC) and immunoassay (ELISA) analysis for inflammatory cytokine (IL-1ß, TNF-α, IL-6) expression were performed. RESULTS: FACS analysis showed in macrophages treated with CTLA4-Ig alone, CTLA4-Ig-DEX and CTLA4-Ig-DEX-MTX a CD86 decrease of almost 35%, versus untreated cells (CNT). After 3 hrs, macrophages treated with DEX alone or with CTLA4-Ig-DEX or CTLA4-Ig-DEX-MTX showed a significant reduction (p<0.05) for all cytokines gene expression, that was still significant for IL-1ß after 24 hrs (p<0.05). After 3 hrs, CTLA4-Ig alone significantly (p<0.05) reduced all cytokine genes; however, after 24 hrs still evident only for TNF-α (p<0.05). After 24 hrs CTLA4-Ig-DEX induced a significant decrease of gene expression (p<0.05) for TNF-α and IL-6, whereas CTLA4-Ig-DEX-MTX induced a decrease (p<0.05) limited to IL-6, versus CNT. Finally, ICC showed, after 24 hrs of CTLA4-Ig-DEX or CTLA4-Ig-DEX-MTX treatment a reduction (p<0.05) of IL-1ß and IL-6 expression, versus CNT; DEX alone reduced only IL-1ß (p<0.05). ELISA analysis confirmed these results. CONCLUSIONS: CTLA4-Ig-DEX and CTLA4-Ig-DEX-MTX combined treatments, decreased at any level the inflammatory cytokine expression more efficiently then monotreatments on activated cultured human macrophages.

CTLA4-Ig/CD86 interactions in cultured human endothelial cells: effects on VEGFR-2 and ICAM1 expression.

OBJECTIVES: Previous studies have reported the presence of CD86 (B7.2) costimulatory molecule on endothelial cells (ECs) and recent data have shown that CTLA4-Ig (abatacept), used as a biological agent in rheumatoid arthritis, interacts with CD86 expressed on different cells involved in synovitis. Therefore, the effects of CTLA4-Ig/CD86 interaction on VEGFR-2 (vascular endothelial growth factor receptor 2) and ICAM1 expression, were evaluated in cultured ECs. METHODS: Activated ECs (γIFN 500 U/ml or IL-17 100 ng/ml), treated with CTLA4-Ig (10, 100, 500 µg/ml) were analysed for CD86, VEGFR-2 and ICAM1 expression, by flow cytometry (FACS), by western blot (WB) and quantitative real time PCR (qRT-PCR). RESULTS: Following CTLA4-Ig treatment (10, 100, 500 µg/ml; 24 hrs), activated ECs decreased their CD86-positivity at FACS: 66, 59, 51%, respectively, versus 68% of untreated cells (cnt) (for γIFN-activated cells) and 42, 47, 46% versus 71% (cnt) (for IL-17-activated ECs). Gamma-IFN-activated ECs, treated with CTLA4-Ig, showed a dose-dependent decrease only for ICAM1 fluorescence. Whereas, WB showed a significant decrease (p<0.05) for both ICAM1 and VEGFR-2 after CTLA4-Ig 500 µg/ml (3 and 24 hrs) and for VEGFR-2 also after CTLA4-Ig 100 µg/ml (3 hrs). QRT-PCR showed a significant decrease (p<0.05) for VEGFR-2 after CTLA4-Ig 500 µg/ml (3 and 24 hrs) and after CTLA4-Ig 100 µg/ml (limited at 3 hrs). QRT-PCR for ICAM1 was negative at 3 and 24 hrs, possibly since it was to late to be detected. CONCLUSIONS: Results support a CTLA4-Ig/CD86 interaction on γIFN and IL-17 activated ECs modulation, in the expression of VEGFR-2 and ICAM1, both relevant for inflammatory and angiogenetic processes, suggesting ECs as a further target for abatacept.

Intracellular NF-kB-decrease and IKBα increase in human macrophages following CTLA4-Ig treatment.

