Human Synovial Lubricin Expresses Sialyl Lewis x Determinant and Has L-selectin Ligand Activity

Lubricin (or proteoglycan 4 (PRG4)) is an abundant mucin-like glycoprotein in synovial fluid (SF) and a major component responsible for joint lubrication. In this study, it was shown that O-linked core 2 oligosaccharides (Galβ1–3(GlcNAcβ1–6)GalNAcα1-Thr/Ser) on lubricin isolated from rheumatoid arthritis SF contained both sulfate and fucose residues, and SF lubricin was capable of binding to recombinant L-selectin in a glycosylation-dependent manner. Using resting human polymorphonuclear granulocytes (PMN) from peripheral blood, confocal microscopy showed that lubricin coated circulating PMN and that it partly co-localized with L-selectin expressed by these cells. In agreement with this, activation-induced shedding of L-selectin also mediated decreased lubricin binding to PMN. It was also found that PMN recruited to inflamed synovial area and fluid in rheumatoid arthritis patients kept a coat of lubricin. These observations suggest that lubricin is able to bind to PMN via an L-selectin-dependent and -independent manner and may play a role in PMN-mediated inflammation.     

Consequences of disease-causing mutations on lubricin protein synthesis, secretion, and post-translational processing

Lubricin, a protein product of the gene PRG4, is a secreted mucin-like proteoglycan that is a major lubricant in articulating joints. Mutations in PRG4 cause the autosomal recessive, human disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome. We developed rabbit polyclonal antibodies against human lubricin to determine the consequence of disease-causing mutations at the protein level and to study the protein's normal post-translational processing. Antiserum generated against an epitope in the amino-terminal portion of lubricin detected protein in wild-type synovial fluid and in conditioned media from wild-type cultured synoviocytes. However, the antiserum did not detect lubricin in synovial fluid or cultured synoviocytes from several patients with frameshift or nonsense mutations in PRG4. Antiserum generated against an epitope in the protein's carboxyl-terminal, hemopexin-like domain identified a post-translational cleavage event in wild-type lubricin, mediated by a subtilisin-like proprotein convertase (SPC). Interestingly, in contrast to wild-type lubricin, one disease-causing mutation that removes the last 8 amino acids of the protein, including a conserved cysteine residue, was not cleaved within the hemopexin-like domain when expressed in COS-7 cells. This suggests that formation of an intrachain disulfide bond is required for SPC-mediated cleavage and that SPC-mediated cleavage is essential to protein function.     

The biology of Lubricin: Near frictionless joint motion

Lubricin is a surface-active mucinous glycoprotein secreted in the synovial joint that plays an important role in cartilage integrity. In healthy joints, lubricin molecules coat the cartilage surface, providing boundary lubrication and preventing cell and protein adhesion. Arthropathy occurring in patients with joint trauma, inflammatory arthritis or genetically mediated lubricin deficiencies have insufficient lubricin to prevent damage to articular cartilage. Recent studies in lubricin null joints indicate that lubricin (Prg4) plays a role in preventing damage to the superficial zone and preservation of chondrocytes. Progress in the production of recombinant forms of lubricin and the successes of lubricin supplementation in small animal models identify rhPRG4 as a potential therapeutic for patients with transient lubricin deficiency in the setting of trauma or autoimmune arthritis.     

Lubricin Protects the Temporomandibular Joint Surfaces from Degeneration

The temporomandibular joint (TMJ) is a specialized synovial joint essential for the mobility and function of the mammalian jaw. The TMJ is composed of the mandibular condyle, the glenoid fossa of the temporal bone, and a fibrocartilagenous disc interposed between these bones. A fibrous capsule, lined on the luminal surface by the synovial membrane, links these bones and retains synovial fluid within the cavity. The major component of synovial fluid is lubricin, a glycoprotein encoded by the gene proteoglycan 4 (Prg4), which is synthesized by chondrocytes at the surface of the articular cartilage and by synovial lining cells. We previously showed that in the knee joint, Prg4 is crucial for maintenance of cartilage surfaces and for regulating proliferation of the intimal cells in the synovium. Consequently, the objective of this study was to determine the role of lubricin in the maintenance of the TMJ. We found that mice lacking lubricin have a normal TMJ at birth, but develop degeneration resembling TMJ osteoarthritis by 2 months, increasing in severity over time. Disease progression in Prg4 ^−/− mice results in synovial hyperplasia, deterioration of cartilage in the condyle, disc and fossa with an increase in chondrocyte number and their redistribution in clusters with loss of superficial zone chondrocytes. All articular surfaces of the joint had a prominent layer of protein deposition. Compared to the knee joint, the osteoarthritis-like phenotype was more severe and manifested earlier in the TMJ. Taken together, the lack of lubricin in the TMJ causes osteoarthritis-like degeneration that affects the articular cartilage as well as the integrity of multiple joint tissues. Our results provide the first molecular evidence of the role of lubricin in the TMJ and suggest that Prg4 ^−/− mice might provide a valuable new animal model for the study of the early events of TMJ osteoarthritis.     

