Bernard Grillet

Cleavage of denatured natural collagen type II by neutrophil gelatinase B reveals enzyme specificity, post-translational modifications in the substrate, and the formation of remnant epitopes in rheumatoid arthritis

During acute inflammation, leukocytes release proteolytic enzymes including matrix metalloproteinases (MMPs), but the physiopathological mechanisms and consequences of this process are not yet fully understood. Neutrophils, the predominant leukocyte type, produce neutrophil collagenase (MMP‐8) and gelatinase B (MMP‐9) but not the tissue inhibitors of MMPs. After stimulation, these cells also activate MMPs chemically. In arthritic diseases, neutrophils undergo great chemoattraction to the synovium, are activated by interleukin‐8, and are stimulated to release gelatinase B in vivo. Production levels and net activities of gelatinase B were found to be absent in degenerative osteoarthritis but significantly increased in rheumatoid arthritis. The cleavage sites in cartilage type II collagen by gelatinase B were determined by a combination of reverse phase high‐performance liquid chromatography, Edman degradation, and mass spectrometry analysis. The analysis revealed the site specificity of proline and lysine hydroxylations and O‐linked glycosylation, the cleavage specificities by gelatinase B, and the preferential absence and presence of post‐translational modifications at P2′ and P5′, respectively. Furthermore, gelatinase B leaves the immunodominant peptides intact, which are known from studies with (autoreactive) T cells. Lysine hydroxylation was detected at a critical position for T‐cell activation. These data lend support to the thesis that extracellular proteolysis and other post‐translational modifications of antigenic peptides may be critical in the establishment and perpetuation of autoimmune processes.—Van den Steen, P.E., Proost, P., Grillet, B., Brand, D.D., Kang, A.H., Van Damme, J., Opdenakker, G. Cleavage of denatured natural collagen type II by neutrophil gelatinase B reveals enzyme specificity, post‐translational modifications in the substrate, and the formation of remnant epitopes in rheumatoid arthritis. FASEB J. 16, 379–389 (2002)

Microbial Toll‐like receptor ligands differentially regulate CXCL10/IP‐10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN‐γ and provide a mechanism for enhanced synovial chemokine levels in septic arthritis

The CXC chemokine IFN‐γ‐inducible protein‐10 (IP‐10/CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T cells and natural killer cells. Peripheral blood mononuclearcells (PBMC) produce low but significant amounts of IP‐10/CXCL10 protein upon stimulation with double‐stranded (ds) RNA, the Toll‐like receptor 3 (TLR3) ligand. IFN‐γ is a superior IP‐10/CXCL10inducer. The bacterial TLR4 and TLR2 ligands, LPS and peptidoglycan (PGN), inhibit IFN‐γ‐ or dsRNA‐dependent IP‐10/CXCL10 production in PBMC, whereas IL‐8/CXCL8 production was enhanced. In fibroblasts a different picture emerges with IFN‐γ inducing moderate and dsRNA provoking strong IP‐10/CXCL10 production. Furthermore, treatment of fibroblasts with IFN‐γ in combination with bacterial LPS or PGN results in a synergistic production of IP‐10/CXCL10 and IL‐8/CXCL8. The synergistic induction of IP‐10/CXCL10 in fibroblasts is reflected by significantly enhanced IP‐10/CXCL10 concentrations in synovial fluids of septic compared to osteoarthritis patients to reach on average higher levels than those of IL‐8/CXCL8. These high amounts of IP‐10/CXCL10 produced by connective tissue fibroblasts not only attract CXCR3 expressing activated Th1 cells and natural killer cells to sites of infection but may also antagonize the CCR3 dependent attraction of Th2 lymphocytes and exert CXCR3‐independent, defensin‐like antibacterial activity.

