From supernatants to cytokines: a personal view on the early history of IL-1, IL-1Ra, TNF and its inhibitor in rheumatology.

Long-term cell cultures developed early in the twentieth century allowed identification in their supernatants of biological mediators subsequently defined as migration factors, interferons, lymphokines, monokines, cytokines and interleukins. In rheumatology, early in the 1930s, synovial cell cultures revealed two major distinct populations, i.e. synovial fibroblasts and monocyte-macrophages. Discovery of the interstitial collagenase (MMP-1) and its role in tissue destruction, such as in rheumatoid arthritis (RA), raised the question of the cellular source for this enzyme. My personal interest in the field was driven by the lack of understanding for the link between tissue destruction and immunology. This triggered our seminal contribution to the field, establishing in 1976-79 at the Arthritis Unit (Massachusetts General Hospital, with SM Krane) that a mononuclear factor (MCF, around 15 kDa) produced by stimulated macrophage, under direct contact with activated T cells, induced large amounts of collagenase and prostaglandin E2 (PGE2, a bone resorbing agent) in human synovial fibroblasts from RA patients. Our original "MCF" biological observations preceded cloning, and recombinant IL-1ß confirmed the biological activity of the purified natural IL-1. Following my return to Geneva in 1980 and searching for a high level of IL-1 in urine and serum of patients with high fever or Still's disease, to our surprise-"a finding of absence"-we found that IL-1 was masked by a factor of approximately 17 kDa and first presented this in 1984 at the Fourth International Lymphokine Workshop. In 1987, before IL-Ra cloning, my co-worker P Seckinger and I demonstrated first-time observation in cytokine biology that the mechanism was due to the inhibition of IL-1 binding the cell surface receptor, leading to the concept of IL-1 receptor antagonist (IL-1Ra). Having reported in 1985 that TNF/cachectin also induced collagenase and PGE2 in human synovial cells, we found that IL-1Ra did not block TNF-α but was due to another inhibitor. As other investigators, we confirmed that this inhibitory factor was a soluble TNF receptor. The years between the 1970s and 1990s were probably the most exciting period in the field of cytokines and cytokine antagonists; it gave rise to two concepts in the cytokine field-one of the receptor antagonist, and the other of soluble receptor antagonists.

A Brief History of IL-1 and IL-1 Ra in Rheumatology.

The history of what, in 1979, was called interleukin-1 (IL-1), orchestrator of leukocyte inter-communication, began many years before then, initially by the observation of fever induction via the endogenous pyrogen (EP) (1974) and then in rheumatology on the role in tissue destruction in rheumatoid diseases via the induction of collagenase and PGE2 in human synovial cells by a mononuclear cell factor (MCF) (1977). Since then, the family has exploded to presently 11 members as well as many membrane-bound and soluble receptor forms. The discovery of a natural Interleukin-1 receptor antagonist (IL-1Ra) in human biological fluids has highlighted the importance of IL-1 and IL-1Ra in human diseases. Evidence delineating its role in autoinflammatory syndromes and the elucidation of the macromolecular complex referred to as "inflammasome" have been instrumental to our understanding of the link with IL-1. At present, the IL-1blockade as therapeutic approach is crucial for many hereditary autoinflammatory diseases, as well as for adult-onset Still's disease, crystal-induced arthropathies, certain skin diseases including neutrophil-triggered skin diseases, Behçet's disease and deficiency of IL-1Ra and other rare fever syndromes. Its role is only marginally important in rheumatoid arthritis and is still under debate with regard to osteoarthritis, type 2 diabetes mellitus, cardiovascular diseases and cancer. This brief historical review focuses on some aspects of IL-1, mainly IL-1ß and IL-Ra, in rheumatology. There are many excellent reviews focusing on the IL-1 family in general or with regard to specific diseases or biological discoveries.

Rheumatoid synovial fibroblasts differentiate into distinct subsets in the presence of cytokines and cartilage.