OBJECTIVES: The transcription factor NF-kB is involved in the expression of several genes linked to the immune response, including those of pro-inflammatory cytokines. We investigated cytokine production and NF-kB expression following CTLA4-Ig (abatacept) treatment of cultured human macrophages. METHODS: Human THP1 cells, differentiated in macrophages, were treated with CTLA4-Ig (100, 500 µg/ml; 3,12,24 hours). Quantitative RT-PCR analysis (qRT-PCR) of mRNA for NF-kB, IKBα and for IL-6, TNF-α, IL-1ß, was performed after 3 and 12 hours from treatment. Western blot (WB) analysis for NF-kB and IKBα was performed after 24 hours from treatment. RESULTS: NF-kB gene expression was significantly downregulated (p<0.05), at 3 and 12 hours from CTLA4-Ig treatment, vs. untreated cells (cnt). IKBα resulted significantly increased vs. cnt (p<0.05), at 12 hours from CTLA4-Ig [500 µg/ml] treatment. After 3 hours, CTLA4-Ig [100 µg/ml] induced a significant decrease of TNF-α and IL-6 (p<0.05), vs. cnt and CTLA4-Ig [500 µg/ml] further reduced TNF-α (p<0.001), vs. cnt. After 12 hours from CTLA4-Ig treatment, a significant downregulation for IL-6 and IL1ß expression (p<0.001), vs. cnt, was still evident. Results were confirmed by WB. CONCLUSIONS: NF-kB pathway seems to be implicated in the CTLA4-Ig modulation of macrophage cytokine expression. NF-kB expression resulted downregulated while its cytoplasmatic inhibitor IKBα was increased.

Identification and quantification of selected inflammatory genes modulated by leflunomide and prednisone treatment in early rheumatoid arthritis patients.

OBJECTIVES: The present study evaluates the effects of combined leflunomide (LEF) and low dose of prednisone therapy, on selected inflammatory gene expression in peripheral blood mononuclear cells (PBMCs) of early rheumatoid arthritis (ERA) patients by gene microarray analysis and quantitative real time-polymerase chain reaction (qRT-PCR). METHODS: Ten ERA patients (mean age 53 ± 10 years) were assigned as untreated (group 1) or pre-treated (group 2) with prednisone (5 mg/day for 3 months) after informed consent and ethics committee approval. Five sex- and age-matched healthy subjects were used as controls (CNT). RNA was extracted by PBMCs, amplified, labelled and hybridised on inflammation DualChip microarray. The expression ratio of 282 inflammatory genes between CNT and ERA patients, before (T0) and after 12 weeks (T1) of combined therapy was detected. qRT-PCR was performed on 7 selected inflammatory RA-related genes (STAT4, MAPK9, HIF1A, MIF, STAT6, NFKB1, TNFRSF1B). RESULTS: At T0, microarray analysis showed 34 altered genes in both ERA groups when compared to CNT (vs. CNT). Seven RA-related genes, investigated in further details, were found up-regulated in group 1 and down-regulated or unchanged in group 2 vs. CNT. At T1, combined therapy induced the down-regulation of these genes in both groups vs. CNT as also confirmed by qRT-PCR performed on selected genes. CONCLUSIONS: Untreated ERA patients seem characterised by up-regulation of specific genes involved both in the resistance/inhibition to apoptosis and in the stimulation of pro-inflammatory cytokine production by immune inflammatory cells. Combined LEF and low dose of prednisone therapy seems to play synergistic effects on down-regulation of these genes.

A functional soluble form of CTLA-4 is present in the serum of celiac patients and correlates with mucosal injury.

Celiac disease (CD) is a multifactorial disorder influenced by environmental, genetic and immunological factors. Increasing evidence showed CTLA-4 gene as an important susceptibility locus for autoimmune disorders. A native soluble cytotoxic T-lymphocyte-associated protein-4 (sCTLA-4), lacking of transmembrane sequence, has been described in several autoimmune diseases. We aimed to evaluate the presence of increased sCTLA-4 concentration in the serum of patients with CD and the possible immunoregulatory function. Blood samples were collected from 160 CD patients; sCTLA-4 levels were evaluated by ELISA, western blot and reverse transcription-PCR. The capability of serum sCTLA-4 to modulate T-lymphocyte proliferation in vitro was evaluated by two-way mixed leukocyte reaction assay. We demonstrated high levels of sCTLA-4 in serum of untreated celiac patients. Additionally, we observed that sCTLA-4 concentrations are related to gluten intake and that a correlation between autoantibodies to tissue transglutaminase and sCTLA-4 concentration exists. Moreover, sCTLA-4 levels correlate with the degree of mucosal damage. Conversely, no correlation between sCTLA4 levels and the HLA-related risk was observed. Finally, we show that sCTLA-4 from sera of CD patients displays functional activities. These results strongly suggest a regulation of sCTLA-4 synthesis depending on the presence or absence of dietary gluten and imply a possible immunomodulatory effect on cytotoxic T lymphocyte functions. In gluten-exposed patients, serum sCTLA-4 levels might provide insight about mucosal injury.

