Anna Scanu

Stimulated T cells generate microparticles, which mimic cellular contact activation of human monocytes: differential regulation of pro- and anti-inflammatory cytokine production by high-density lipoproteins.

Imbalance in cytokine homeostasis plays an important part in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. We demonstrated that T cells might exert a pathological effect through direct cellular contact with human monocytes/macrophages, inducing a massive up‐regulation of the prototypical proinflammatory cytokines IL‐1β and TNF. This mechanism that might be implicated in chronic inflammation is specifically inhibited by high‐density lipoproteins (HDL). Like many other stimuli, besides proinflammatory cytokines, the contact‐mediated activation of monocytes induces the production of cytokine inhibitors such as the secreted form of the IL‐1 receptor antagonist (sIL‐1Ra). The present study demonstrates that stimulated T cells generate microparticles (MP) that induce the production of TNF, IL‐1β, and sIL‐1Ra in human monocytes; the production of TNF and IL‐1β but not that of sIL‐1Ra is inhibited in the presence of HDL. The results were similar when monocytes were stimulated by whole membranes of T cells or soluble extracts of the latter. This suggests that MP carry similar monocyte‐activating factors to cells from which they originate. Thus, by releasing MP, T cells might convey surface molecules similar to those involved in the activation of monocytes by cellular contact. By extension, MP might affect the activity of cells, which are usually not in direct contact with T cells at the inflammatory site. Furthermore, this study demonstrates that HDL exert an anti‐inflammatory effect in nonseptic activation of human monocytes, not only by inhibiting the production of IL‐1β and TNF but also, by leaving sIL‐1Ra production unchanged.

Synovial effusion and synovial fluid biomarkers in psoriatic arthritis to assess intraarticular tumor necrosis factor-α blockade in the knee joint.

IntroductionThe purpose of this study was theevaluation of synovial effusion (SE), synovial fluid (SF) and synovial tissue (ST) biomarkers in relation to disease activity indexes to assess the response to intraarticular (IA) tumor necrosis factor (TNF)-α blockers in psoriatic arthritis (PsA).MethodsSystemic and local disease activity indexes (disease activity score (DAS); the Ritchie articular index (mRAI), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP); Thompson articular (THOMP) and joint articular (KJAI)-Index ) and ST samples were assessed at baseline, throughout treatment, and during the follow-up in 14 patients affected with PsA who underwent IA injections (0.5 ml to 12.5 mg) in the knee joint of etanercept (E) or placebo (P) once every two weeks for a 10-week period. Total SF white blood cell (WBC) counts (WBC/μl) and SF cytokine/chemokine (CK/CCK) levels were measured before IA-E at baseline, after IA-E, and as long as there were adequate amounts of SF for knee aspiration (post). Characterization of synovial mononuclear cell infiltration and synovial vessels was carried out in 8 out of 14 knees by staining serial sections of synovial tissue biopsies for CD45, CD3, CD68, CD31 and CD105.ResultsAt baseline, CRP and/or ESR were significantly correlated with SF-CK (interleukin- (IL-)1β, IL-1Ra, IL-6, IL-8) and CCK (CCL3). Post-IA injections, there was a decrease in SE in the knees in which aspiration following IA-E injection was possible as well as a significant reduction in SF WBC/μl and in SF-CK (IL-1β, IL-1Ra, IL-6 and IL-22). Pre- and post-IA-E injections, there were significant correlations between ST markers and SF-CK (IL-1β with CD45; IL-1β and IL-6 with CD31) and between SF-CCK (CCL4 and CCL3 with CD3). At the end of the study, there was a significant reduction in disease activity indexes (CRP, DAS, RAI, THOMP, KJAI) as well as in the ST markers (CD45; CD3).ConclusionsSynovial effusion regression is a reliable indicator of the response to IA TNF-α blockers in PsA patients as it is confirmed by the correlation between SF biomarkers to disease activity and synovial tissue inflammation.

Cytokine levels in human synovial fluid during the different stages of acute gout: role of transforming growth factor β1 in the resolution phase.