BACKGROUND: We investigated two distinct synovial fibroblast populations that were located preferentially in the lining or sub-lining layers and defined by their expression of either podoplanin (PDPN) or CD248, and explored their ability to undergo self-assembly and transmigration in vivo. METHODS: Synovial fibroblasts (SF) were cultured in vitro and phenotypic changes following stimulation with interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-ß1 were examined. To examine the phenotype of SF in vivo, a severe combined immunodeficiency (SCID) human-mouse model of cartilage destruction was utilised. RESULTS: SF in the lining layer in rheumatoid arthritis (RA) expressed high levels of PDPN compared to the normal synovium, whereas CD248 expression was restricted to sub-lining layer cells. TNF-α or IL1 stimulation in vitro resulted in an increased expression of PDPN. In contrast, stimulation with TGF-ß1 induced CD248 expression. In the SCID human-mouse model, rheumatoid SF recapitulated the expression of PDPN and CD248. Fibroblasts adjacent to cartilage expressed PDPN, and attached to, invaded, and degraded cartilage. PDPN+ CD248- SF preceded the appearance of PDPN- CD248+ cells in contralateral implants. CONCLUSIONS: We have identified two distinct SF populations identified by expression of either PDPN or CD248 which are located within different anatomical compartments of the inflamed synovial membrane. These markers discriminate between SF subsets with distinct biological properties. As PDPN-expressing cells are associated with early fibroblast migration and cartilage erosion in vivo, we propose that PDPN-expressing cells may be an attractive therapeutic target in RA.

Blocking interferon γ reduces expression of chemokines CXCL9, CXCL10 and CXCL11 and decreases macrophage infiltration in ex vivo cultured arteries from patients with giant cell arteritis.

BACKGROUND: Interferon γ (IFNγ) is considered a seminal cytokine in the pathogenesis of giant cell arteritis (GCA), but its functional role has not been investigated. We explored changes in infiltrating cells and biomarkers elicited by blocking IFNγ with a neutralising monoclonal antibody, A6, in temporal arteries from patients with GCA. METHODS: Temporal arteries from 34 patients with GCA (positive histology) and 21 controls were cultured on 3D matrix (Matrigel) and exposed to A6 or recombinant IFNγ. Changes in gene/protein expression were measured by qRT-PCR/western blot or immunoassay. Changes in infiltrating cells were assessed by immunohistochemistry/immunofluorescence. Chemotaxis/adhesion assays were performed with temporal artery-derived vascular smooth muscle cells (VSMCs) and peripheral blood mononuclear cells (PBMCs). RESULTS: Blocking endogenous IFNγ with A6 abrogated STAT-1 phosphorylation in cultured GCA arteries. Furthermore, selective reduction in CXCL9, CXCL10 and CXCL11 chemokine expression was observed along with reduction in infiltrating CD68 macrophages. Adding IFNγ elicited consistent opposite effects. IFNγ induced CXCL9, CXCL10, CXCL11, CCL2 and intracellular adhesion molecule-1 expression by cultured VSMC, resulting in increased PBMC chemotaxis/adhesion. Spontaneous expression of chemokines was higher in VSMC isolated from GCA-involved arteries than in those obtained from controls. Incubation of IFNγ-treated control arteries with PBMC resulted in adhesion/infiltration by CD68 macrophages, which did not occur in untreated arteries. CONCLUSIONS: Our ex vivo system suggests that IFNγ may play an important role in the recruitment of macrophages in GCA by inducing production of specific chemokines and adhesion molecules. Vascular wall components (ie, VSMC) are mediators of these functions and may facilitate progression of inflammatory infiltrates through the vessel wall.

Ex Vivo Signaling Protein Mapping in T Lymphocytes in the Psoriatic Arthritis Joints.