Sex hormones modulate the effects of Leflunomide on cytokine production by cultures of differentiated monocyte/macrophages and synovial macrophages from rheumatoid arthritis patients.

Immune response is greater in females than in males and lymphocytes/monocytes from female subjects (or tested in vitro with estrogens) show higher immune/inflammatory reactivity. In order to test in vitro the interactions between 17beta-estradiol (E2--10(-9) M), testosterone (T--10(-8) M) and the antiproliferative/immune suppressive drug Leflunomide metabolite A77 1726 (LEF-M--30 microM) employed in rheumatoid arthritis (RA), their combined effects were evaluated on inflammatory cytokine (CK) expression/production in cultures of differentiated macrophages (M) (from activated THP-1 monocytes) and primary cultures of RA synovial macrophages (SM). TNFalpha, IL-6 and TGFbeta were detected by immunocytochemistry (ICC), Western blot analysis (WB) and reverse transcriptase-polymerase chain reaction (RT-PCR). The ICC, WB and RT-PCR showed a significant down-regulation induced by LEF-M on CK expression by cultured M when compared to untreated cells (IL-6 p < 0.01, TNFalpha p < 0.001, TGFbeta p < 0.01). At ICC analysis E2 increased CK expression, whereas T decreased the expression, confirmed by WB and RT-PCR (range between p < 0.05 and p < 0.001). LEF-M treatment significantly downregulated the CK expression in E2/T treated M: the effect was more significant in LEF-M plus T-treated cells versus controls (range between p < 0.01 and p < 0.001). Concerning the RA SM, the results were replicated (range between p < 0.05 and p < 0.001). E2 seems to contrast, but T seems to synergize the LEF-M activity. Results might support a stronger therapeutical efficacy, at least for LEF, in male RA patients, as already reported by clinical evidences.

Effects of CTLA4-Ig treatment on circulating fibrocytes and skin fibroblasts from the same systemic sclerosis patients: an in vitro assay.

BACKGROUND: Systemic sclerosis (SSc) is characterized by vasculopathy and progressive fibrosis. CTLA4-Ig (abatacept) is able to interact with the cell surface costimulatory molecule CD86 and downregulate the target cell. The aim of this study was to evaluate the in-vitro effects of CTLA4-Ig treatment on circulating fibrocytes and skin fibroblasts isolated from the same SSc patient. METHODS: Circulating fibrocytes and skin fibroblasts were obtained from eight SSc patients with "limited" cutaneous involvement and from four healthy subjects (HSs). Samples were analyzed by fluorescence-activated cell sorter analysis (FACS) at baseline (T0) and after 8 days of culture (T8) for CD45, collagen type I (COL I), CXCR4, CD14, CD86, and HLA-DRII expression. Circulating fibrocytes were treated for 3 h and skin fibroblasts for 24/48 h with CTLA4-Ig (10, 50, 100, 500 µg/ml). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for CD86, COL I, FN, TGFß, αSMA, S100A4, CXCR2, CXCR4, CD11a, and Western blotting was performed for COL I and FN. RESULTS: Using qRT-PCR, the T8-cultured SSc circulating fibrocytes which had not been treated with CTLA4-Ig showed higher gene expression for CD86, αSMA, S100A4, TGFß, and COL I compared with HS circulating fibrocytes. Interestingly, αSMA/COL I gene expression was significantly lower only in the SSc circulating fibrocytes treated with CTLA4-Ig for 3 h (p < 0.01, p < 0.05). On the contrary, no effects were observed for either SSc or HS skin fibroblasts after CTLA4-Ig treatment. COL I and FN protein expression was unchanged in both SSc and HS skin fibroblasts by Western blot. CONCLUSIONS: Circulating fibrocytes seem to be more responsive to CTLA4-Ig treatment than skin fibroblasts from the same SSc patient, likely due to their higher expression of CD86. CTLA4-Ig treatment might downregulate the fibrotic process in SSc patients by downregulating the fibrocytes, circulating progenitor cells.