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

Objective

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.

Methods

For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.

Results

In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage.

Conclusion

Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.     

The molecular structure and lubricating activity of lubricin isolated from bovine and human synovial fluids.

Lubricin was isolated from bovine ankle, metacarpophalangeal and knee and human knee synovial fluids. The lubricins isolated from the bovine joint fluids had the same amino acid and carbohydrate compositions, but differences were observed in the relative molecular masses. The Mr values of bovine metacarpophalangeal and ankle lubricin determined by light-scattering measurements were about 200 000, whereas values of 132 000 and 143 000 were obtained for the bovine knee lubricin. The human knee lubricin had a similar carbohydrate composition to bovine knee lubricin except for the higher glucosamine content, and the amino acid composition differed slightly. The human sample had a lower glutamic acid content and a leucine/isoleucine ratio of 2:1 compared with 1:1 in the bovine. The Mr value of the human knee lubricin (166 000) was also lower than that of the bovine metacarpophalangeal and ankle samples. The Mr value of the bovine knee lubricin determined by sedimentation-equilibrium measurements was 171 000. The length measurements determined by electron microscopy and also the sedimentation measurements showed considerable polydispersity and indicate that the degree of extension of lubricin molecules can vary. Friction measurements showed that the human knee synovial-fluid lubricin had equivalent lubricating ability in a test system in vitro to that observed for lubricin isolated from normal bovine synovial fluids. The lubricating ability of lubricin was concentration-dependent, and each lubricin sample was able to act as a lubricant in vitro in an equivalent manner to whole synovial fluid at concentrations that are thought to occur in vivo.     

Interaction of lubricin with type II collagen surfaces: Adsorption,friction, and normal forces

One of the major constituents of the synovial fluid that is thought to be responsible for chondroprotection and boundary lubrication is the glycoprotein lubricin (PRG4); however, the molecular mechanisms by which lubricin carries out its critical functions still remain largely unknown. We hypothesized that the interaction of lubricin with type II collagen, the main component of the cartilage extracellular matrix, results in enhanced tribological and wear properties. In this study, we examined: (i) the molecular details by which lubricin interacts with type II collagen and how binding is related to boundary lubrication and adhesive interactions; and (ii) whether collagen structure can affect lubricin adsorption and its chondroprotective properties. We found that lubricin adsorbs strongly onto denatured, amorphous, and fibrillar collagen surfaces. Furthermore, we found large repulsive interactions between the collagen surfaces in presence of lubricin, which increased with increasing lubricin concentration. Lubricin attenuated the large friction and also the long-range adhesion between fibrillar collagen surfaces. Interestingly, lubricin adsorbed onto and mediated the frictional response between the denatured and native amorphous collagen surfaces equally and showed no preference on the supramolecular architecture of collagen. However, the coefficient of friction was lowest on fibrillar collagen in the presence of lubricin. We speculate that an important role of lubricin in mediating interactions at the cartilage surface is to attach to the cartilage surface and provide a protective coating that maintains the contacting surfaces in a sterically repulsive state.     

Mass spectrometry assays of plasma biomarkers to predict radiographic progression of knee osteoarthritis

Introduction

Biomarkers to identify osteoarthritis (OA) patients at risk for disease progression are needed. As part of a proteomic analysis of knee synovial fluid from normal and OA patients, differentially expressed proteins were identified that could represent potential biomarkers for OA. This study aimed to use mass spectrometry assays to identify representative peptides from several proteins in synovial fluid and peripheral blood, and assess their levels as biomarkers of OA progression.

Methods

Multiplexed high throughput selected reaction monitoring (SRM) assays were developed to measure tryptic peptides representative of 23 proteins in matched serum and synovial fluid samples from late OA subjects at the time of joint replacement. Subsequently plasma samples from the baseline visit of 173 subjects in an observational OA cohort were tested by SRM for peptides from nine of these proteins: afamin, clusterin, cartilage oligomeric matrix protein, hepatocyte growth factor, kallistatin, insulin-like growth factor binding protein, acid labile subunit, lubricin, lumican, and pigment epithelium-derived factor. Linear regression was used to determine the association between the peptide biomarker level at baseline and change in joint space width (ΔJSW) from baseline to 30 months, adjusting for age and sex.