TLR ligands and cytokines induce CXCR3 ligands in endothelial cells: enhanced CXCL9 in autoimmune arthritis

CXC chemokines are potent attractants of neutrophil granulocytes, T cells or natural killer cells. Toll-like receptors (TLR) recognize microbial components and are also activated by endogenous molecules possibly implicated in autoimmune arthritis. In contrast to CXC chemokine ligand 8 (CXCL8), no CXC chemokine receptor 3 (CXCR3) ligand (ie CXCL9, CXCL10 and CXCL11) was induced by bacterial TLR ligands in human microvascular endothelial cells (HMVEC). However, peptidoglycan (PGN), double-stranded (ds) RNA or lipopolysaccharide (LPS) (TLR2, TLR3 or TLR4 ligands, respectively) synergized with interferon-γ (IFN-γ) at inducing CXCL9 and CXCL10. In contrast, enhanced CXCL11 secretion was only obtained when IFN-γ was combined with TLR3 ligand. Furthermore, flagellin, loxoribine and unmethylated CpG oligonucleotide (TLR5, TLR7 and TLR9 ligands, respectively) did not enhance IFN-γ-dependent CXCR3 ligand production in HMVEC. In analogy with TLR ligands, tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β), in combination with IFN-γ, synergistically induced CXCL9 and CXCL11 in HMVEC and human fibroblasts, two fundamental cell types delineating the joint cavity. Etanercept, a humanized soluble recombinant p75 TNF-receptor/IgG1Fc fusionprotein, neutralized synergistic CXCL9 production induced by TNF-α plus IFN-γ, but not synergy between IFN-γ and the TLR ligands PGN or LPS. Synovial chemokine concentrations exemplify the fysiopathological relevance of the observed in vitro chemokine production patterns. In synovial fluids of patients with spondylarthropathies (ie ankylosing spondylitis or psoriatic arthritis) or rheumatoid arthritis, significantly enhanced CXCL9, but not CXCL11 levels, were detected compared to concentrations in synovial fluids of patients with metabolic crystal-induced arthritis. Thus, CXCL9 is an important chemokine in autoimmune arthritis.

Synergistic induction of CXCL9 and CXCL11 by Toll‐like receptor ligands and interferon‐γ in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids

The synovial cavity constitutes the ideal stage to study the interplay between microbial Toll‐like receptor (TLR) ligands and cytokines. Infiltrated leukocytes and synovial fibroblasts produce cytokine‐ and chemokine‐induced proteases for remodeling the extracellular matrix. The regulation of chemokine function for attraction and activation of leukocytes constitutes a key feature in host immunity and resolution of inflammation after infection. Enhanced levels of the CXC chemokine ligand (CXCL9)/monokine induced by interferon‐γ (IFN‐γ) and CXCL11/IFN‐inducible T cell α chemoattractant, two chemoattractants for activated T cells and natural killer cells, and ligands for CXC chemokine receptor 3 (CXCR3) were detected in the synovial fluid of septic arthritis compared with osteo‐ and crystal arthritis patients. In vitro, IFN‐γ and TLR3 ligation by double‐stranded RNA (dsRNA) induced the expression of CXCL9 and CXCL11 in leukocytes and skin‐muscle fibroblasts, whereas ligation of TLR2, TLR4, TLR5, and TLR9 by peptidoglycan (PGN), lipopolysaccharide (LPS), flagellin, and unmethylated CpG oligonucleotides, respectively, did not. PGN and LPS, but not unmethylated CpG oligonucleotides, even inhibited IFN‐γ‐induced CXCL9 and CXCL11 expression in leukocytes. In sharp contrast, in fibroblasts, the TLR ligands PGN, dsRNA, LPS, and flagellin synergized with IFN‐γ for the production of CXCL9 and CXCL11. Although TLR ligands stimulate leukocytes to produce CXCL8/interleukin‐8 during the early innate defense, they contribute less to the production of CXCR3 ligands, whereas fibroblasts are important sources of CXCR3 ligands. These results illustrate the complex interaction between cytokines and TLR ligands in infection.

