Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

Lubricin, encoded by the gene PRG4, is the principal lubricant in articulating joints. We immunized mice genetically deficient for lubricin (Prg4-/-) with purified human lubricin, and generated several mAbs. We determined each mAb’s binding epitope, sensitivity, and specificity using biologic samples and recombinant lubricin sub-domains, and we also developed a competition ELISA assay to measure lubricin in synovial fluid and blood. We found the mAbs all recognized epitopes containing O-linked oligosaccharides conjugated to the peptide motif KEPAPTTT. By western blot, the mAbs detected lubricin in 1 μl of synovial fluid from several animal species, including human. The mAbs were specific for lubricin since they did not cross-react with other synovial fluid constituents from patients with camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP), who genetically lack this protein. The competition ELISA detected lubricin in blood samples from healthy individuals but not from patients with CACP, indicating blood can be used in a diagnostic test for patients suspected of having CACP. Lubricin epitopes in blood do not represent degradation fragments from synovial fluid. Therefore, although blood lubricin levels did not differentiate patients with inflammatory joint disease from healthy controls, epitope-specific anti-lubricin mAbs could be useful for monitoring disease activity in synovial fluid.     

Increasing expression of substance P and calcitonin gene-related peptide in synovial tissue and fluid contribute to the progress of arthritis in developmental dysplasia of the hip

IntroductionDevelopmental dysplasia of the hip (DDH) is a common musculoskeletal disorder that has pain and loss of joint function as major pathological features. In the present study, we explored the mechanisms of possible involvement and regulation of substance P (SP) and calcitonin gene-related peptide (CGRP) in the pathological and inflammatory processes of arthritis in DDH.MethodsBlood, synovial tissue and fluid samples were collected from patients diagnosed with different severities of DDH and from patients with femoral neck fracture. Levels of SP, CGRP and inflammatory cytokines in synovium and synovial fluid (SF) in the different groups were evaluated by immunohistochemistry, real-time PCR and enzyme-linked immunosorbent assay (ELISA). Correlations between neuropeptides and inflammatory cytokines in SF were evaluated by partial correlation analysis. The proinflammatory effects of SP and CGRP on synoviocytes obtained from patients with moderate DDH were investigated in vitro by real-time PCR and ELISA. The mechanisms of those effects were evaluated by Western blot analysis and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) DNA binding assay.ResultsSignificantly increased levels of neuropeptides and inflammatory cytokines were observed in synovium and SF from patients in the severe DDH group compared with the moderate DDH and control groups. In moderate DDH samples, SP in SF correlated with tumor necrosis factor (TNF)-α, and CGRP in SF correlated with TNF-α and interleukin (IL)-10. In the severe DDH group, SP in SF correlated with interleukin (IL)-1β, TNF-α and IL-10. CGRP in SF correlated with TNF-α. Additionally, SP might have had obvious proinflammatory effects on synoviocytes through the activation of NF-κB.ConclusionsThe upregulation of SP and CGRP in synovium and SF might participate in the inflammatory process of arthritis in DDH. The activation of the NF-κB pathway seems indispensable in the proinflammatory effect of SP on synoviocytes. This original discovery may indicate a potential clinical drug target and the development of innovative therapies for DDH.     

Synergistic Effects of Ethanol and Isopentenyl Pyrophosphate on Expansion of γδ T Cells in Synovial Fluid from Patients with Arthritis

Low to moderate ethanol consumption has been associated with protective effects in autoimmune diseases such as rheumatoid arthritis, RA. An expansion of γδ T cells induced by isopentenyl pyrophosphate, IPP, likewise seems to have a protective role in arthritis. The aim of this project was to test the hypothesis that low doses of ethanol can enhance IPP-induced expansion of synovial fluid γδ T cells from patients with arthritis and may thereby potentially account for the beneficial effects of ethanol on symptoms of the arthritic process. Thus, mononuclear cells from synovial fluid (SF) from 15 patients with arthritis and from peripheral blood (PB) from 15 healthy donors were stimulated with low concentrations of ethanol and IPP for 7 days in vitro. IPP in combination with ethanol 0.015%, 2.5 mM, equivalent to the decrease per hour in blood ethanol concentration due to metabolism, gave a significantly higher fractional expansion of SF γδ T cells compared with IPP alone after 7 days (ratio 10.1+/−4.0, p<0.0008, n = 12) in patients with arthritis. Similar results were obtained for PB γδ T cells from healthy controls (ratio 2.0+/−0.4, p<0.011, n = 15). The augmented expansion of γδ T cells in SF is explained by a higher proliferation (p = 0.0034, n = 11) and an increased survival (p<0.005, n = 11) in SF cultures stimulated with IPP plus ethanol compared to IPP alone. The synergistic effects of IPP and ethanol indicate a possible allosteric effect of ethanol. Similar effects could be seen when stimulating PB with ethanol in presence of risedronate, which has the ability to increase endogenous levels of IPP. We conclude that expansion of γδ T cells by combinatorial drug effects, possibly in fixed-dose combination, FDC, of ethanol in the presence of IPP might give a protective role in diseases such as arthritis.     

