heparitin-sulfate and Arthritis--Rheumatoid

Topics: Angiogenesis Inhibitors; Arthritis, Rheumatoid; Endothelial Growth Factors; Extracellular Matrix; Fibroblast Growth Factor 2; Glucuronidase; Glycosaminoglycans; Growth Substances; Heparitin Sulfate; Humans; Joints; Lymphokines; Neovascularization, Pathologic; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Syndecan-4 (sdc4) is a cell-anchored proteoglycan that consists of a transmembrane core protein and glucosaminoglycan (GAG) side chains. Binding of soluble factors to the GAG chains of sdc4 may result in the dimerisation of sdc4 and the initiation of downstream signalling cascades. However, the question of how sdc4 dimerisation and signalling affects the response of cells to inflammatory stimuli is unknown.. Sdc4 immunostaining was performed on rheumatoid arthritis (RA) tissue sections. Interleukin (IL)-1 induced extracellular signal-regulated kinases (ERK) phosphorylation and matrix metalloproteinase-3 production was investigated. Il-1 binding to sdc4 was investigated using immunoprecipitation. IL-1 receptor (IL1R1) staining on wild-type, sdc4 and IL1R1 knockout fibroblasts was performed in fluorescence-activated cell sorting analyses. A blocking sdc4 antibody was used to investigate sdc4 dimerisation, IL1R1 expression and the histological paw destruction in the human tumour necrosis factor-alpha transgenic mouse.. We show that in fibroblasts, the loss of sdc4 or the antibody-mediated inhibition of sdc4 dimerisation reduces the cell surface expression of the IL-1R and regulates the sensitivity of fibroblasts to IL-1. We demonstrate that IL-1 directly binds to sdc4 and in an IL-1R-independent manner leads to its dimerisation. IL-1-induced dimerisation of sdc4 regulates caveolin vesicle-mediated trafficking of the IL1R1, which in turn determines the responsiveness to IL-1. Administration of antibodies (Ab) against the dimerisation domain of sdc4, thus, strongly reduces the expression IL1R1 on arthritic fibroblasts both in vitro and an animal model of human RA.. Collectively, our data suggest that Ab that specifically inhibit sdc4 dimerisation may support anti-IL-1 strategies in diseases such as inflammatory arthritis.

Topics: Animals; Antibodies, Blocking; Arthritis, Rheumatoid; Dimerization; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Knockout Techniques; Heparitin Sulfate; Hindlimb; Humans; Interleukin-1; Interleukin-1beta; MAP Kinase Signaling System; Matrix Metalloproteinase 3; Mice; Mice, Transgenic; NIH 3T3 Cells; Osteoarthritis; Phosphorylation; Protein Transport; Receptors, Interleukin-1 Type I; Signal Transduction; Syndecan-4; Synovial Membrane; Tumor Necrosis Factor-alpha

Heparan sulfate (HS) is a complex and highly variable polysaccharide, expressed ubiquitously on the cell surface as HS proteoglycans (HSPGs), and found in the extracellular matrix as free HS fragments. Its heterogeneity due to various acetylation and sulfation patterns endows a multitude of functions. In animal tissues, HS interacts with a wide range of proteins to mediate numerous biological activities; given its multiple roles in inflammation processes, characterization of HS in human serum has significant potential for elucidating disease mechanisms. Historically, investigation of HS was limited by its low concentration in human serum, together with the complexity of the serum matrix. In this study, we used a modified mass spectrometry method to examine HS disaccharide profiles in the serum of 50 women with rheumatoid arthritis (RA), and compared our results to 51 sera from healthy women. Using various purification methods and online LC-MS/MS, we discovered statistically significant differences in the sulfation and acetylation patterns between populations. Since early diagnosis of RA is considered important in decelerating the disease's progression, identification of specific biomolecule characterizations may provide crucial information towards developing new therapies for suppressing the disease in its early stages. This is the first report of potential glycosaminoglycan biomarkers for RA found in human sera, while acknowledging the obvious fact that a larger population set, and more stringent collection parameters, will need to be investigated in the future.

Topics: Acetylation; Arthritis, Rheumatoid; Biomarkers; Chromatography, Liquid; Disaccharides; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Molecular Structure; ROC Curve; Serum; Tandem Mass Spectrometry

IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases.

Topics: Amino Acid Substitution; Animals; Anti-Inflammatory Agents; Arthritis, Rheumatoid; Binding Sites; Cattle; Drug Design; Drug Evaluation, Preclinical; Guanidine; Heparitin Sulfate; Humans; Interleukin-8; Male; Mice; Mice, Inbred C57BL; Mutagenesis, Site-Directed; Protein Binding; Protein Denaturation; Receptors, Interleukin-8A

Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, proinflammatory processes, and proliferation of fibroblast-like cells. Abnormalities in these processes are primary features of rheumatoid arthritis (RA) in synovial tissues. Tissue destruction in joints causes the accumulation of large quantities of free hyaluronic acid (HA) in RA synovial fluid. The present study was conducted to investigate the effects of HA and several other glycosaminoglycans on antithrombin, a plasma inhibitor of thrombin. Various glycosaminoglycans, including HA, chondroitin sulfate, keratan sulfate, heparin, and heparan, were incubated with human antithrombin III in vitro. The residual activity of antithrombin was determined using a thrombin-specific chromogenic assay. HA concentrations ranging from 250 to 1000 mug/ml significantly blocked the ability of antithrombin to inhibit thrombin in the presence of Ca2+ or Fe3+, and chondroitin A, B and C also reduced this ability under the same conditions but to a lesser extent. Our study suggests that the high concentration of free HA in RA synovium may block antithrombin locally, thereby deregulating thrombin activity to drive the pathogenic process of RA under physiological conditions. The study also helps to explain why RA occurs and develops in joint tissue, because the inflamed RA synovium is uniquely rich in free HA along with extracellular matrix degeneration. Our findings are consistent with those of others regarding increased coagulation activity in RA synovium.

Topics: Antithrombin III; Arthritis, Rheumatoid; Autoimmune Diseases; Calcium; Chondroitin Sulfates; Chromogenic Compounds; Dermatan Sulfate; Glycosaminoglycans; Heparin; Heparitin Sulfate; Humans; Hyaluronic Acid; Iron; Keratan Sulfate; Synovial Fluid; Thrombin

To identify and characterize which endothelial heparan sulfate proteoglycans (HSPGs) bind the chemokine CXCL8 (interleukin-8) in human rheumatoid arthritis (RA) and nonrheumatoid synovia.. CXCL8 binding to endothelial HSPGs in RA and nonrheumatoid synovia was determined by heparinase treatment followed by an in situ binding assay and autoradiography. Endothelial HSPGs were characterized by immunohistochemical analysis and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Phosphatidyinositol-specific phospholipase C (PI-PLC) and antibodies to HSPGs were used in in situ binding experiments to identify which HSPGs bound CXCL8.. The expression of heparan sulfate on microvascular endothelial cells was demonstrated in RA and nonrheumatoid synovia. Using antibodies to syndecan-1-4 and glypican-1, -3, and -4, the selective expression of syndecan-3 by endothelial cells was detected in RA and nonrheumatoid synovia. In addition, RT-PCR showed the presence of syndecan-3 messenger RNA in endothelial cells extracted from RA and nonrheumatoid synovia. (125)I-CXCL8 bound to venular endothelial cells; treatment with heparinases I and III significantly reduced this binding in RA but not nonrheumatoid synovia. (125)I-CXCL8 binding was not reduced after treatment with PI-PLC, which cleaves glycosyl phosphatidylinositol linkages, suggesting that CXCL8 did not bind to glypicans. Treatment of synovia with a syndecan-3 antibody reduced CXCL8 binding to RA but not nonrheumatoid endothelial cells; however, no reduction in binding was observed with syndecan-2 or glypican-4 antibodies.. Our results show the selective induction of a CXCL8 binding site on endothelial syndecan-3 in RA synovium. This site may be involved in leukocyte trafficking into RA synovial tissue.

Topics: Adult; Aged; Arthritis, Rheumatoid; Binding Sites; Case-Control Studies; Endothelial Cells; Female; Heparitin Sulfate; Humans; Immunohistochemistry; Interleukin-8; Male; Membrane Glycoproteins; Microcirculation; Middle Aged; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Syndecan-3; Synovial Membrane

Urinary glycosaminoglycans (GAG) and heparan sulfate (HS) are considered to be markers of early renal involvement. This study was undertaken to demonstrate their excretion patterns in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) with and without arthritis. Serum creatinine and urinary GAG, HS, microalbumin, and creatinine measurements were made in 51 biopsy-proven lupus nephritis (LN) cases, 12 RA patients, and 21 healthy controls. Urinary GAG and HS levels were higher in the LN and RA groups than in controls. Heparan sulfate excretions and SLE disease activity index (SLEDAI) scores were no different between SLE patients with classes 1 and 2 (group A) and those with classes 3, 4, and 5 (group B) renal involvement. However, GAG and microalbumin excretions were significantly high in the latter. There were no differences in GAG and HS excretions between normoalbuminuric, microalbuminuric, and macroproteinuric SLE patients or between those with and without arthritis. In conclusion, urinary GAG and HS, being unrelated to the presence of arthritis, are independent markers of LN. Extrarenal causes or subclinical renal involvement may be responsible in RA due to their increased excretion in these patients.