We assessed signaling protein mapping in total T cells, to analyze the proportions of T regulatory (Treg) and TCD4+ effector (Teff) cell phenotypes, and the respective interleukin 6Rα (IL-6Rα) expression in the inflammatory microenvironment of synovial fluid (SF) of patients with sustained psoriatic arthritis (PsA). Our approach was to measure the IL-6 level in SF using a multiplex bead immunoassay. Reverse-phase protein array was used to assess Janus kinase (JAK) 1 and JAK2, extra-cellular regulated kinase (ERK) 1 and 2, protein kinase Cδ (PKCδ), signal transducer and activator and transcription (STAT) 1, STAT3, and STAT5 phosphoproteins in total T cell lysates from SF of patients with PsA. Frequencies of CD4+IL-17A-F+IL-23+ CD4+ Th cells producing IL-17A and IL-17F (Th17) and CD4+CD25high intracellular forkhead box transcription factor+ (FOXP3+) phenotypes, and the percentage of Treg- and Teff- cells were quantified in SF and matched peripheral blood (PB) of patients with PsA and PB of healthy controls (HC) by flow cytometry. Our results were the following: In PsA SF samples, a coordinate increase of JAK1, ERK1/2, STAT1, STAT3, and STAT5 phosphoproteins was found in total T cells in SF of PsA; where IL-6 levels were higher than in PB from HC. Expanded CD4+IL-17A-F+IL-23+ Th17, CD4+ CD25- Teff- and CD4+CD25(high) FoxP3+Treg subsets, showing similar levels of enhanced IL-6Rδ expression, were confined to PsA joints. In our studies, the transcriptional network profile identified by ex vivo signaling protein mapping in T lymphocytes in PsA joints revealed the complex interplay between IL-1, IL-6, and IL-23 signaling and differentiation of Th17 cells and CD4+Tregs in sustained joint inflammation in PsA.

Transcriptional network profile on synovial fluid T cells in psoriatic arthritis.

The objective of the study was to quantify the transcriptional profile, as the main T cell lineage-transcription factors on synovial fluid (SF) T cells, in relation to SF cytokines and T cell frequencies (%) of psoriatic arthritis (PsA) patients. Reverse phase protein array was employed to identify interleukin (IL)-23Rp19-, FOXP3- and related orphan receptor gamma T (RORγt)- protein and Janus associated tyrosine kinases 1 (JAK1), signal transducer and activator and transcription 1 (STAT1), STAT3 and STAT5 phosphoproteins in total T cell lysates from SF of PsA patients. IL-1ß, IL-2, IL-6, IL-21 and interferon (INF)-γ were measured using a multiplex bead immunoassay in SF from PsA patients and peripheral blood (PB) from healthy controls (HC). Frequencies of CD4(+)CD25(-), CD4(+)CD25(high) FOXP3(+) and CD4(+)CD25(high) CD127(low) Treg, and either mean fluorescence intensity (MFI) of FOXP3(+) on CD4(+) Treg or MFI of classic IL-6 receptor (IL-6R) α expression on CD4(+)CD25(-) helper/effector T cells (Th/eff) and Treg cells, were quantified in SF of PsA patients and in PB from HC by flow cytometry (FC). In PsA SF samples, IL-2, IL-21 and IFN-γ were not detectable, whereas IL-6 and IL-1ß levels were higher than in SF of non-inflammatory osteoarthritis patients. Higher levels of IL-23R-, FOXP3- and RORγt proteins and JAK1, STAT1, STAT3 and STAT5 were found in total T cells from SF of PsA patients compared with PB from HC. Direct correlations between JAK1 Y1022/Y1023 and STAT5 Y694, and STAT3 Y705 and IL6, were found in SF of PsA patients. Increased proportion of CD4(+)CD25(high) FOXP3(+) and CD4(+)CD25(high) CD127(low) Treg cells and brighter MFI of IL-6Rα were observed both on CD4(+)CD25(high)- and CD4(+)CD25(-) T cells in PsA SF. The study showed a distinctive JAK1/STAT3/STAT5 transcriptional network on T cells in the joint microenvironment, outlining the interplay of IL-6, IL-23, IL-1ß and γC cytokines in the polarization and plasticity of Th17 and Treg cells, which might participate in the perpetuation of joint inflammation in PsA patients.

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

Looking 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.

Blockade of intra-articular TNF in peripheral spondyloarthritis: its relevance to clinical scores, quantitative imaging and synovial fluid and synovial tissue biomarkers.