Articular Joint Lubricants during Osteoarthritis and Rheumatoid Arthritis Display Altered Levels and Molecular Species

Background

Hyaluronic acid (HA), lubricin, and phospholipid species (PLs) contribute independently or together to the boundary lubrication of articular joints that is provided by synovial fluid (SF). Our study is the first reporting quantitative data about the molecular weight (MW) forms of HA, lubricin, and PLs in SF from cohorts of healthy donors, patients with early (eOA)- or late (lOA)-stage osteoarthritis (OA), and patients with active rheumatoid arthritis (RA).

Methods

We used human SF from unaffected controls, eOA, lOA, and RA. HA and lubricin levels were measured by enzyme-linked immunosorbent assay. PLs was quantified by electrospray ionization tandem mass spectrometry. Fatty acids (FAs) were analyzed by gas chromatography, coupled with mass spectrometry. The MW distribution of HA was determined by agarose gel electrophoresis.

Results

Compared with control SF, the concentrations of HA and lubricin were lower in OA and RA SF, whereas those of PLs were higher in OA and RA SF. Moreover, the MW distribution of HA shifted toward the lower ranges in OA and RA SF. We noted distinct alterations between cohorts in the relative distribution of PLs and the degree of FA saturation and chain lengths of FAs.

Conclusions

The levels, composition, and MW distribution of all currently known lubricants in SF—HA, lubricin, PLs—vary with joint disease and stage of OA. Our study is the first delivering a comprehensive view about all joint lubricants during health and widespread joint diseases. Thus, we provide the framework to develop new optimal compounded lubricants to reduce joint destruction.     

Focal adhesion kinase is required for synovial fibroblast invasion,but not murine inflammatory arthritis

Introduction

Synovial fibroblasts invade cartilage and bone, leading to joint destruction in rheumatoid arthritis. However, the mechanisms that regulate synovial fibroblast invasion are not well understood. Focal adhesion kinase (FAK) has been implicated in cellular invasion in several cell types, and FAK inhibitors are in clinical trials for cancer treatment. Little is known about the role of FAK in inflammatory arthritis, but, given its expression in synovial tissue, its known role in invasion in other cells and the potential clinical availability of FAK inhibitors, it is important to determine if FAK contributes to synovial fibroblast invasion and inflammatory arthritis.

Methods

After treatment with FAK inhibitors, invasiveness of human rheumatoid synovial fibroblasts was determined with Matrigel invasion chambers. Migration and focal matrix degradation, two components of cellular invasion, were assessed in FAK-inhibited rheumatoid synovial fibroblasts by transwell assay and microscopic examination of fluorescent gelatin degradation, respectively. Using mice with tumor necrosis factor α (TNFα)–induced arthritis in which fak could be inducibly deleted, invasion and migration by FAK-deficient murine arthritic synovial fibroblasts were determined as described above and arthritis was clinically and pathologically scored in FAK-deficient mice.

Results

Inhibition of FAK in human rheumatoid synovial fibroblasts impaired cellular invasion and migration. Focal matrix degradation occurred both centrally and at focal adhesions, the latter being a novel site for matrix degradation in synovial fibroblasts, but degradation was unaltered with FAK inhibitors. Loss of FAK reduced invasion in murine arthritic synovial fibroblasts, but not migration or TNFα-induced arthritis severity and joint erosions.

Conclusions

FAK inhibitors reduce synovial fibroblast invasion and migration, but synovial fibroblast migration and TNFα-induced arthritis do not rely on FAK itself. Thus, inhibition of FAK alone is unlikely to be sufficient to treat inflammatory arthritis, but current drugs that inhibit FAK may inhibit multiple factors, which could increase their efficacy in rheumatoid arthritis.     

