Oxidative modification of inflammatory synovial fluid immunoglobulin G

The release of highly reactive oxygen-derived products by activated phagocytic cells in inflammatory foci plays a major role in defense mechanisms against infection and in the generation of tissue injury. Oxidative modification of proteins in inflammatory foci may give rise to products that contribute to the perpetuation of inflammation. The present studies analyze the oxidative alterations of inflammatory synovial fluid immunoglobulin G (IgG). IgG was purified from the synovial fluids of five patients with rheumatoid arthritis and two patients with acute gouty arthritis by three sequential steps: gel filtration chromatography, immunoaffinity chromatography, and a final gel filtration chromatography step under dissociative conditions (4 M guanidine). The resulting protein peaks of > 150 kDa (pool I-1), 150 kDa (pools I-2 and II-2), and < 150 kDa (pools I-3 and II-3) were tested for the presence of a fluorescence profile distinctive of oxidized proteins, the peak corresponding to monomer IgG (pool II-2) was subjected to quantitative amino acid analysis, and the results were compared with a standard preparation of normal IgG oxidized with HOCl. In addition, the protein pools were tested for the presence of lipid peroxide products. The results indicate that a portion of the affinity-purified IgG formed high-molecular-mass covalently cross-linked aggregates as evidenced by its presence in the >150-kDa pool after dissociative gel filtration chromatography. Moreover, this fraction and the pool corresponding to monomer IgG (pool II-2) exhibited the fluorescence profile characteristic of oxidized proteins. Amino acid analysis of the monomer IgG fraction revealed decreases in the content of histidine, methionine, tyrosine, and cystsine, which were similar to the alterations measured in normal IgG oxidized by HOCl. The synovial fluid and standard oxidized IgG showed the presence of oxidative by-products of tyrosine (monochlorotyrosine) and cysteine (cysteic acid). The synovial fluid IgG yielded a novel component that was not present in the standard control or oxidized IgG. This component was partially identified by mass spectrometry. Finally, the smaller peptide fraction isolated from affinity-purified synovial fluid IgG (pools I-3 and II-3) only after the gel filtration chromatography step under dissociative conditions exhibited evidence of oxidative damage and the presence of high concentrations of thiobarbituric acid-reactive material (TBAR). These observations suggest that oxidative processes in inflammatory foci generate products derived from protein and lipids that may contribute to the self-perpetuation of inflammation.     

Isolation and Characterization of Amyloid-Related Serum Protein SAA as a Low Molecular Weight Protein

With direct immunoprecipitation or gel filtration under dissociating conditions, amyloid-related serum protein SAA has been isolated as a low molecular weight protein from the serum of two patients with rheumatoid arthritis but without known amyloidosis. The isolated protein SAA showed antigenic identity and an amino acid composition that was similar, but not identical, with isolated fibril protein AA. Molecular weight estimations suggest that protein SAA is approximately 50% larger than protein AA and has a molecular weight of 14,000-15,000 daltons. Preliminary results indicate that protein SAA from a patient with amyloidosis has a similar small molecular weight subunit.     

A chemotactic inhibitor in synovial fluid.

Synovial fluid was found to contain an inhibitor of neutrophil chemotaxis. The activity of this inhibitor was masked in native synovial fluid, but could be detected in fluid in which complement had been deactivated by mild heating. The inhibitor was most effective against the chemotactic activity of zymosan-activated serum (C5ades arg). It had little effect when N-formyl-methionyl-leucyl-phenylalanine served as chemoattractant. Inhibition was not the result of a direct effect on the neutrophils, since incubation of cells with synovial fluid did not alter their chemotactic response. The inhibitory activity was destroyed by boiling the synovial fluid or treating it with trypsin, suggesting that it is a protein (or proteins); it was not affected by hyaluronidase treatment. Gel filtration revealed that the inhibitor was present in native as well as decomplemented synovial fluid, and that its molecular weight was in the vicinity of 25,000. It is proposed that this inhibitory activity plays a role in the regulation of the inflammatory response in joints.     