Results

In the matched cohort, 17 proteins could be identified in synovial fluid and 16 proteins were detected in serum. For the progression cohort, the average age was 62 and average ΔJSW over 30 months was 0.68 mm. A high correlation between different peptides from individual proteins was observed, indicating our assays correctly measured their target proteins. Peptides representative of clusterin, lumican and lubricin showed statistically significant associations with joint space narrowing after adjustment for age and sex. Partial R² values showed clusterin FMETVAEK and lubricin LVEVNPK peptide biomarkers explains about 2 to 3% of the variability of ΔJSW, similar to that explained by age. A biomarker score combining normalized data for both lubricin and clusterin peptides increased the model R² to 0.079.

Conclusions

Our results suggest that when combined, levels of peptides representative of clusterin and lubricin in plasma are as predictive of OA progression as age. Replication of these findings in other prospective OA cohorts is planned.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0456-6) contains supplementary material, which is available to authorized users.     

Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint

Introduction

A major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response. Specifically, this study aims to demonstrate that the surrogate marker CCP2 can capture ACPAs that bind to autoantigens expressed in vivo in the major inflammatory lesions of RA (that is, in the rheumatoid joint).

Methods

Plasma (n = 16) and synovial fluid (n = 26) samples were collected from RA patients with anti-CCP2 IgG levels of above 300 AU/mL. Total IgG was isolated on Protein G columns and subsequently applied to CCP2 affinity columns. Purified anti-CCP2 IgG was analyzed for reactivity and specificity by using the CCPlus® ELISA, in-house peptide ELISAs, Western blot, and immunohisto-/immunocytochemistry.

Results

Approximately 2% of the total IgG pool in both plasma and synovial fluid was CCP2-reactive. Purified anti-CCP2 reactive antibodies from different patients showed differences in binding to CCP2 and differences in binding to citrullinated peptides from α-enolase, vimentin, fibrinogen, and collagen type II, illustrating different ACPA fine-specificity profiles. Furthermore, the purified ACPA bound not only in vitro citrullinated proteins but, more importantly, in vivo-generated epitopes on synovial fluid cells and synovial tissues from patients with RA.

Conclusions

We have isolated ACPAs from plasma and synovial fluid and demonstrated that the CCP2 peptides, frequently used in diagnostic ELISAs, de facto act as surrogate antigens for at least four different, well-characterized, largely non-cross-reactive, ACPA fine specificities. Moreover, we have determined the concentration and proportion of CCP2-reactive IgG molecules in rheumatoid plasma and synovial fluid, and we have shown that the purified ACPAs can be used to detect both in vitro- and in vivo-generated citrullinated epitopes by various techniques. We anticipate that these antibodies will provide us with new opportunities to investigate the potential pathogenic effects of human ACPAs.     

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.     

Extra-virgin olive oil diet and mild physical activity prevent cartilage degeneration in an osteoarthritis model: an in vivo and in vitro study on lubricin expression

Mediterranean diet includes a relatively high fat consumption mostly from monounsaturated fatty acids mainly provided by olive oil, the principal source of culinary and dressing fat. The beneficial effects of olive oil have been widely studied and could be due to its phytochemicals, which have been shown to possess anti-inflammatory properties. Lubricin is a chondroprotective glycoprotein and it serves as a critical boundary lubricant between opposing cartilage surfaces. A joint injury causes an initial flare of cytokines, which decreases lubricin expression and predisposes to cartilage degeneration such as osteoarthritis. The aim of this study was to evaluate the role of extra-virgin olive oil diet and physical activity on inflammation and expression of lubricin in articular cartilage of rats after injury. In this study we used histomorphometric, histological, immunocytochemical, immunohistochemical, western blot and biochemical analysis for lubricin and interleukin-1 evaluations in the cartilage and in the synovial fluid. We report the beneficial effect of physical activity (treadmill training) and extra-virgin olive oil supplementation, on the articular cartilage. The effects of anterior cruciate ligament transection decrease drastically the expression of lubricin and increase the expression of interleukin-1 in rats, while after physical activity and extra-virgin olive oil supplemented diet, the values return to a normal level compared to the control group. With our results we can confirm the importance of the physical activity in conjunction with extra-virgin olive oil diet in medical therapy to prevent osteoarthritis disease in order to preserve the articular cartilage and then the entire joint.     

Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

Introduction

Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients.

Methods

An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined.

Results

Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the citrullination process to occur.

Conclusions

We present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients.     