5-Lipoxygenase Inhibitors Attenuate TNF-α-Induced Inflammation in Human Synovial Fibroblasts

The lipoxygenase isoform of 5-lipoxygenase (5-LOX) is reported to be overexpressed in human rheumatoid arthritis synovial tissue and involved in the progress of inflammatory arthritis. However, the detailed mechanism of how 5-lipoxygenase regulates the inflammatory response in arthritis synovial tissue is still unclear. The aim of this study was to investigate the involvement of lipoxygenase pathways in TNF-α-induced production of cytokines and chemokines. Human synovial fibroblasts from rheumatoid patients were used in this study. 5-LOX inhibitors and shRNA were used to examine the involvement of 5-LOX in TNF-α-induced cytokines and chemokines expression. The signaling pathways were examined by Western Blotting or immunofluorescence staining. The effect of 5-LOX inhibitor on TNF-α-induced chemokine expression and paw edema was also explored in vivo in C57BL/6 mice. Treatment with 5-LOX inhibitors significantly decreased TNF-α-induced pro-inflammatory mediators including interleukin-6 (IL-6) and monocyte chemo-attractant protein-1 (MCP-1) in human synovial fibroblasts. Knockdown of 5-LOX using shRNA exerted similar inhibitory effects. The abrogation of NF-κB activation was involved in the antagonizing effects of these inhibitors. Furthermore, 5-LOX inhibitor decreased TNF-α-induced up-regulation of serum MCP-1 level and paw edema in mouse model. Our results provide the evidence that the administration of 5-LOX inhibitors is able to ameliorate TNF-α-induced cytokine/chemokine release and paw edema, indicating that 5-LOX inhibitors may be developed for therapeutic treatment of inflammatory arthritis.     

MiR-125a-3p inhibits cell proliferation and inflammation responses in fibroblast-like synovial cells in rheumatoid arthritis by mediating the Wnt/β-catenin and NF-κB pathways via targeting MAST3

Objectives:To explore the role and mechanism of miR-125a-3p in rheumatoid arthritis (RA) progression.Methods:The RA-tissues and fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS) were used in this study. qRT-PCR, western blot and ELISA assay were performed to detect the expression levels of IL-6, IL-β and ΤΝF-α. Dual-luciferase reporter gene assay was used to observe the binding effect of miR-125a-3p and MAST3, and CCK-8 was used to observe the effect of miR-125a-3p on the proliferation of RA-FLS.Results:miR-125a-3p was significantly downregulated in the RA-tissues and RA-FLS, and miR-125a-3p could inhibit the proliferation and reduce the inflammation response of RA-FLS. Besides, MAST3 was found as a target of miR-125a-3p, and increased MAST3 could reverse the effects of miR-125a-3p on RA-FLS including decreased proliferation, reduced inflammation level and the inactivation of Wnt/β-catenin and NF-κB pathways.Conclusions:This study suggests that miR-125a-3p could inactivate the Wnt/β-catenin and NF-κB pathways to reduce the proliferation and inflammation response of RA-FLS via targeting MAST3.     

Liposomal Targeting of Prednisolone Phosphate to Synovial Lining Macrophages during Experimental Arthritis Inhibits M1 Activation but Does Not Favor M2 Differentiation

Background

To determine the effects of liposomal targeting of prednisolone phosphate (Lip-PLP) to synovial lining macrophages on M1 and M2 polarization in vitro and during experimental arthritis.

Material and Methods

Experimental arthritis (antigen and immune complex induced) was elicited in mice and prednisolone containing liposomes were given systemically. Synovium was investigated using microarray analysis, RT-PCR and histology. Bone–marrow macrophages were stimulated towards M1 using LPS and IFNγ before treatment by PLP-liposomes. M1 and M2 markers were determined using RT-PCR.

Results

Microarray analysis of biopsies of inflamed synovium during antigen induced arthritis (AIA) showed an increased M1 signature characterized by upregulation of IL-1β, IL-6 and FcγRI starting from day 1 and lasting up until day 7 after arthritis induction. The M2 signature remained low throughout the 7 day course of arthritis. Treatment of AIA with intravenously delivered Lip-PLP strongly suppressed joint swelling and synovial infiltration whereas colloidal gold containing liposomes exclusively targeted the macrophages within the inflamed synovial intima layer. In vitro studies showed that Lip-PLP phagocytosed by M1 macrophages resulted in a suppression of the M1 phenotype and induction of M2 markers (IL-10, TGF-β, IL-1RII, CD163, CD206 and Ym1). In vivo, Lip-PLP treatment strongly suppressed M1 markers (TNF-α, IL-1β, IL-6, IL-12p40, iNOS, FcγRI, Ciita and CD86) after local M1 activation of lining macrophages with LPS and IFN-γ and during experimental AIA and immune complex arthritis (ICA). In contrast, M2 markers were not significantly upregulated in antigen-induced arthritis and down regulated in immune complex arthritis.