The role of hyaluronic acid in joint stability--a hypothesis for hip dysplasia and allied disorders

The concentration of hyaluronic acid (HA) and proteins in synovial fluids of hip and shoulder joints of a variety of canine breeds has been investigated. In the Australian Kelpie, a working dog with a low incidence of hip dysplasia, shoulder synovial fluid viscosity and HA concentration were higher than in similar joints of Alsatians in which hip dysplasia is relatively common. Moreover, the HA levels and viscosity in shoulder fluids of animals with clinically defined hip dysplasia were substantially lower than in all other breeds studied. On the basis of these findings, we propose that hip dysplasia and other joint abnormalities may arise as a consequence of a deficiency in the levels of HA in synovial fluids.     

透明质酸钠治疗骨性关节炎的临床与实验研究

目的 探讨透明质酸钠关节腔注射对骨性关节炎的临床疗效、安全耐受性及对血清与滑膜液中ＩＬ－１、ＩＬ＿６水平的影响。方法 取３４例有膝关节腔积液的一关节炎患者随机分成两组,分别给予关节腔注身南酸钠与非甾体药治疗,观察治疗前后关节休息痛、活动痛、压痛、肿胀与功能改善程度,并分析其安全耐受性;同时测定前后血清与 膜液中ＩＬ－１、ＩＬ－６水平变化。结果 治疗组关节各种症状均有改善,尤以活动痛与肿胀改善显著,     

Antikeratin antibodies in synovial fluid in rheumatoid arthritis

Serum and synovial fluid of 20 patients with classical or definite rheumatoid arthritis (RA) were tested for antikeratin antibodies (AKA) by indirect immunofluorescence using rat esophagus as antigen. AKA were found in 80% of the RA patients, in serum as well as in synovial fluid. None of the 54 serum control patients were AKA positive in serum. None of the 17 synovial fluid control patients were AKA positive in synovial fluid. F(ab)'2 fragments prepared from AKA positive RA serum retained antibody activity. AKA belonged to the IgG class of immunoglobulins. Corrected for the lower IgG content in synovial fluid, AKA constituted a higher percentage of the IgG in synovial fluid than in serum. This could imply a possibility of local production of AKA in the joint.     

Rheologic changes in the synovial fluid of patients with gonarthritis induced by intraarticular infiltration of hyaluronic acid.

The aim of the present study was to evaluate possible changes in synovial fluid viscosity in gonarthritic patients treated with intraarticular infiltration of hyaluronic acid. Thirty patients with radiologically proven (Stage III Kellgren) serious gonarthrosis, local pain and functional limitation were enrolled. All patients had reported at least 4-5 episodes of hydrarthrosis during the previous 12 months. Therefore, two different phases of their illness could be observed: a relatively silent phase of hydrarthrosis and a symptomatic phase. According to the protocol, one sample of synovial fluid was collected for evaluation of baseline viscosity (pretreatment); immediately afterwards, intraarticular administration of high molecular weight sodium hyaluronate (one 20 mg vial/week for 3 weeks) was initiated. During the entire treatment period and for 3 weeks following the end of treatment, intraarticular synovial fluid samples (one sample per week for 3 treatment weeks followed by a further 3 weeks as control) were collected to perform rheologic assessment and viscosimetric analysis. The results of this preliminary study show that exogenous administration of high molecular weight sodium hyaluronate induced normalization of hyaluronic acid viscosity values in patients with high and low baseline hyaluronic acid viscosity values.     

Serum hyaluronic acid in patients with disseminated neoplasm.