Effects of selexipag and its active metabolite in contrasting the profibrotic myofibroblast activity in cultured scleroderma skin fibroblasts.

BACKGROUND: Myofibroblasts contribute to fibrosis through the overproduction of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated in systemic sclerosis (SSc) by the activation of fibrogenic intracellular signaling transduction molecules, including extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt). Selexipag is a prostacyclin receptor agonist synthesized for the treatment of pulmonary arterial hypertension. The study investigated the possibility for selexipag and its active metabolite (ACT-333679) to downregulate the profibrotic activity in primary cultures of SSc fibroblasts/myofibroblasts and the fibrogenic signaling molecules involved. METHODS: Fibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3rd culture passage and then either maintained in normal growth medium (untreated cells) or independently treated with different concentrations of selexipag (from 30 µM to 0.3 µM) or ACT-333679 (from 10 µM to 0.1 µM) for 48 h. Protein and gene expressions of α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), COL-1, and FN were investigated by western blotting and quantitative real-time PCR. Erk1/2 and Akt phosphorylation was investigated in untreated and ACT-333679-treated cells by western botting. RESULTS: Selexipag and ACT-333679 significantly reduced protein synthesis and gene expression of α-SMA, S100A4, and COL-1 in cultured SSc fibroblasts/myofibroblasts compared to untreated cells, whereas FN was significantly downregulated at the protein level. Interestingly, ACT-333679 significantly reduced the phosphorylation of Erk1/2 and Akt in cultured SSc fibroblasts/myofibroblasts. CONCLUSIONS: Selexipag and mainly its active metabolite ACT-333679 were found for the first time to potentially interfere with the profibrotic activity of cultured SSc fibroblasts/myofibroblasts at least in vitro, possibly through the downregulation of fibrogenic Erk1/2 and Akt signaling molecules.

Correction: Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages.

[This corrects the article DOI: 10.1371/journal.pone.0166433.].

Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages.

BACKGROUND: Alternatively activated (M2) macrophages are phenotypically characterized by the expression of specific markers, mainly macrophage scavenger receptors (CD204 and CD163) and mannose receptor-1 (CD206), and participate in the fibrotic process by over-producing pro-fibrotic molecules, such as transforming growth factor-beta1 (TGFbeta1) and metalloproteinase (MMP)-9. Endothelin-1 (ET-1) is implicated in the fibrotic process, exerting its pro-fibrotic effects through the interaction with its receptors (ETA and ETB). The study investigated the possible role of ET-1 in inducing the transition from cultured human macrophages into M2 cells. METHODS: Cultured human monocytes (THP-1 cell line) were activated into macrophages (M0 macrophages) with phorbol myristate acetate and subsequently maintained in growth medium (M0-controls) or treated with either ET-1 (100nM) or interleukin-4 (IL-4, 10ng/mL, M2 inducer) for 72 hours. Similarly, primary cultures of human peripheral blood monocyte (PBM)-derived macrophages obtained from healthy subjects, were maintained in growth medium (untreated cells) or treated with ET-1 or IL-4 for 6 days. Both M0 and PBM-derived macrophages were pre-treated with ET receptor antagonist (ETA/BRA, bosentan 10-5M) for 1 hour before ET-1 stimulation. Protein and gene expression of CD204, CD206, CD163, TGFbeta1 were analysed by immunocytochemistry, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Gene expression of interleukin(IL)-10 and macrophage derived chemokine (CCL-22) was evaluated by qRT-PCR. MMP-9 production was investigated by gel zymography. RESULTS: ET-1 significantly increased the expression of M2 phenotype markers CD204, CD206, CD163, IL-10 and CCL-22, and the production of MMP-9 in both cultures of M0 and PBM-derived macrophages compared to M0-controls and untreated cells. In cultured PBM-derived macrophages, ET-1 increased TGFbeta1 protein and gene expression compared to untreated cells. The ET-1-mediated effects were contrasted by ETA/BRA treatment in both cultured cell types. CONCLUSION: ET-1 seems to induce the M2 phenotype in cultured human macrophages, a process apparently contrasted by the action of the ETA/BRA, suggesting possible clinical implications in those fibrotic diseases characterized by increased ET-1 concentrations, such as systemic sclerosis but also type 2 diabetes.