OBJECTIVES: This open-label study is based on a translational approach with the aim of detecting changes in the clinical condition as well as in imaging and synovial biological markers in both synovial fluid (SF) and synovial tissue (ST) in peripheral spondyloarthritis (SpA) patients following intra-articular (IA) blockade of TNF-α by serial etanercept injections. METHODS: Twenty-seven SpA patients with resistant knee synovitis underwent four biweekly IA injections of etanercept (E) (12.5 mg). The primary outcome of Thompson's Knee Index (THOMP), and secondary outcomes of Knee Joint Articular Index (KJAI), C-reactive protein (CRP), HAQ-Disability Index (HAQ-DI), maximal synovial thickness (MST) according to ultrasonography (US) and contrast-enhanced magnetic resonance (C+MR) imaging, ST-CD45+ mononuclear cells (MNC) and ST-CD31+ vessels, IL-1ß, IL-1Ra and IL-6 levels in SF were assessed at baseline and at the end of the study. RESULTS: At the study end, clinical and imaging outcomes as well as ST and SF biological markers were significantly reduced compared to baseline. There were significant correlations between clinical, imaging and biological markers (CRP with either THOMP, or KJAI, or HAQ-DI or SF-IL-1Ra; US-MST with KJAI, ST-CD45+ with either THOMP, or KJAI, or ST-CD31+, or SF-IL-1ß; SF-IL-6 with either THOMP, or KJAI, or SF-IL-1ß, or IL-1Ra). CONCLUSIONS: The proof of concept study revealed early improvement either in local and systemic clinical scores, in synovial thickness measures by C+MR and US, or expression of synovial biological markers. CD45+, CD31+ in ST and IL-6 and IL-1ß in SF may be considered potential biomarkers of the peripheral SpA response to IA TNF-α blocking.

Synovial biomarkers in psoriatic arthritis.

OBJECTIVE: To find candidate biomarkers of psoriatic arthritis (PsA). A panel of synovial fluid (SF) and synovial tissue (ST) biomarkers was analyzed in patients with resistant peripheral PsA, in relation to clinical and imaging outcomes of synovitis response following serial intraarticular (IA) etanercept injections (12.5 mg). METHODS: Fourteen PsA patients with resistant knee joint synovitis were treated with 4 IA etanercept injections in a single knee joint, once every 2 weeks. Primary outcome (Thompson's knee index: THOMP) and secondary outcomes were assessed at baseline and end of study: C-reactive protein, Knee Joint Articular Index (KJAI), Health Assessment Questionnaire disability index, maximal synovial thickness (MST) by gray-scale ultrasonography, contrast-enhanced magnetic resonance imaging (C+MRI), ST-cluster differentiation (CD)45+ mononuclear cell, ST-CD31+ vessels, and ST-CD105+ angiogenic endothelial cells, along with levels of SF interleukin 1ß (IL-1ß), IL-1 receptor antagonist (Ra), and IL-6. RESULTS: At the end of the study, clinical and imaging outcomes, ST and SF biological markers were significantly reduced compared to baseline. There was a significant association between IL-6 and either THOMP or KJAI; between either ST-CD31+ or ST-CD105+ or ST-CD45+; between ST and SF biomarkers expression (CD45+ and IL-1ß) and between ST-CD45+ and both KJAI and MRI-MST. Comparing pre- versus post-IA etanercept injection changes (Δ), Δ IL-1ß was significantly correlated with both Δ IL-6 and with Δ IL-1Ra and Δ IL-6 with Δ IL-1Ra. CONCLUSION: The association to disease activity and the changes following IA treatment indicate that ST-CD45+ and ST-CD31+, along with SF-IL-6 and SF-IL-1ß, may represent candidate biomarkers of the knee synovitis response to IA tumor necrosis factor-α blockade.

Contact with stimulated T cells up-regulates expression of peptidylarginine deiminase 2 and 4 by human monocytes.