Objectives To determine the most relevant parameters in synovial fluid (SF) during the various stages of acute gout. Methods SFs from 38 gouty patients were analysed for white blood cell (WBC) count, percentage of polymorphonuclear cells (PMNs) and levels of interleukin 1β (IL-1β), IL-6, IL-8, tumour necrosis factorα (TNFα) and transforming growth factor β1 (TGFβ1). Patients were divided into three groups according to the length of time since onset of the attack: phase I (0–48 h), phase II (days 3–4) and phase III (days 5–7). Results Levels of WBCs were similar in SFs from phases I and II, while phase III showed the lowest WBC count. Percentages of PMNs were raised in all SFs. None of the cytokines analysed differed between phases I and II except for TGFβ1, which was higher in phase II. IL-1β, IL-6 and TNFα were higher in group 1 than in group 3. Levels of all the cytokines assessed, with the exception of TGFβ1, were significantly lower in phase III than in phase II IL-1β, p<0.05; IL-6, p<0.01; IL-8, p<0.001; TNFα, p<0.05).TGFβ1 levels were highest in SFs from phase III. Conclusion Cytokine levels in SFs may change depending on the different stages of acute gout, highlighting the role of TGFβ1 in the resolution of gout.

Gout as autoinflammatory disease: New mechanisms for more appropriated treatment targets

Gout is probably one of the oldest diseases affecting men. This is not surprising especially for the active role that innate immunity seems to play in its pathogenesis. It is fascinating to observe as this ancestral mechanism of defense feels that microcrystals are a danger, quite similar to infectious agents. New advances have revealed that at the base of the powerful inflammatory reaction stimulated by monosodium urate crystals there are many complexes cellular mechanisms, mainly involving inflammasome and toll-like receptors. Subsequently, there is an early increase of proinflammatory cytokines responsible for the acute attack, followed in few days by their reduction along with an increase of anti-inflammatory cytokines, probably main actors of the resolution phase. New targets have also been identified for the reduction of hyperuricemia, the prerequisite of gout, in order to prevent new attacks and the deposition of urate crystals in and around the joints. All these aspects, leading to deeper insight, have suggested new treatments, some of which are already available while others are likely to become available in the near future.

High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

IntroductionTo investigate whether monosodium urate (MSU) crystals induce the production of CCL2 (monocyte chemoattractant protein-1; MCP-1) in human fibroblast-like synoviocytes (FLS) and whether this mechanism would be affected by high-density lipoproteins (HDL).MethodsHuman FLS isolated from synovial tissue explants were stimulated with MSU crystals (0.01 to 0.5 mg/ml) or interleukin (IL)-1β (10 pg/ml) in the presence or absence of HDL (50 and 100 μg/ml). The production and expression of CCL2 was evaluated with ELISA, confocal microscopy, immunofluorescence microscopy, chemotaxis assay, and real-time quantitative PCR.ResultsExposure of FLS to MSU crystals induced CCL2 accumulation in culture medium in a dose- and time-dependent manner, reaching a plateau at 50 to 75 μg/ml MSU crystals and 20 to 24 hours. Although low, the induced CCL2 levels were sufficient to trigger mononuclear cell migration. In resting FLS, CCL2 was localized in small cytoplasmic vesicles whose number diminished with MSU crystal stimulation. Concomitantly, MSU crystals triggered the induction of CCL2 mRNA expression. All these processes were inhibited by HDL, which cause a 50% decrease in CCL2 mRNA levels and a dose-dependent inhibition of the release of CCL2. Similar results were obtained when FLS were pretreated with HDL and washed before activation by MSU crystals or IL-1β, suggesting a direct effect of HDL on the FLS activation state.ConclusionsThe present results demonstrate that MSU crystals induce FLS to release CCL2 that is stored in vesicles in resting conditions. This mechanism is inhibited by HDL, which may limit the inflammatory process by diminishing CCL2 production and, in turn, monocytes/macrophages recruitment in joints. This study confirms the antiinflammatory functions of HDL, which might play a part in the limitation of acute gout attack.