Hyaluronic acid concentrations were measured by a laser nephelometric assay in serum samples from 50 patients with advanced disseminated neoplasm and 50 healthy controls matched for age and sex. The identity of hyaluronic acid was confirmed by a combination of electrophoretic and enzymatic techniques. The mean serum hyaluronic acid concentration for the control group was 1.09 mg/l, with a range of 0-4 mg/l. The mean concentration for patients with neoplastic disease was 10.38 mg/l, with a range of 0-100 mg/l. Sixty two per cent of the patients with disseminated neoplasm had serum hyaluronic acid concentrations above the control range. There was no correlation between the increased concentration of hyaluronic acid and tumour type, serum bilirubin, serum alkaline phosphatase, or serum urea concentrations. There was a higher incidence of hypercalcaemia in patients with increased hyaluronic acid concentrations, but the correlation between hyaluronic acid and calcium concentrations was not significant. In view of the possible role of hyaluronic acid in cellular differentiation and morphogenesis the finding of increased hyaluronic acid concentrations in patients with advanced neoplastic disease may be of fundamental importance in cancer biology.     

Synovial fluids from patients with rheumatoid arthritis induce the differentiation of human promyelocytic leukemia cell line HL 60]

Bone marrow abnormalities have been found to play a role in the pathogenesis of rheumatoid arthritis (RA). Recent studies have also confirmed the presence of undifferentiated hematopoietic progenitor cells as well as the expression of stem cell factor in the synovial membranes in RA. The present study investigates whether RA synovial fluids contain factors that can induce differentiation of CD 14 positive/HLA-DR positive cells from undifferentiated hematopoietic cells. Synovial fluid specimens from 18 patients with RA and from 10 control patients, including patients with osteoarthritis and Behcet's disease, were studied. Human promyelocytic leukemia cell line HL 60 (5 x 10(4)/well) were cultured in the presence or absence of the synovial fluids for 5 days, after which the expression of CD 14 and HLA-DR was examined by flow cytometry. The induction of differentiation of CD 14 positive/HLA-DR positive cells or HLA-DR positive cells from HL 60 cells was significantly enhanced more in the presence of synovial fluids from RA patients than in the presence of those of control patients. However, the sera from the RA patients could not induce the differentiation of CD 14 positive/HLA-DR positive cells or HLA-DR positive cells from HL 60 cells. Most cytokines found in RA synovial fluid could not induce the differentiation of HL 60 cells. Of note, treatment of synovial fluids with hyaluronidase significantly decreased or abrogated their capacity to induce the differentiation of HLA-DR positive cells from HL 60. There was no significant difference in the concentration of hyaluronic acid in the synovial fluid between the RA patients and the control patients. These results indicate that there are factors that can induce differentiation of HLA-DR positive cells from undifferentiated hematopoietic cells in the synovial fluid of RA. The data also suggest that such differentiation factors might be related with qualitative abnormality of hyaluronic acid.     

Isolation and Characterization of Porcine Mannan-Binding Proteins of Different Size and Ultrastructure

The authors report on the purification and characterization of mannan-binding proteins (MBP) isolated from porcine serum. The MBPs were purified by use of PEG precipitation, affinity chromatography on mannan-Sepharose, protein A- and anti-porcine IgM-Sepharose followed by gel filtration. The MBP proteins were collagenase sensitive and showed γ₁-γ₂-electrophoretic mobility. The MBP designated pMBP-28 had a molecular mass of 28 kDa when analysed on SDS-PAGE under reducing conditions and eluted corresponding to a molecular mass of approximately 700 kDa on gel filtration chromatography. Electron micrographs of pMBP-28 revealed an oligomeric protein similar to rodent MBP-A and human MBP but with a predominance of penta- and hexameric molecules. Another protein designated pMBP-27 was composed of peptides of 27 kDa and had an M_r of 300–350 kDa on gel filtration chromatography. Electron microscopy of pMBP-27 showed dimer and trimer molecules; the trimers without distinct stalk regions. The N-terminal 26 (pMBP-27) and 24 (MBP-28) amino acid residues showed 54% and 58% identity with human MBP. pMBP-28 showed a higher degree of sequence similarity to rat and mouse MBP-A (60% identity) than to mouse and rat MBP-C (41–45% identity). Both pMBPs exhibited Ca²⁺-dependent binding to D-mannose immobilized on agarose but no significant binding to N-acetyl-D-glucosamine- or fucose-agarose. The results further suggested the presence of a third pMBP which copurified with pMBP-27 but this protein was not sequenced.     