OBJECTIVE: The antigenic targets of the rheumatoid arthritis (RA)-associated autoantibodies to "citrullinated" proteins are generated following citrullination by a peptidylarginine deiminase (PAD). Of the five PAD isotypes, two - PAD2 and PAD4 - are expressed in RA synovial tissue. Within this tissue, the activation of macrophages and fibroblasts mediated by T-cell contact or driven by cytokines plays a prominent part in the pathogenesis. We wanted to determine whether cytokine stimulation and contact with T cells could play a role in PAD expression by peripheral blood monocytes and fibroblastic synoviocytes. METHODS: Human monocytes and T lymphocytes were derived from the blood of healthy donors. HUT-78 cells and T lymphocytes were stimulated with PHA and PMA. Human synovial fibroblasts were isolated after surgical synoviectomy. The expression of PAD was determined by real-time PCR and immunoblot. RESULTS: In monocytes, the PADI2 and PADI4 mRNAs were transiently up-regulated by contact with stimulated HUT-78 and/or T lymphocytes. Positive modulation of the PAD2 and PAD4 proteins were also observed upon contact with stimulated HUT-78 T cells. Stimulation by IL-1ß or IFN-ß did not modify the PADI2 and PADI4 mRNAs, but enhanced PAD4 protein expression. No isotype of PAD was detected at the mRNA or protein level in resting or stimulated synovial fibroblasts. CONCLUSION: Contact between stimulated T cells and monocyte-macrophages or cytokine-activated monocyte-macrophages constitutes a highly likely source of PAD2 and PAD4, which are observed in inflamed synovial tissues. In contrast, it is most unlikely that fibroblastic synoviocytes contribute to PAD expression in rheumatoid synovial membranes.

Serum IL-6 and IL-21 are associated with markers of B cell activation and structural progression in early rheumatoid arthritis: results from the ESPOIR cohort.

OBJECTIVE: To identify a specific pattern of serum cytokines that correlates with the diagnosis, activity and severity of rheumatoid arthritis (RA) in patients with early RA as well as with the level of serum markers of B cell activation. METHODS: Serum interleukin (IL)-1ß, IL-1 receptor antagonist (IL1-Ra), IL-2, IL-4, IL-6, IL-10, IL-17, IL-21, monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor α and interferon γ levels were measured in the (ESPOIR) Etude et Suivi des POlyarthrites Indifférenciées Récentes early arthritis cohort, which included patients with at least two swollen joints for >6 weeks and <6 months, and no previous corticosteroids or disease-modifying antirheumatic drugs. Serum cytokine levels were compared between patients who met the 1987 American College of Rheumatology criteria for RA (n=578) or had undifferentiated arthritis (UA, n=132) at the 1-year follow-up visit. RESULTS: Serum IL-6 and IL-21 were the only cytokines that discriminated RA from UA on univariate analysis. IL-6 level was associated with RA, whereas erythrocyte sedimentation rate and C-reactive protein were not. Higher proportions of rheumatoid factor and anticyclic citrullinated protein (CCP) positivity, levels of markers of B cell activation, and a higher frequency of rapid radiographic progression were observed in patients with RA with detectable IL-6 or IL-21. Multivariate analysis associated IL-6 and anti-CCP levels with radiographic erosions at enrolment with 1-year radiographic progression. CONCLUSION: Serum IL-6 concentration is greater in RA than in UA. Increase in serum IL-6 and IL-21 levels is associated with markers of B cell activation, and IL-6 is associated with radiographic progression in patients with RA.

Cytokine levels in human synovial fluid during the different stages of acute gout: role of transforming growth factor ß1 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 1ß (IL-1ß), IL-6, IL-8, tumour necrosis factorα (TNFα) and transforming growth factor ß1 (TGFß1). 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 TGFß1, which was higher in phase II. IL-1ß, IL-6 and TNFα were higher in group 1 than in group 3. Levels of all the cytokines assessed, with the exception of TGFß1, were significantly lower in phase III than in phase II IL-1ß, p<0.05; IL-6, p<0.01; IL-8, p<0.001; TNFα, p<0.05).TGFß1 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 TGFß1 in the resolution of gout.