Effects of macitentan and its active metabolite on cultured human systemic sclerosis and control skin fibroblasts.

OBJECTIVE: To investigate the effects of the endothelin 1 (ET-1) receptor antagonists (ETRA) macitentan, its active metabolite ACT-132577, and bosentan on myofibroblast activation and extracellular matrix production induced by ET-1 in cultured systemic sclerosis (SSc) and control skin fibroblasts. METHODS: Fibroblasts were obtained from skin biopsies of 6 patients with SSc and 5 healthy subjects. Some cultured cells were untreated or treated with macitentan, ACT-132577, or bosentan alone (10 µM). Other cultured cells were treated with ET-1 alone (100 nM) or with ETRA, and after 1 h, also with ET-1. After 48 h of treatment, myofibroblast activation was investigated to evaluate the α-smooth muscle actin (α-SMA) expression by immunofluorescence; type I collagen (COL-1) and fibronectin (FN) were investigated by immunocytochemistry, Western blotting, and quantitative real-time PCR (qRT-PCR). Statistical analysis was performed by the nonparametric Mann-Whitney U test. RESULTS: In cultured SSc skin fibroblasts, only the treatment with macitentan significantly reduced the basal level of α-SMA expression (p = 0.03 vs untreated cells). Macitentan also significantly reduced the basal level of COL-1 synthesis, similarly to bosentan (p < 0.05 vs untreated cells). Macitentan or ACT-132577 antagonized the ability of ET-1 to further induce α-SMA expression (p = 0.03), COL-1, and FN synthesis (p = 0.03, p = 0.005); bosentan showed similar effects. These results obtained by immunofluorescence and immunocytochemistry were confirmed by Western blotting and qRT-PCR. The downregulatory effects exerted by ETRA were observed also in cultured human control skin fibroblasts. CONCLUSION: Macitentan and ACT-132577 seem to downregulate in vitro the profibrotic myofibroblast phenotype induced by ET-1 in cultured human SSc skin fibroblasts.

Effects of estrogens on extracellular matrix synthesis in cultures of human normal and scleroderma skin fibroblasts.

To investigate the effects of 17beta-estradiol (E2) on extracellular matrix (ECM) protein synthesis (collagen type I, fibronectin, and laminin) using cultures of normal and scleroderma (SSc) skin fibroblasts. Primary fibroblasts cultures, obtained from skin biopsies of six female voluntary subjects and three female SSc patients, were treated for 24 h with E2 (10(-10)M) alone or in combination with tamoxifene (TAM, 10(-7)M) as an estrogen receptor (ER) antagonist. ECM protein synthesis was analyzed by immunocytochemistry and Western blotting. E2 induced a significant increase of fibronectin, collagen type I, and laminin synthesis both in normal (P < 0.01, P < 0.05, P < 0.01, respectively) and SSc fibroblasts (P < 0.001, P < 0.05, P < 0.001, respectively) when compared to untreated fibroblasts. TAM induced a significant decrease of ECM protein synthesis when compared to E2-treated TAM-untreated fibroblasts. This study seems to support important modulatory effects of E2 in the fibrotic progression of the SSc process via ER interactions.

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 16alpha-hydroxylated estrogens, as observed in rheumatoid arthritis synovial fluids, is an unfavorable sign in synovial inflammation. Since 17beta-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.

Estrogens interfere with leflunomide modulation of cytokine production by human activated monocytes.