Extracorporeal shockwave-induced expression of lubricin in tendons and septa

Lubricin, a lubricating glycoprotein that facilitates tendon gliding, is upregulated by mechanical as well as biochemical stimuli, prompting this study of its induction by extracorporeal shockwave therapy (ESWT). The objective of this study was to characterize and quantify the effect of ESWT on lubricin expression in tendons and septa in a rat model. Hindlimbs of six rats were treated with low-dose ESWT and those of another six with high-dose ESWT, using contralateral limbs as controls. After 4 days, resected samples were processed for immunolocalization of lubricin using a purified monoclonal antibody. ESWT was found to increase lubricin expression in both low-dose and high-dose ESWT-treated tendons and also in septa. Lubricin expression generally increased with increasing dose of ESWT. Increased lubricin expression may contribute to the beneficial effects of ESWT in providing pain and symptom relief in musculoskeletal disorders by decreasing erosive wear.     

Apolipoprotein-A1 as a Damage-Associated Molecular Patterns Protein in Osteoarthritis: Ex Vivo and In Vitro Pro-Inflammatory Properties

Osteoarthritis (OA) is associated with a local inflammatory process. Dyslipidemia is known to be an underlying cause for the development of OA. Therefore, lipid and inflammatory levels were quantified ex vivo in blood and synovial fluid of OA patients (n=29) and compared to those of rheumatoid arthritis (RA) patients (n=27) or healthy volunteers (HV) (n=35). The role of apolipoprotein A-I (ApoA1) was investigated in vitro on inflammatory parameters using human joint cells isolated from cartilage and synovial membrane obtained from OA patients after joint replacement. Cells were stimulated with ApoA1 in the presence or not of serum amyloid A (SAA) protein and/or lipoproteins (LDL and HDL) at physiological concentration observed in OA synovial fluid. In our ex vivo study, ApoA1, LDL-C and total cholesterol levels were strongly correlated to each other inside the OA joint cavity whereas same levels were not or weakly correlated to their corresponding serum levels. In OA synovial fluid, ApoA1 was not as strongly correlated to HDL as observed in OA serum or in RA synovial fluid, suggesting a dissociative level between ApoA1 and HDL in OA synovial fluid. In vitro, ApoA1 induced IL-6, MMP-1 and MMP-3 expression by primary chondrocytes and fibroblast-like synoviocytes through TLR4 receptor. HDL and LDL attenuated joint inflammatory response induced by ApoA1 and SAA in a ratio dependent manner. In conclusion, a dysregulated lipidic profile in the synovial fluid of OA patients was observed and was correlated with inflammatory parameters in the OA joint cavity. Pro-inflammatory properties of ApoA1 were confirmed in vitro.     

Acute-Phase Serum Amyloid A in Osteoarthritis: Regulatory Mechanism and Proinflammatory Properties

Objective

To determine if serum amyloid A (A-SAA) could be detected in human osteoarthritic (OA) joints and further clarify if high A-SAA level in joints result from a local production or from a diffusion process from abnormally elevated plasma concentration. Regulatory mechanism of A-SAA expression and its pro-inflammatory properties were also investigated.

Methods

A-SAA levels in serum and synovial fluid of OA (n = 29) and rheumatoid arthritis (RA) (n = 27) patients were measured and compared to matched-healthy volunteers (HV) (n = 35). In vitro cell cultures were performed on primary joint cells provided from osteoarthritis patients. Regulatory mechanisms were studied using Western-blotting, ELISA and lentiviral transfections.

Results

A-SAA was statistically increased in OA plasma patients compared to HV. Moreover, A-SAA level in OA plasma and synovial fluid increased with the Kellgren & Lauwrence grade. For all OA and RA patients, A-SAA plasma level was higher and highly correlated with its corresponding level in the synovial fluid, therefore supporting that A-SAA was mainly due to the passive diffusion process from blood into the joint cavity. However, A-SAA expression was also observed in vitro under corticosteroid treatment and/or under IL-1beta stimuli. A-SAA expression was down-regulated by PPAR-γ agonists (genistein and rosiglitazone) and up-regulated by TGF-β1 through Alk1 (Smad1/5) pathway. RhSAA induced proinflammatory cytokines (IL-6, IL-8, GRO-α and MCP-1) and metalloproteinases (MMP-1, MMP-3 and MMP-13) expression in FLS and chondrocytes, which expression was downregulated by TAK242, a specific TLR4 inhibitor.

Conclusion

Systemic or local A-SAA expression inside OA joint cavity may play a key role in inflammatory process seen in osteoarthritis, which could be counteracted by TLR4 inhibition.     