Synovial fluid inhibits killing of Staphylococcus aureus by neutrophils.

Serum in the extracellular environment promotes neutrophil bactericidal activity apart from its opsonizing properties. We examined the effect of non-inflammatory osteoarthritic synovial fluid on serum-mediated neutrophil killing of Staphylococcus aureus. This was done to evaluate the effect of synovial fluid on neutrophil bactericidal activity independent of opsonin concentration. With an initial inoculum of 5 X 10(6) CFU/ml, 1.47 +/- 0.14% bacteria survived after 120 min of incubation with 10% serum and neutrophils. In contrast, 4.07 +/- 0.33% bacteria survived after incubation in serum plus synovial fluid (P less than 0.001). This inhibitory effect was directly related to the concentration of synovial fluid in the incubation mixture. Increasing the concentration of synovial fluid resulted in an increased percent survival. Studies utilizing preopsonized bacteria and radiolabeled organisms demonstrated that synovial fluid did not interfere with opsonization or phagocytosis. Intracellular bactericidal activity was assayed separately from phagocytosis by utilizing a brief ingestion period followed by the removal of extracellular bacteria by either differential centrifugation or lysostaphin treatment. The reincubation of cells and associated bacteria with serum or serum plus synovial fluid revealed that synovial fluid significantly inhibited the promoting effect of serum on neutrophil bactericidal activity. After 60 min of incubation with 10% serum, 13.0 +/- 1.2% bacteria survived, whereas 21.5 +/- 2.3% survived after incubation in serum plus synovial fluid (P less than 0.005). Superoxide production was not affected by the presence of synovial fluid. These findings suggest that the inhibitory effect of synovial fluid is due to an interaction between synovial fluid and the serum factors that promote intracellular killing.     

Use of two-dimensional gel electrophoresis to measure changes in synovial fluid proteins from patients with rheumatoid arthritis treated with antibody to CD4

Synovial fluid proteins from microliter volumes of synovial fluid were resolved by two-dimensional polyacrylamide gel electrophoresis and detected by silver staining to investigate the feasibility of using two-dimensional (2D) electrophoresis in the clinical research setting and provide global disease information of disease progression. Several hundred proteins could be resolved as spots, many of which displayed the characteristic pattern of plasma-derived glycoproteins. The lowest level of detection was approximately 0.2 ng from a total of 50 microg of protein loaded. Most of the proteins could be identified on the basis of pI and molecular weight when compared with plasma protein maps on the World Wide Web. Unknown proteins were characterized by mass spectrometry of tryptic digests and by comparison with peptide databases. Synovial fluids from patients with rheumatoid arthritis were analyzed using this technique. Each subject received a fixed dose of antibody to CD4 as part of a phase II clinical trial to determine the efficacy of this immunosuppressive treatment in modifying disease activity. Synovial fluid was removed at day 0, followed by administration of antibody. Subsequent removal of synovial fluid and additional administration of antibody were carried out at different times thereafter. Changes in levels of acute-phase proteins were quantified by densitometry of silver-stained 2D polyacrylamide gels. Other parameters of disease progression such as serum C-reactive protein and physician's global assessment of clinical condition were used for comparison. In this way, changes in acute-phase proteins towards normal levels, as measured by 2D polyacrylamide gel electrophoresis, could be correlated with clinical improvement and conventional clinical chemistry measurements. Thus, the system can be used for quantitative analysis of protein expression in sites of autoimmune disease activity such as the synovial fluid of rheumatoid arthritis patients.     

Preferential immune response to virion surface glycoproteins by caprine arthritis-encephalitis virus-infected goats.