Conclusion

This study clearly shows that systemic treatment with PLP-liposomes selectively targets synovial lining macrophages and inhibits M1 activation. In contrast to in vitro findings, PLP-liposomes do not cause a shift of synovial lining macrophages towards M2.     

Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization,KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients.     

Linking Power Doppler Ultrasound to the Presence of Th17 Cells in the Rheumatoid Arthritis Joint

Background

Power Doppler ultrasound (PDUS) is increasingly used to assess synovitis in Rheumatoid Arthritis (RA). Prior studies have shown correlations between PDUS scores and vessel counts, but relationships with T cell immunopathology have not been described.

Methodology/Principal Findings

PBMC were isolated from healthy controls (HC) or RA patients and stimulated ex vivo with PMA and ionomycin for 3 hours in the presence of Golgistop. Paired synovial fluid (SF) or synovial tissue (ST) were analysed where available. Intracellular expression of IL-17, IFNγ, and TNFα by CD4+ T cells was determined by flow cytometry. Synovial blood flow was evaluated by PDUS signal at the knees, wrists and metacarpophalangeal joints of RA patients. Serum, SF and fibroblast culture supernatant levels of vascular endothelial growth factor-A (VEGF-A) were measured by ELISA. The frequency of IL17+IFNγ-CD4+ T cells (Th17 cells) was significantly elevated in peripheral blood (PB) from RA patients vs. HC (median (IQR) 0.5 (0.28–1.59)% vs. 0.32 (0.21–0.54)%, p = 0.005). Th17 cells were further enriched (mean 6.6-fold increase) in RA SF relative to RA PB. Patients with active disease had a higher percentage of IL-17+ T cells in ST than patients in remission, suggesting a possible role for Th17 cells in active synovitis in RA. Indeed, the percentage of Th17 cells, but not Th1, in SF positively correlated with CRP (r = 0.51, p = 0.04) and local PDUS-defined synovitis (r = 0.61, p = 0.002). Furthermore, patients with high levels of IL-17+CD4+ T cells in SF had increased levels of the angiogenic factor VEGF-A in SF. Finally, IL-17, but not IFNγ, increased VEGF-A production by RA synovial fibroblasts in vitro.

Conclusions/Significance

Our data demonstrate a link between the presence of pro-inflammatory Th17 cells in SF and local PDUS scores, and offer a novel immunological explanation for the observation that rapid joint damage progression occurs in patients with persistent positive PDUS signal.     

Gene expression and activity of cartilage degrading glycosidases in human rheumatoid arthritis and osteoarthritis synovial fibroblasts

Introduction

Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints.

Methods

Expressions of β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. β-D-Glucuronidase, β-D-glucosaminidase and β-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity.

Results

According to our data, β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the β-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-β1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFα, IL-1β and IL-17.

Conclusions

According to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.     

Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

This study examines the role of L-selectin in monocyte adhesion to arterial endothelium, a key pathogenic event of atherosclerosis. Using a nonstatic (rotation) adhesion assay, we observed that monocyte binding to bovine aortic endothelium at 4°C increased four to nine times upon endothelium activation with tumor necrosis factor (TNF)-α. mAb-blocking experiments demonstrated that L-selectin mediates a major part (64 ± 18%) of monocyte attachment. Videomicroscopy experiments performed under flow indicated that monocytes abruptly halted on 8-h TNF-α–activated aortic endothelium, ~80% of monocyte attachment being mediated by L-selectin. Flow cytometric studies with a L-selectin/IgM heavy chain chimeric protein showed calcium-dependent L-selectin binding to cytokine-activated and, unexpectedly, unactivated aortic cells. Soluble L-selectin binding was completely inhibited by anti–L-selectin mAb or by aortic cell exposure to trypsin. Experiments with cycloheximide, chlorate, or neuraminidase showed that protein synthesis and sulfate groups, but not sialic acid residues, were essential for L-selectin counterreceptor function. Moreover, heparin lyases partially inhibited soluble L-selectin binding to cytokine-activated aortic cells, whereas a stronger inhibition was seen with unstimulated endothelial cells, suggesting that cytokine activation could induce the expression of additional ligand(s) for L-selectin, distinct from heparan sulfate proteoglycans. Under flow, endothelial cell treatment with heparinase inhibited by ~80% monocyte attachment to TNF-α–activated aortic endothelium, indicating a major role for heparan sulfate proteoglycans in monocyte–endothelial interactions. Thus, L-selectin mediates monocyte attachment to activated aortic endothelium, and heparan sulfate proteoglycans serve as arterial ligands for monocyte L-selectin.     