Topics: Adolescent; Arthritis, Rheumatoid; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Lupus Erythematosus, Systemic; Lupus Nephritis; Male; Middle Aged

The localization and the nature of glycosaminoglycans (GAGs) in the synovial membrane in 30 patients with rheumatoid arthritis (RA) involving 40 knees were studied by newly developed histochemical methods. To detect the acidic glycoconjugates, sensitized diamine procedures were employed based upon high and low iron diamine stainings. To identify the various molecular species of the GAGs, enzyme (chondroitinase ABC and B, testicular hyaluronidase and keratanase) digestion and chemical modification (nitrous acid treatment) procedures were performed prior to the diamine stainings. The sensitized diamine methods could clearly stain the acidic glycoconjugates contained in the synovial tissue components in shades of brown to black, and could detect the precise distribution patterns of the GAGs. The results obtained in the present study confirmed that the tissue in RA synovial membranes contained various amounts of each GAG molecular species such as dermatan sulfate, chondroitin sulfate A/C, hyaluronic acid and heparan sulfate. Furthermore, the distribution patterns of dermatan and chondroitin sulfates in the diseased synovial tissues were pathophysiologically interesting; in the inflammatory areas, the molecular species of GAGs was primarily dermatan sulfate, whereas in the fibrotic areas, it was mainly chondroitin sulfate A/C. Such results appear to be useful for pathophysiological studies on the synovial tissues of RA.

Topics: Adult; Aged; Arthritis, Rheumatoid; Chondroitin Sulfates; Dermatan Sulfate; Female; Glycosaminoglycans; Heparitin Sulfate; Histocytochemistry; Histological Techniques; Humans; Hyaluronic Acid; Knee Joint; Male; Middle Aged; Synovial Membrane

Systemic lupus erythematosus (SLE) is an autoimmune disease which involves the basement membranes of blood vessels in multiple organs. An important component of the microvasculature is vascular heparan sulfate proteoglycan (HSPG). In this study, we investigated the presence in SLE and other immune disease sera of autoantibodies to purified vascular HSPG. Our data demonstrate that antibody to HSPG is found primarily in SLE sera, and not in sera from controls or patients with other immune diseases. The titer of antibody to HSPG correlated with complement depletion in SLE sera. Antibody to HSPG was frequently found in high titer in SLE patients with renal and neurologic involvement. These studies indicate that our assay for antibody to vascular HSPG detects a pathologically relevant autoantibody in SLE sera. The implications of our findings for pathogenesis of vascular autoimmunity are discussed, including the relationship of anti-vascular HSPG antibody to anti-DNA and antiphospholipid antibodies.

Topics: Administration, Topical; Anti-Inflammatory Agents; Antibody Specificity; Arthritis, Rheumatoid; Basement Membrane; Blotting, Western; Chondroitin Sulfates; Collagen; Complement System Proteins; Dose-Response Relationship, Immunologic; Enzyme-Linked Immunosorbent Assay; Fibronectins; Heparin; Heparitin Sulfate; Humans; Hyaluronic Acid; Laminin; Lupus Erythematosus, Systemic; Muscle, Smooth, Vascular; Polymyalgia Rheumatica; Proteoglycans; Scleroderma, Systemic; Sjogren's Syndrome

Human neutrophil elastase was purified to homogeneity as two isozymes named E1 and E2. The isozymes degraded Type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan similarly to each other. The degradation of such basement membrane components by elastase may assist the extravasation of neutrophils in the process of inflammation. Among the substrates tested, only type V collagen, which is susceptible to neutrophil gelatinase, was resistant to elastase. This broad substrate specificity of the enzyme may also contribute to tissue destruction at the sites of inflammation. We produced a monoclonal antibody against the purified enzyme and applied it to immunohistochemical studies. In bronchopneumonia and polyarteritis nodosa, elastase was associated with the cleaved elastic fibers, indicating that the enzyme really destroys tissue in vivo. In the exudates of rheumatoid joint, elastase was stained as diffuse fine granules. Immunohistochemical studies with the monoclonal antibody will provide a complementary way to disclose the mechanism of diseases related to neutrophil infiltration.

Topics: Antibodies, Monoclonal; Arthritis, Rheumatoid; Basement Membrane; Bronchopneumonia; Chondroitin Sulfate Proteoglycans; Collagen; Extracellular Matrix; Fibronectins; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Hydrolysis; Inflammation; Isoenzymes; Laminin; Neutrophils; Pancreatic Elastase; Polyarteritis Nodosa; Substrate Specificity

Human synovial cells in culture are known to synthesize hyaluronic acid, but the production of sulfated glycosaminoglycans (GAG) has received less attention. Using 14C-glucosamine as a precursor, GAG content was studied in the medium, trypsin-solubilized pericellular layer, and cell residue fraction of cultured synovial cells derived from the synovial membranes of nonrheumatoid and rheumatoid joints. Over 90% of the total non-dialyzable counts appeared in the culture medium, for the most part in hyaluronic acid. The remaining nondialyzable counts were cell-associated, almost equally divided between the pericellular layer and cell residues. In these fractions, only part of the counts were in GAG susceptible to testicular hyaluronidase digestion, and GAG were significantly lower in the cell residue of the rheumatoid synovial cells compared to the nonrheumatoid cells. Analysis of the chondroitinase ABC and AC digestion products of these GAG indicated the presence of chondroitin-4 and -6 sulfates, and dermatan sulfate, but not heparan sulfate. Similar findings with respect to the identity of the GAG in nonrheumatoid and rheumatoid synovial cell culture media were obtained with 35SO4 as a precursor.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Glycosaminoglycans; Heparitin Sulfate; Humans; Synovial Membrane