Interleukin-6: from identification of the cytokine to development of targeted treatments.

Interleukin-6 (IL-6) was identified based on extensive research conducted simultaneously on a variety of topics ranging from hepatocyte production of acute-phase proteins to plasmacytoma growth. IL-6 is a cytokine produced by a broad array of cell types and can exert its effects on virtually all cells. IL-6 can induce cell signaling not only via the classic pathway involving the transmembrane receptor IL-6Rα (restricted cellular expression) associated with gp130 (ubiquitous and responsible for signal transmission), but also via the soluble receptor IL-6Rα, which binds to IL-6 and induces a signal mediated by the ubiquitous gp130 molecule (transsignaling). IL-6 is deregulated in many inflammatory and autoimmune diseases, including rheumatoid arthritis (RA). By virtue of its multiple effects, IL-6 is involved in the various phases of RA development, including the acute phase, immuno-inflammatory phase, and destructive phase. IL-6 has an impact on the many pathogenic factors identified in RA and, consequently, holds promise for targeted treatments. However, anti-IL-6 monoclonal antibodies evaluated as IL-6 antagonists, instead, increased the half-life of the cytokine. In contrast, monoclonal antibody (tocilizumab) to transmembrane and soluble IL-6Rα has been found effective in patients with RA. Tocilizumab is now indicated for the treatment of adults with RA who have failed at least one synthetic disease-modifying antirheumatic drug or TNFα antagonist.

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

INTRODUCTION: The purpose of this study was the evaluation 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). METHODS: Systemic 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. RESULTS: At baseline, CRP and/or ESR were significantly correlated with SF-CK (interleukin- (IL-)1ß, 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-1ß, IL-1Ra, IL-6 and IL-22). Pre- and post-IA-E injections, there were significant correlations between ST markers and SF-CK (IL-1ß with CD45; IL-1ß 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). CONCLUSIONS: Synovial 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.

Blockade of T cell contact-activation of human monocytes by high-density lipoproteins reveals a new pattern of cytokine and inflammatory genes.

BACKGROUND: Cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes and is likely to play a substantial part in chronic/sterile inflammatory diseases. High-density lipoproteins (HDL) specifically inhibit the production of pro-inflammatory cytokines induced by T cell contact. METHODOLOGY/PRINCIPAL FINDINGS: To further elucidate the pro-inflammatory functions of cellular contact with stimulated T cells and its inhibition by HDL, we carried out multiplex and microarray analyses. Multiplex analysis of monocyte supernatant revealed that 12 out of 27 cytokines were induced upon contact with stimulated T cells, which cytokines included IL-1Ra, G-CSF, GM-CSF, IFNgamma, CCL2, CCL5, TNF, IL-1beta, IL-6, IL-8, CCL3, and CCL4, but only the latter six were inhibited by HDL. Microarray analysis showed that 437 out of 54,675 probe sets were enhanced in monocytes activated by contact with stimulated T cells, 164 probe sets (i.e., 38%) being inhibited by HDL. These results were validated by qPCR. Interestingly, the cytokines induced by T cell contact in monocytes comprised IL-1beta, IL-6 but not IL-12, suggesting that this mechanism might favor Th17 polarization, which emphasizes the relevance of this mechanism to chronic inflammatory diseases and highlights the contrast with acute inflammatory conditions that usually involve lipopolysaccharides (LPS). In addition, the expression of miR-155 and production of prostaglandin E(2)-both involved in inflammatory response-were triggered by T cell contact and inhibited in the presence of HDL. CONCLUSIONS/SIGNIFICANCE: These results leave no doubt as to the pro-inflammatory nature of T cell contact-activation of human monocytes and the anti-inflammatory functions of HDL.

Therapeutic targets in rheumatoid arthritis: the interleukin-6 receptor.