Irreversible Heavy Chain Transfer to Hyaluronan Oligosaccharides by Tumor Necrosis Factor-stimulated Gene-6

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).     

Prolonged, granulocyte-macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

Introduction

A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (T_H17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect T_H17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17.

Methods

IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFα or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3''-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry.

Results

T_H17-expressing CD4⁺ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFα (RASF_IL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte–macrophage colony-stimulating factor from RASF_IL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-κB pathways showed a requirement for both signalling pathways in RASF_IL-17/TNF-mediated neutrophil rescue.

Conclusion

The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFα activation of synovial fibroblasts. T_H17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.     

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 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).

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-)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).

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.     

Functional role of IL-22 in psoriatic arthritis

Introduction

Interleukin-22 (IL-22) is a cytokine of IL-10 family with significant proliferative effect on different cell lines. Immunopathological role of IL-22 has been studied in rheumatoid arthritis (RA) and psoriasis. Here we are reporting the functional role of IL-22 in the inflammatory and proliferative cascades of psoriatic arthritis (PsA).

Method

From peripheral blood and synovial fluid (SF) of PsA (n = 15), RA (n = 15) and osteoarthritis (OA, n = 15) patients, mononuclear cells were obtained and magnetically sorted for CD3⁺ T cells. Fibroblast like synoviocytes (FLS) were isolated from the synovial tissue of PsA (n = 5), RA (n = 5) and OA (n = 5) patients. IL-22 levels in SF and serum were measured by enzyme linked immunosorbent assay (ELISA). Proliferative effect of human recombinant IL-22 (rIL-22) on FLS was assessed by MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a yellow tetrazole) and CFSE dilution (Carboxyfluorescein succinimidyl ester) assays. Expression of IL-22Rα1 in FLS was determined by western blot.

Results

IL-22 levels were significantly elevated in SF of PsA patients (17.75 ± 3.46 pg/ml) compared to SF of OA (5.03 ± 0.39 pg/ml), p < 0.001. In MTT and CFSE dilution assays, rIL-22 (MTT, OD: 1.27 ± 0.06) induced significant proliferation of FLS derived from PsA patients compared to media (OD: 0.53 ± 0.02), p < 0.001. In addition, rIL-22 induced significantly more proliferation of FLS in presence of TNF-α. IL-22Rα1 was expressed in FLS of PsA, RA and OA patients. Anti IL-22R antibody significantly inhibited the proliferative effect of rIL-22. Further we demonstrated that activated synovial T cells of PsA and RA patients produced significantly more IL-22 than those of OA patients.

Conclusion

SF of PsA patients have higher concentration of IL-22 and rIL-22 induced marked proliferation of PsA derived FLS. Moreover combination of rIL-22 and TNF-α showed significantly more proliferative effect on FLS. IL-22Rα1 was expressed in FLS. Successful inhibition of IL-22 induced FLS proliferation by anti IL-22R antibody suggests that blocking of IL-22/IL-22R interaction may be considered as a novel therapeutic target for PsA.     

Natural killer cells and natural killer T cells in Lyme arthritis

Introduction

Natural killer (NK) and natural killer T (NKT) cells provide a first line of defense against infection. However, these cells have not yet been examined in patients with Lyme arthritis, a late disease manifestation. Lyme arthritis usually resolves with antibiotic treatment. However, some patients have persistent arthritis after spirochetal killing, which may result from excessive inflammation, immune dysregulation and infection-induced autoimmunity.

Methods

We determined the frequencies and phenotypes of NK cells and invariant NKT (iNKT) cells in paired peripheral blood (PB) and synovial fluid (SF) samples from eight patients with antibiotic-responsive arthritis and fifteen patients with antibiotic-refractory arthritis using flow cytometry and cytokine analyses.