JAK/STAT/PKCδ molecular pathways in synovial fluid T lymphocytes reflect the in vivo T helper-17 expansion in psoriatic arthritis

AbstractLooking to the sustained psoriatic arthritis (PsA) joint as a model of local human inflammation, this study was designed to assess the T lymphocyte signal transduction pathways potentially involved in this chronic immune-mediated inflammatory process, as characterized by direct ex vivo analysis of T helper (Th)-17 T effector (Teff) cell phenotypes in synovial fluid (SF) and peripheral blood (PB) of clinically active PsA patients. The reverse-phase protein arrays (RPPA) technique was employed to identify STAT3, STAT1, JAK1, JAK2, PKCδ and ERK1/2 phosphoprotein levels on total T cell lysates in SF samples of PsA patients. Frequencies of T CD4+IL-17A-F+ and T CD4+IL-23R+ Th17 cells were quantified in SF and matched PB of PsA patients by flow cytometry and compared with PB of healthy controls (HC). Increased levels of JAK1, STAT3, STAT1 and PKCδ phosphoproteins were found in SF T cells of PsA patients, compared with PB of HC. The expansion of T CD4+IL-17A-F+ cells, as well as of T CD4+ cells expressing IL-23Rp19 (T CD4+ IL-23R+), considered as the pathogenic phenotype of effector Th17 cells, was found to be confined to the joints of PsA patients, as the frequencies of both populations were significantly higher in SF than in matched PB, or in PB of HC. In conclusion, T lymphocyte signal transduction pathway mapping revealed an enhanced activation of JAK1/STAT3/STAT1 and PKCδ phosphoproteins that may drive the local inflammatory process, characterized by the in vivo expansion of T CD4+IL-17A-F+ and T CD4+IL-23R+ Th17 Teff cells in SF of clinically active joints of PsA patients.

A comparative study of serum and synovial fluid lipoprotein levels in patients with various arthritides.

UNLABELLED The aim of this study was to investigate apolipoprotein (apo) A-I, apo B, lipoprotein (Lp) (a), HDL-cholesterol (C), LDL-C, triglycerides (TG) and total cholesterol (TC) values in the serum and synovial fluid (SF) of untreated rheumatoid arthritis (RA), psoriatic arthritis (PsA), and osteoarthritis (OA) patients. METHODS Paired SF and serum samples were collected simultaneously from 14 patients with RA, 14 with PsA, and 16 with OA and tested for apo A-I, apo B, HDL-C, LDL-C, Lp(a), TC and TG. Serum C reactive protein (CRP) and amyloid A (SAA) levels were also determined. RESULTS The inflammatory arthritis patients had higher SF lipid levels with the exception of HDL. Reflecting increased synovial permeability, the lipid SF/serum ratio was always higher in RA and PsA with respect to OA patients. The positive correlation between serum and SF apo A-I, apo B, HDL-C, TG, and Lp(a) levels confirmed that there is lipoprotein diffusion into the SF. RA and PsA patients had lower concentrations of all serum lipids except for Lp(a) with respect to OA patients. The levels in the RA patients were similar to those in healthy matched controls, while the PsA patients had significantly lower apo A-I and HDL levels and higher apo B and LDL values. CONCLUSIONS Lipid diffusion into the joint cavity, which largely depends on the degree of inflammation, may contribute to modulating local inflammatory processes.

HDL Interfere with the Binding of T Cell Microparticles to Human Monocytes to Inhibit Pro-Inflammatory Cytokine Production

Background Direct cellular contact with stimulated T cells is a potent mechanism that induces cytokine production in human monocytes in the absence of an infectious agent. This mechanism is likely to be relevant to T cell-mediated inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. Microparticles (MP) generated by stimulated T cells (MPT) display similar monocyte activating ability to whole T cells, isolated T cell membranes, or solubilized T cell membranes. We previously demonstrated that high-density lipoproteins (HDL) inhibited T cell contact- and MPT-induced production of IL-1β but not of its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra). Methodology/Principal Findings Labeled MPT were used to assess their interaction with monocytes and T lymphocytes by flow cytometry. Similarly, interactions of labeled HDL with monocytes and MPT were assessed by flow cytometry. In parallel, the MPT-induction of IL-1β and sIL-1Ra production in human monocytes and the effect of HDL were assessed in cell cultures. The results show that MPT, but not MP generated by activated endothelial cells, bond monocytes to trigger cytokine production. MPT did not bind T cells. The inhibition of IL-1β production by HDL correlated with the inhibition of MPT binding to monocytes. HDL interacted with MPT rather than with monocytes suggesting that they bound the activating factor(s) of T cell surface. Furthermore, prototypical pro-inflammatory cytokines and chemokines such as TNF, IL-6, IL-8, CCL3 and CCL4 displayed a pattern of production induced by MPT and inhibition by HDL similar to IL-1β, whereas the production of CCL2, like that of sIL-1Ra, was not inhibited by HDL. Conclusions/Significance HDL inhibit both MPT binding to monocytes and the MPT-induced production of some but not all cytokines, shedding new light on the mechanism by which HDL display their anti-inflammatory functions.