Rheumatoid arthritis (RA) prevalence is greater in females than in males, supporting estrogens as modulators of immune response. Leflunomide (LEF) is employed in the RA treatment. We studied the combinatory effects of LEF active metabolite A77 1726 (LEF-M) and 17beta-estradiol (E2) on inflammatory cytokine production by cultured macrophages obtained from activated human monocytes (THP-1 cells). Macrophages were cultured with LEF-M alone and in combination with E2. IL-6, TNF-alpha, and TGF-beta were evaluated by immunocytochemistry (ICC), Western blot (WB), and reverse transcriptase-polymerase chain reaction (RT-PCR). ICC, as well as WB and RT-PCR, showed that LEF-M, in respect to untreated cells, significantly downregulated the cytokine production (IL-6 P < 0.01, TNF-alphaP < 0.001, TGF-betaP < 0.01). On the contrary, E2 increased the cytokine production, a result that was significantly reversed when LEF-M was subsequently added (IL-6, TNF-alpha, TGF-betaP < 0.001 vs. E2). E2 seems to contrast the LEF-M activity. These results might support a more efficient therapeutical effect of LEF in male with respect to female RA patients.

Sex hormones and leflunomide treatment of human macrophage cultures: effects on apoptosis.

In order to establish the possible gender influence on the activity of leflunomide (LEF) in rheumatoid arthritis (RA), we evaluated the proapoptotic activity of the active LEF metabolite A77 1726 (LEF-M), in combination with sex hormones, on cultures of human macrophages. In particular, we focussed our investigation on the triggering phase of the apoptosis. Cultures of macrophages from activated THP-1 cells and from RA synovial tissues were treated with LEF-M alone [30muM] or in presence of 17beta-estradiol (E2) (10(-9)M) or testosterone (T) (10-(8)M) for 24 hours. FAS, FAS-L, FADD (Fas-Associated via Death Domain) and FLICE (FADD-Like Interleukin-1 beta Converting Enzyme) were evaluated by immunocytochemistry (ICC), Western blot (WB) and reverse transcriptase-multiplex polymerase chain reaction (RT-MPCR). Regarding macrophages from THP-1 cells (M), the ICC showed that LEF-M exerted a significant up-regulation on all investigated apoptotic proteins, when compared to untreated cells (control) (p<0.001). On the contrary, E2 significantly increased FAS-L positivity (p<0.05) and down-regulated FADD (p<0.01), while others apoptotic proteins were not modulated when compared to control. Regarding RA synovial macrophages (SM), the ICC showed that LEF-M exerted a significant up-regulation on FAS and FAS-L when compared to control (p<0.001). On the contrary, E2 down-regulated significantly FAS-L (p<0.001), while FAS was not modulated respect to control. T significantly increased the apoptotic proteins in all conditions. The results of the present study suggests a less efficient therapeutic effect of LEF in female patients, due to the contrasting action of estrogens on LEF- induced apoptosis.

Raynaud's phenomenon and plasma endothelin: correlations with capillaroscopic patterns in systemic sclerosis.

OBJECTIVE: We evaluated endothelin (ET)-1 plasma levels and some clinical measures in patients with primary Raynaud's phenomenon (PRP), and in patients with systemic sclerosis (SSc) and secondary RP (SRP), in the latter according to their different nailfold videocapillaroscopy (NVC) patterns of microangiopathy (early, active, and late). METHODS: Ninety-nine patients with SSc, 49 with PRP, and 45 control subjects were studied. NVC was performed in all patients to distinguish the pattern of microvascular damage, and the morphological alterations were scored by a semiquantitative rating scale. ET-1 plasma levels were evaluated in all individuals by ELISA. RESULTS: ET-1 plasma levels were significantly higher (p=0.001) in patients with both PRP and SRP, compared to controls. A significant positive correlation (p=0.03) was found between ET-1 plasma levels and SRP duration, but not between ET-1 plasma levels and PRP duration. Significant correlations were observed in patients with SSc between ET-1 plasma levels and clinical measures (e.g., digital ulcers), as well as the score value of single NVC measures, such as the number of capillaries, "ramified" capillaries, and enlarged capillaries (p<0.05). Finally, the highest ET-1 plasma levels were found in patients with SSc showing the late pattern of microangiopathy when compared to the early pattern (p=0.03) and to controls (p=0.003). CONCLUSION: Highest ET-1 plasma levels were detected in the more advanced stage of the SSc microangiopathy, namely the late NVC pattern, characterized by capillary loss and increased tissue fibrosis; this might support the involvement of ET-1 in the progression of the microvascular/fibrotic SSc damage.