Results

In antibiotic-responsive patients, who were seen during active infection, high frequencies of CD56bright NK cells were found in SF, the inflammatory site, compared with PB (P <0.001); at both sites, a high percentage of cells expressed the activation receptor NKG2D and the chaperone CD94, a low percentage expressed inhibitory killer immunoglobulin-like receptors (KIR), and a high percentage produced IFN-γ. In antibiotic-refractory patients, who were usually evaluated near the conclusion of antibiotics when few if any live spirochetes remained, the phenotype of CD56bright cells in SF was similar to that in patients with antibiotic-responsive arthritis, but the frequency of these cells was significantly less (P = 0.05), and the frequencies of CD56dim NK cells tended to be higher. However, unlike typical NKdim cells, these cells produced large amounts of IFN-γ, suggesting that they were not serving a cytotoxic function. Lastly, iNKT cell frequencies in the SF of antibiotic-responsive patients were significantly greater compared with that of antibiotic-refractory patients where these cells were often absent (P = 0.003).

Conclusions

In patients with antibiotic-responsive arthritis, the high percentage of activated, IFN-γ-producing CD56bright NK cells in SF and the presence of iNKT cells suggest that these cells still have a role in spirochetal killing late in the illness. In patients with antibiotic-refractory arthritis, the frequencies of IFN-γ-producing CD56bright and CD56dim NK cells remained high in SF, even after spirochetal killing, suggesting that these cells contribute to excessive inflammation and immune dysregulation in joints, and iNKT cells, which may have immunomodulatory effects, were often absent.     

The O-glycomap of Lubricin,a Novel Mucin Responsible for Joint Lubrication,Identified by Site-specific Glycopeptide Analysis