RA is a chronic, debilitating disease in which articular inflammation and joint destruction are accompanied by systemic manifestations including anaemia, fatigue and osteoporosis. IL-6 is expressed abundantly in the SF of RA patients and is thought to mediate many of the local and systemic effects of this disease. Unlike a number of other cytokines, IL-6 can activate cells through both membrane-bound (IL-6R) and soluble receptors (sIL-6R), thus widening the number of cell types responsive to this cytokine. Indeed, trans-signalling, where IL-6 binds to the sIL-6R, homodimerizes with glycoprotein 130 subunits and induces signal transduction, has been found to play a key role in acute and chronic inflammation. Elevated levels of IL-6 and sIL-6R in the SF of RA patients can increase the risk of joint destruction and, at the joint level, IL-6/sIL-6R can stimulate pannus development through increased VEGF expression and increase bone resorption as a result of osteoclastogenesis. Systemic effects of IL-6, albeit through conventional or trans-signalling, include regulation of acute-phase protein synthesis, as well as hepcidin production and stimulation of the hypothalamo-pituitary-adrenal axis, the latter two actions potentially leading to anaemia and fatigue, respectively. This review aims to provide an insight into the biological effects of IL-6 in RA, examining how IL-6 can induce the articular and systemic effects of this disease.

Synovial tissue rank ligand expression and radiographic progression in rheumatoid arthritis: observations from a proof-of-concept randomized clinical trial of cytokine blockade.

The objective of the study was to evaluate synovial tissue receptor activator of nuclear factor-κß ligand (RANKL) and osteoprotegerin (OPG) as biomarkers of disease activity, progressive joint damage, and therapeutic response, during cytokine blockade in rheumatoid arthritis (RA). Patients with active RA entered a randomized open-label 12-month study of anakinra 100 mg/day, administered as monotherapy or in combination with pegsunercept 800 µg/kg twice weekly. Arthroscopic synovial tissue biopsies were obtained at baseline, at 4 weeks and at the final time point. Following immunohistochemical staining, RANKL and OPG expression was quantified using digital image analysis. Radiographic damage was evaluated using the van der Heijde modification of the Sharp scoring system. Twenty-two patients were randomized. Baseline expression of RANKL, but not OPG, correlated significantly with baseline CRP levels (r = 0.61, P < 0.01). While a significant reduction in OPG expression following treatment was observed in clinical responders at the final time point (P < 0.05 vs. baseline), RANKL levels did not change, and the RANKL:OPG ratio remained unaltered, even at the highest levels of clinical response. When potential predictors of radiographic outcome were evaluated, baseline RANKL expression correlated with erosive progression at 1 year (r = 0.71, P < 0.01). Distinct, though related, pathophysiologic processes mediate joint inflammation and destruction in RA. Elevated synovial tissue RANKL expression is associated with progressive joint erosion, and may be independent of the clinical response to targeted therapy. The potential therapeutic importance of modulating RANKL in RA is highlighted, if radiographic arrest is to be achieved.

Synovial tissue and serum biomarkers of disease activity, therapeutic response and radiographic progression: analysis of a proof-of-concept randomised clinical trial of cytokine blockade.

OBJECTIVES: To evaluate synovial tissue and serum biomarkers of disease activity, therapeutic response and radiographic progression during biological therapy for rheumatoid arthritis (RA). METHODS: Patients with active RA entered a randomised study of anakinra 100 mg/day, administered as monotherapy or in combination with pegsunercept 800 microg/kg twice a week. Arthroscopic synovial tissue biopsies were obtained at baseline and two further time points. Following immunohistochemical staining, selected mediators of RA pathophysiology were quantified using digital image analysis. Selected mediators were also measured in the serum. RESULTS: Twenty-two patients were randomly assigned: 11 received monotherapy and 11 combination therapy. American College of Rheumatology 20, 50 and 70 response rates were 64%, 64% and 46% with combination therapy and 36%, 9% and 0% with monotherapy, respectively. In synovial tissue, T-cell infiltration, vascularity and transforming growth factor beta (TGFbeta) expression demonstrated significant utility as biomarkers of disease activity and therapeutic response. In serum, interleukin 6 (IL-6), matrix metalloproteinase (MMP) 1, MMP-3 and tissue inhibitor of metalloproteinase 1 (TIMP-1) were most useful in this regard. An early decrease in serum levels of TIMP-1 was predictive of the later therapeutic outcome. Pretreatment tissue levels of T-cell infiltration and the growth factors vascular endothelial growth factor/TGFbeta, and serum levels of IL-6, IL-8, MMP-1, TIMP-1, soluble tumour necrosis factor receptor types I and II and IL-18 correlated with radiographic progression. CONCLUSIONS: Synovial tissue analysis identified biomarkers of disease activity, therapeutic response and radiographic progression. Biomarker expression in tissue was independent of the levels measured in the serum.