Selective induction of CCL18/PARC by staphylococcal enterotoxins in mononuclear cells and enhanced levels in septic and rheumatoid arthritis

Chemokines are mediators of innate and acquired immunity. CCL18, also designated pulmonary and activation‐regulated chemokine (PARC), dendritic cell‐derived CC chemokine‐1 (DC‐CK1), alternative macrophage activation‐associated CC chemokine‐1 (AMAC‐1) and macrophage inflammatory protein‐4 (MIP‐4), was for the first time isolated from peripheral blood mononuclear cells (PBMC) and biochemically characterized. We found that CCL18/PARC protein is spontaneously secreted by PBMC and is selectively induced in PBMC by staphylococcal enterotoxins (SEA, SEB) and IL‐4, but not by IFN‐γ andthe CXCL8/IL‐8 inducers lipopolysaccharide (LPS) or Concanavalin A. Human fibroblasts, chondrocytes and endothelial cells did not produce CCL18/PARC in response to inflammatory mediators such as measles virus, double‐stranded RNA, LPS or IL‐1β, whereas up to 150 ng/ml of CCL2/MCP‐1 was induced under these conditions. In synovial fluids from septic and rheumatoid arthritis patients, fourfold‐enhanced CCL18/PARC levels (150 ng/ml) were detected compared to those in crystal‐induced arthritis and osteoarthritis. In septic arthritis, the synovial levels of CCL18/PARC were fivefold higher than those of CXCL8/IL‐8. Immunochemistry revealed CD68+ monocytes/macrophages as the main CCL18/PARC‐producing cell type in both PBMC and arthritic synovial tissue. In addition, CD1a+ blood dendritic cells expressed CCL18/PARC. These findings suggest that monocytic cells respond to Gram‐positive bacterial infection by the production of CCL18/PARC in the synovial cavity.

Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies

Leukocyte infiltration during acute and chronic inflammation is regulated by exogenous and endogenous factors, including cytokines, chemokines and proteases. Stimulation of fibroblasts and human microvascular endothelial cells with the inflammatory cytokines interleukin-1β (IL-1β) or tumour necrosis factor alpha (TNF-α) combined with either interferon-α (IFN-α), IFN-β or IFN-γ resulted in a synergistic induction of the CXC chemokine CXCL10, but not of the neutrophil chemoattractant CXCL8. In contrast, simultaneous stimulation with different IFN types did not result in a synergistic CXCL10 protein induction. Purification of natural CXCL10 from the conditioned medium of fibroblasts led to the isolation of CD26/dipeptidyl peptidase IV-processed CXCL10 missing two NH2-terminal residues. In contrast to intact CXCL10, NH2-terminally truncated CXCL10(3–77) did not induce extracellular signal-regulated kinase 1/2 or Akt/protein kinase B phosphorylation in CXC chemokine receptor 3-transfected cells. Together with the expression of CXCL10, the expression of membrane-bound CD26/dipeptidyl peptidase IV was also upregulated in fibroblasts by IFN-γ, by IFN-γ plus IL-1β or by IFN-γ plus TNF-α. This provides a negative feedback for CXCL10-dependent chemotaxis of activated T cells and natural killer cells. Since TNF-α and IL-1β are implicated in arthritis, synovial concentrations of CXCL8 and CXCL10 were compared in patients suffering from crystal arthritis, ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis. All three groups of autoimmune arthritis patients (ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis) had significantly increased synovial CXCL10 levels compared with crystal arthritis patients. In contrast, compared with crystal arthritis, only rheumatoid arthritis patients, and not ankylosing spondylitis or psoriatic arthritis patients, had significantly higher synovial CXCL8 concentrations. Synovial concentrations of the neutrophil chemoattractant CXCL8 may therefore be useful to discriminate between autoimmune arthritis types.

Health-related quality of life and functional ability in patients with early arthritis during remission steered treatment: results of the IMPROVED study.

IntroductionThe aim of this study was to investigate patient reported outcomes (PROs) of functional ability and health related quality of life (HRQoL) in patients with early (rheumatoid) arthritis during one year of remission steered treatment.MethodsIn this study, 610 patients with early rheumatoid arthritis (RA) or undifferentiated arthritis (UA) were treated with methotrexate (MTX) and tapered high dose of prednisone. Patients in early remission (Disease Activity Score (DAS) <1.6 after 4 months) tapered prednisone to zero and when in persistent remission, also tapered MTX. Patients not in early remission were randomized to either MTX + hydroxychloroquine + sulphasalazine + prednisone (arm 1) or to MTX + adalimumab (arm 2). Every 4 months, patients filled out the Health Assessment Questionnaire (HAQ) and the McMaster Toronto Arthritis Patient Preference Questionnaire (MACTAR), the Short Form 36 (SF-36) and visual analogue scales (VAS). Change scores were compared between treatment groups. The association with achieving remission was analyzed using linear mixed models.ResultsDuring year 1, patients who achieved early remission had the most improvement in PROs with scores comparable to the general population. Patients in the randomization arms showed less improvement. Scores were comparable between the arms. There was a significant association between achieving remission and scores of HAQ, MACTAR and physical HRQoL.ConclusionsIn early arthritis, PROs of functional ability and HRQoL after one year of remission steered treatment reach normal values in patients who achieved early remission. In patients not in early remission, who were randomized to two strategy arms, PROs improved less, with similar scores in both treatment arms.Trial registrationsISRCTN11916566 and EudraCT2006-006186-16