The lubricative, heavily glycosylated mucin-like synovial glycoprotein lubricin has previously been observed to contain glycosylation changes related to rheumatoid and osteoarthritis. Thus, a site-specific investigation of the glycosylation of lubricin was undertaken, in order to further understand the pathological mechanisms involved in these diseases. Lubricin contains an serine/threonine/proline (STP)-rich domain composed of imperfect tandem repeats (EPAPTTPK), the target for O-glycosylation. In this study, using a liquid chromatography–tandem mass spectrometry approach, employing both collision-induced and electron-transfer dissociation fragmentation methods, we identified 185 O-glycopeptides within the STP-rich domain of human synovial lubricin. This showed that adjacent threonine residues within the central STP-rich region could be simultaneously and/or individually glycosylated. In addition to core 1 structures responsible for biolubrication, core 2 O-glycopeptides were also identified, indicating that lubricin glycosylation may have other roles. Investigation of the expression of polypeptide N-acetylgalactosaminyltransferase genes was carried out using cultured primary fibroblast-like synoviocytes, a cell type that expresses lubricin in vivo. This analysis showed high mRNA expression levels of the less understood polypeptide N-acetylgalactosaminyltransferase 15 and 5 in addition to the ubiquitously expressed polypeptide N-acetylgalactosaminyltransferase 1 and 2 genes. This suggests that there is a unique combination of transferase genes important for the O-glycosylation of lubricin. The site-specific glycopeptide analysis covered 82% of the protein sequence and showed that lubricin glycosylation displays both micro- and macroheterogeneity. The density of glycosylation was shown to be high: 168 sites of O-glycosylation, predominately sialylated, were identified. These glycosylation sites were focused in the central STP-rich region, giving the domain a negative charge. The more positively charged lysine and arginine residues in the N and C termini suggest that synovial lubricin exists as an amphoteric molecule. The identification of these unique properties of lubricin may provide insight into the important low-friction lubricating functions of lubricin during natural joint movement.Human diarthrodial joints are surrounded by synovial fluid (SF),¹ a dense extracellular matrix fluid composed of proteins, glycoproteins, hyaluronic acid, proteoglycans, and phospholipids (1). During movement, the cartilage surfaces of the articulating joints slide over each other with an extremely low coefficient of friction that ranges from 0.0005 to 0.04 (2) and handle pressures up to ∼200 atm (3). In a healthy state, the joint surface and SF constitute a system of reduced friction that results in lifelong lubrication and wear resistance, primarily due to biolubricating molecules such as hyaluronic acid and lubricin (4). Human synovial lubricin is encoded by the proteoglycan 4 (Prg4) gene (5, 6) and is synthesized by fibroblast-like synoviocytes (FLSs) and superficial zone chondrocytes. Its 1404-amino-acid sequence contains a central mucin-like domain consisting of 59 imperfectly repeated sequences of EPAPTTPK. The O-glycosylation (in particular core 1 and sialylated core 1) of lubricin is suggested to be responsible for its lubricating properties (7), as the removal of these residues results in the loss of boundary lubrication. The molecule has also been suggested to play a key role in protecting the cartilage surface from excessive adsorption of proteins and cells (8).Arthritis results in the loss of this joint surface, leading to severe pain and a restricted range of motion. The two most common arthritic diseases, osteoarthritis (OA) and rheumatoid arthritis (RA), have different mechanisms of degradation. RA is an autoimmune systemic high inflammatory disease that increases the friction between articulating cartilage surfaces, resulting in degradation of the joint (9), whereas OA is a result of mechanical stress (10). Degeneration of the cartilage can be detected from proteoglycan fragments in the SF (11, 12). Because of the limited efficacy of available treatments, particularly for OA, understanding the biological factors related to arthritis is essential.The joints of arthritis patients, both RA and OA, have shown a down-regulation of expression and changes in glycosylation of lubricin (13). Studies using OA animal models suggest that there is a relationship between pathogenesis and the down-regulation of lubricin (9, 14, 15). This decrease in lubricin expression exacerbates the disease by accelerating the joint destruction, suggesting that certain characteristics of lubricin may be indicators of disease progression in RA and OA. Given the critical nature of lubricin glycosylation, we initiated a site-specific glycopeptide characterization of the lubricin mucin-like domain using liquid chromatography–tandem mass spectrometry with both collision-induced and electron-transfer dissociation fragmentation methods (LC-CID/ETD-MS²) after tryptic digestion of both intact and partly de-glycosylated lubricin.Collision-induced dissociation–tandem mass spectrometry (CID-MSⁿ) of O-linked (and N-linked) glycopeptides is capable of generating sequence information both for the attached glycan (in MS²) and for the de-glycosylated peptide (in MS³), but it lacks the site-specific information of the modified amino acids (16). This is due to extensive glycosidic bond cleavage of the precursor ion in MS² producing B/C and Y/Z ions (Domon and Costello carbohydrate fragmentation nomenclature (17)). In addition, the identification of the modified amino acids is even more difficult for peptides containing several Ser/Thr residues because of the lack of a consensus sequence for mucin-type O-glycosylation. Electron-capture dissociation and ETD are fragmentation techniques used for the site-specific characterization of protein post-translational modifications including phosphorylation (18) and glycosylation (19). Both techniques induce cleavage of the N-Cα bonds of the peptide backbone, producing c- and z-type fragment ions, while leaving the post-translational modification unaffected.In order to understand the biosynthesis of O-linked glycoproteins, one needs to link site localization of glycosylation to the expression of enzymes responsible for GalNAc-type (or mucin-type) O-glycosylation. This is necessary because the prediction of the site of GalNAc-type O-glycosylation is difficult. One reason for this is the large, redundant UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase (ppGalNAc T) gene family containing 20 gene-encoded isoenzymes, all possessing unique and/or overlapping substrate specificities (20, 21). These ppGalNAc Ts transfer GalNAc from the sugar nucleotide donor UDP-GalNAc to the hydroxyl groups of Ser and Thr residues in the proteins traversing the Golgi/endoplasmic reticulum. Altered protein O-glycosylation, suggested to be due to changes in the expression of distinct ppGalNAc Ts, has been reported in various disease states, including ulcerative colitis and cancer (21, 22). Thus, the connection of site-specific O-glycosylation with the responsible ppGalNAc Ts is important for understanding the functions of lubricin, as site-specific O-glycosylation has been shown to regulate the functions of proteins (23, 24) and may be involved in the pathological transformation of the joint in arthritis diseases.Although the type of glycosylation present on lubricin has been investigated previously, the site-specific glycopeptide characterization, including the analysis of the glycan types at these locations, was investigated for the first time in this study. In order to understand the nature of glycoproteins, it is essential to not only define the protein component or the glycan characteristics, but also understand how these two essential components interact. The macro- (different site occupation) and micro-heterogeneity (different glycan structure at each site) provided a heterogeneous mixture of lubricin O-linked glycopeptides that might help to explain the extraordinary properties of lubricin and how it can function as a lubricating agent in a demanding environment.