Immunological determinants of clinical outcome in Peruvian patients with tegumentary leishmaniasis treated with pentavalent antimonials.

The mechanisms linking the immune response to cutaneous and mucosal leishmaniasis (CL and ML, respectively) lesions and the response to treatment are incompletely understood. Our aims were to prospectively assess, by quantitative reverse transcription-PCR, the levels of mRNA for gamma interferon, tumor necrosis factor alpha, interleukin-10 (IL-10), IL-4, and IL-13, as well as the presence of T cells (CD2) and macrophages (CD68), in CL and ML lesions and to follow their changes in response to treatment with pentavalent antimonials. The leishmanin skin test (LST) was performed on all CL and ML patients before treatment. The patient population included individuals living in areas of Peru where the disease is endemic, i.e., 129 with CL and 43 with ML. Compared to CL patients, the LST induration size was larger, the levels of all cytokine mRNAs but IL-10 were higher, T-cell mRNA was similar, and macrophage mRNA was lower in ML patients. The proportion of CL patients with an LST induration size of >8 mm was higher among responders to treatment. In CL, the pretreatment levels of cytokine mRNAs did not discriminate between responders and nonresponders; however, treatment was more often accompanied by a reduction in the levels of T-cell and cytokine mRNAs in responders than in nonresponders. Furthermore, the production of cytokines per T cell and macrophage decreased with treatment but IL-10 production remained high in nonresponders. Overall, these findings point to complex relationships among New World Leishmania parasites, skin and mucosal immune responses, and treatment outcome. The persistence of high levels of IL-10 in CL is characteristically associated with a poor response to treatment.

Prostaglandin E2 synergistically with interleukin-23 favors human Th17 expansion.

Microenvironment molecular cues direct T helper (Th) cell differentiation; however, Th17 fate determination is still imprecisely understood in humans. To assess the role of prostaglandin E(2) (PGE(2)) in Th expansion, we activated peripheral blood mononuclear cells by CD3 cross-linking. In the presence of exogenous PGE(2), peripheral blood mononuclear cells produced higher interleukin-17 (IL-17), C-C chemokine ligand 20 (CCL20)/macrophage inflammatory protein 3alpha (MIP-3alpha), CXC chemokine ligand 8 (CXCL8)/IL-8, and lower interferon-gamma and IL-22 levels than in control cultures. Exogenous PGE(2) and IL-23 synergized in inducing IL-17, whereas indomethacin and IL-23 blockade drastically reduced IL-17 but not interferon-gamma production. Furthermore, IL-1 but not tumor necrosis factor was absolutely required for IL-17 production. PGE(2) doubled the frequency of CD4+ T cells producing IL-17 and within the CD4+ subset enhanced C-C chemokine receptor 6 (CCR6) and CCR4 while decreasing CXC chemokine receptor 3 (CXCR3) expression. Furthermore, in CD4+ T-cell lines, the production of IL-17 segregated with the CCR6+ subset. In the presence of CCR6+ compared with CXCR3+ Th cells, monocytes/macrophages produced much higher levels of matrix metalloproteinase-1, -3, and -9 but similar levels of CXCL10 and IL-1beta. These results identify PGE(2) and IL-23 as participating in the expansion of CD4+ T cells endowed with high IL-17 production capacity, which in turn favors monocyte production of mediators important for host defense and tissue destruction.