Functional Phenotype of Synovial Monocytes Modulating Inflammatory T-Cell Responses in Rheumatoid Arthritis (RA)

Monocytes function as crucial innate effectors in the pathogenesis of chronic inflammatory diseases, including autoimmunity, as well as in the inflammatory response against infectious pathogens. Human monocytes are heterogeneous and can be classified into three distinct subsets based on CD14 and CD16 expression. Although accumulating evidence suggests distinct functions of monocyte subsets in inflammatory conditions, their pathogenic roles in autoimmune diseases remain unclear. Thus, we investigated the phenotypic and functional characteristics of monocytes derived from synovial fluid and peripheral blood in RA patients in order to explore the pathogenic roles of these cells. In RA patients, CD14⁺CD16⁺, but not CD14^dimCD16⁺, monocytes are predominantly expanded in synovial fluid and, to a lesser degree, in peripheral blood. Expression of co-signaling molecules of the B7 family, specifically CD80 and CD276, was markedly elevated on synovial monocytes, while peripheral monocytes of RA and healthy controls did not express these molecules without stimulation. To explore how synovial monocytes might gain these unique properties in the inflammatory milieu of the synovial fluid, peripheral monocytes were exposed to various stimuli. CD16 expression on CD14⁺ monocytes was clearly induced by TGF-β, although co-treatment with IL-1β, TNF-α, or IL-6 did not result in any additive effects. In contrast, TLR stimulation with LPS or zymosan significantly downregulated CD16 expression such that the CD14⁺CD16⁺ monocyte subset could not be identified. Furthermore, treatment of monocytes with IFN-γ resulted in the induction of CD80 and HLA-DR expression even in the presence of TGF-β. An in vitro assay clearly showed that synovial monocytes possess the unique capability to promote Th1 as well as Th17 responses of autologous peripheral CD4 memory T cells. Our findings suggest that the cytokine milieu of the synovial fluid shapes the unique features of synovial monocytes as well as their cardinal role in shaping inflammatory T-cell responses in RA.     

Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-DRB1 shared epitope alleles

Introduction

Fibronectin is one of the most abundant proteins present in the inflamed joint. Here, we characterized the citrullination of fibronectin in the joints of rheumatoid arthritis (RA) patients and studied the prevalence, epitope specificity and human leukocyte antigen (HLA) association of autoantibodies against citrullinated fibronectin in RA.

Methods

Citrullinated residues in fibronectin isolated from RA patient synovial fluid were identified by mass spectrometry. The corresponding citrullinated and non-citrullinated peptides were synthesized and used to analyze the presence of autoantibodies to these peptides in RA sera and sera from other diseases and healthy controls by ELISA. The data were compared with risk factors like shared epitope HLA alleles and smoking, and with clinical features.

Results

Five citrullinated residues were identified in fibronectin from RA synovial fluid. RA sera reacted in a citrulline-dependent manner with two out of four citrullinated fibronectin peptides, one of which contains two adjacent citrulline residues, in contrast to non-RA sera, which were not reactive. The most frequently recognized peptide (FN-Cit_1035,1036, LTVGLTXXGQPRQY, in which × represents citrulline) was primarily targeted by anti-CCP (cyclic citrullinated peptide) 2-positive RA patients. Anti-FN-Cit_1035,1036 autoantibodies were detected in 50% of established anti-CCP2-positive RA patients and in 45% of such patients from a early arthritis clinic. These antibodies appeared to be predominantly of the immunoglobulin G (IgG) isotype and to be associated with HLA shared epitope alleles (odds ratio = 2.11).

Conclusions

Fibronectin in the inflamed synovia of RA patients can be citrullinated at least at five positions. Together with the flanking amino acids, three of these citrullinated residues comprise two epitopes recognized by RA autoantibodies. Anti-citrullinated fibronectin peptide antibodies are associated with HLA shared epitope alleles.     

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.     

Elastoviscous Transitions of Articular Cartilage Reveal a Mechanism of Synergy between Lubricin and Hyaluronic Acid

When lubricated by synovial fluid, articular cartilage provides some of the lowest friction coefficients found in nature. While it is known that macromolecular constituents of synovial fluid provide it with its lubricating ability, it is not fully understood how two of the main molecules, lubricin and hyaluronic acid, lubricate and interact with one another. Here, we develop a novel framework for cartilage lubrication based on the elastoviscous transition to show that lubricin and hyaluronic acid lubricate by distinct mechanisms. Such analysis revealed nonspecific interactions between these molecules in which lubricin acts to concentrate hyaluronic acid near the tissue surface and promotes a transition to a low friction regime consistent with the theory of viscous boundary lubrication. Understanding the mechanics of synovial fluid not only provides insight into the progression of diseases such as arthritis, but also may be applicable to the development of new biomimetic lubricants.