Human gelatinase B, a marker enzyme in rheumatoid arthritis, is inhibited by D-penicillamine: Anti-rheumatic activity by protease inhibition

SummaryThe direct and indirect inhibitory potential of D-penicillamine toward human neutrophil and synovial fluid gelatinase B, a marker enzyme for disease severity in RA, was investigated. Gelatinase and plasminogen activator activities were assessed by SDS-polyacrylamide gel electrophoresis zymography. D-penicillamine significantly inhibits purified and synovial fluid gelatinase Bin vitro at concentrations attainablein vivo and also blocksin vitro plasminogen activation. Protease inhibition may be a mechanism of action for D-penicillamine as DMARD.

Gelatinase B Participates in Collagen II Degradation and Releases Glycosylated Remnant Epitopes in Rheumatoid Arthritis

Rheumatoid arthritis is an autoimmune disease characterized by chronic inflammation of the joints. It is associated with the activation of autoreactive T-cells and with production of autoantibodies. The main auto-antigen is collagen type II, which is a major constituent of the cartilage in the joint. The inflammation causes cartilage degradation, hyperplasia of synovial membranes, accumulation of excessive synovial fluid, and, in the worst cases, bone erosion. The exact aetiology is not known, but it is clear that inflammatory reactions and auto-antibodies, which activate the complement cascade, are main causes of the cartilage degradation. Inhibition of inflammation by interference with some of the main pro-inflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a) has proven to be beneficial and constitutes the basis of currently approved treatments. Matrix metalloproteinases are a family of neutral Zn2+-dependent endoproteases, which share a number of homologous domains (Nagase and Woessner, 1999). These domains are the Zn2+-containing active site, kept inactive by a propeptide in the pro-enzyme form, and a hemopexin domain (Fig. 1). The latter domain is present in most MMPs except for matrilysins (MMP-7 and -26) and cysteine-array MMP (CA-MMP or MMP-23). Several MMPs contain additional domains, such as a carboxyterminal membrane anchor in membrane-type MMPs (MT-MMPs), a fibronectin-like domain in gelatinases A and B (MMP-2 and -9), and a unique mucin-type domain in gelatinase B. The latter domain is often named collagen type

Do we need to lower the cut point of the 2010 ACR/EULAR classification criteria for diagnosing rheumatoid arthritis?

OBJECTIVE In this study we aimed to evaluate the effect of lowering the cut point of the 2010 criteria to identify more patients with RA among early inflammatory arthritis patients. METHODS We included early arthritis patients from the Rotterdam Early Arthritis Cohort with at least one joint with clinical synovitis and symptoms for <1 year, with no other explanation for their symptoms. The demographic and clinical characteristics of each patient were recorded at baseline. Patients were classified as case or non-case at the 1-year follow-up by the definition used in the development of the 2010 criteria (MTX initiation). To assess the diagnostic performance of the 2010 criteria, the sensitivity and specificity at each cut point were determined. RESULTS We included 557 patients in our analysis. At the 1-year follow-up, 253 patients (45%) were classified as case (MTX use). In the group of patients who scored 0-5 points (n = 328), 98 patients (30%) were classified as case (MTX use). The sensitivity and specificity of the 2010 criteria using the cut point of 6 were 61% and 76%, respectively. With the cut point of 5, the sensitivity would increase to 76% and the specificity would decrease to 68%. CONCLUSION By lowering the cut point of the 2010 criteria from 6 to 5 points, we were able to identify 15% more RA patients at the cost of 8% more false-positive patients.