Molecular pathways involved in synovial cell inflammation and tumoral proliferation in diffuse pigmented villonodular synovitis.

Diffuse-type tenosynovial giant cell tumors, also known as pigmented villonodular synovitis, are unique mesenchymal lesions that arise from the synovial tissue of the joints. They are predominantly intraarticular, aggressive, infiltrative processes, characterized by both inflammatory or neoplastic properties and local destructive progression. The pattern of synovial gene and protein expressions in pigmented villonodular synovitis, similar to those in activated macrophages in rheumatoid arthritis, and the phenotype of multinucleated giant cells, characteristic of osteoclasts, suggest that there is a common autocrine mechanism in osteoclast differentiation in both diseases and indicate the potential utility of tumor necrosis factor (TNF)-alpha blockade. High synovial colony stimulating factor 1 (CSF1) messenger RNA (m RNA) expression in pigmented villonodular synovitis, unrelated to a chromosomal translocation involving CSF1 locus, may indicate that there is a synergic paracrine loop mediated by TNF-alpha and CSF1, as shown in both inflammatory and neoplastic conditions. The effects of a new therapeutic approach consisting in intraarticular TNF-alpha blockade were studied in four pigmented villonodular synovitis knees. Knee injections produced a rapid reduction in clinical and sonographic indexes and immunohistological alterations, confirmed by arthroscopic synovectomy. A delayed relapse in one of the four knees and unaltered synovial CSF1 expression were other important findings. In the light of these observations, CSF1/CSF1R interaction probably represents a more sensible therapeutic target than TNF-alpha blockade in the diffuse form of pigmented villonodular synovitis.

Serological markers in psoriatic arthritis: promising tools

The aim of this study was to identify specific biomarkers that could be used to screen for psoriatic arthritis (PsA), as well as to assess disease activity and treatment outcome in affected patients. Forty-three outpatients considered eligible for anti-TNF-α treatment (etanercept 50 mg/week) were enrolled. Serum samples of vascular endothelial growth factor (VEGF), metalloproteinase-3 (MMP3), pentraxin 3 (PTX3), and high-sensitive C-reactive protein (hs-CRP) were collected at baseline (t0) and after 6 (t6), 12 (t12), and 24 months (t24) of treatment. Baseline values were compared with those of a group of healthy controls matched for age and sex. Disease activity scores and functional tests (DAS28, BASDAI, PASI, BASFI, HAQ, VAS pain, and VAS patient global disease activity) after treatment were found to be significantly different from baseline values. At baseline, MMP3, hs-CRP and VEGF values in the PsA-patients were found to be significantly higher with respect to levels in the controls. There were no differences in the PTX3 values. MMP3 was significantly lower at t6 (P < 0.0001), t12 (P < 0.0001) and t24 (P < 0.0001). hs-CRP and VEGF were significantly lower, respectively, at t12 (P < 0.01; P < 0.05) and t24 (P < 0.05; P < 0.01). PTX3 was significantly higher at t24 (P < 0.05). A correlation was found between MMP3 and hs-CRP (r = 0.45, P = 0.0005). MMP3, hs-CRP, and VEGF appear to be useful for the early detection of PsA and to monitor disease progression. The rise in PTX3 did not appear to be linked to the inflammatory state of the disease but might be an expression of the atherosclerotic process frequently observed in PsA.