Six months after inoculation with caprine arthritis-encephalitis virus, the serum and synovial fluid of virus-infected goats had antibodies to [35S]methionine-labeled viral proteins with apparent molecular weights of 125,000, 90,000, 28,000, and 15,000. The 125,000-, 90,000-, and 15,000-molecular-weight methionine-labeled proteins were identified as virion surface glycoproteins by lactoperoxidase iodination and galactose oxidase-boro[3H]hydride reduction labeling techniques. Radioimmunoassay antibody titers to purified p28, the most abundant viral structural protein, averaged 1:182 in synovial fluid and 1:67 in serum 6 months after inoculation. High dilutions of serum and synovial fluid reacted with gp90 and gp125 electroblotted onto nitrocellulose paper from polyacrylamide gels. Anti-gp90 activity was detected at dilutions with an immunoglobulin G content of 0.02 to 11 micrograms, whereas antibody to p28, when detectable on Western blots, was present in samples with an immunoglobulin G content of 0.1 to 2 mg, representing 100- to 1,000-fold-greater titers of antibody to the surface glycoprotein. Synovial fluids often contained more anti-gp90 antibody than did sera. Immunoprecipitation of lactoperoxidase-iodinated virus confirmed the presence of high antibody titers to the two virion surface glycoproteins. Because antiviral gp90 and gp125 antibody is abundant in the synovial fluid of infected goats, it probably contributes to the high immunoglobulin G1 concentrations seen at this site 6 months after caprine arthritis-encephalitis virus infection.     

Use of Two-Dimensional Gel Electrophoresis To Measure Changes in Synovial Fluid Proteins from Patients with Rheumatoid Arthritis Treated with Antibody to CD4

Synovial fluid proteins from microliter volumes of synovial fluid were resolved by two-dimensional polyacrylamide gel electrophoresis and detected by silver staining to investigate the feasibility of using two-dimensional (2D) electrophoresis in the clinical research setting and provide global disease information of disease progression. Several hundred proteins could be resolved as spots, many of which displayed the characteristic pattern of plasma-derived glycoproteins. The lowest level of detection was approximately 0.2 ng from a total of 50 μg of protein loaded. Most of the proteins could be identified on the basis of pI and molecular weight when compared with plasma protein maps on the World Wide Web. Unknown proteins were characterized by mass spectrometry of tryptic digests and by comparison with peptide databases. Synovial fluids from patients with rheumatoid arthritis were analyzed using this technique. Each subject received a fixed dose of antibody to CD4 as part of a phase II clinical trial to determine the efficacy of this immunosuppressive treatment in modifying disease activity. Synovial fluid was removed at day 0, followed by administration of antibody. Subsequent removal of synovial fluid and additional administration of antibody were carried out at different times thereafter. Changes in levels of acute-phase proteins were quantified by densitometry of silver-stained 2D polyacrylamide gels. Other parameters of disease progression such as serum C-reactive protein and physician's global assessment of clinical condition were used for comparison. In this way, changes in acute-phase proteins towards normal levels, as measured by 2D polyacrylamide gel electrophoresis, could be correlated with clinical improvement and conventional clinical chemistry measurements. Thus, the system can be used for quantitative analysis of protein expression in sites of autoimmune disease activity such as the synovial fluid of rheumatoid arthritis patients.     

Detection of superficial zone protein in human and animal body fluids by cross-species monoclonal antibodies specific to superficial zone protein.

In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.     

Protein-mediated boundary lubrication in arthroplasty

Wear of articulated surfaces can be a major lifetime-limiting factor in arthroplasty. In the natural joint, lubrication is effected by the body's natural synovial fluid. Following arthroplasty, and the subsequent reformation of the synovial membrane, a fluid of similar composition surrounds the artificial joint. Synovial fluid contains, among many other constituents, a substantial concentration of the readily adsorbing protein albumin. The ability of human serum albumin to act as a boundary lubricant in joint prostheses has been investigated using a pin-on-disc tribometer. Circular dichroism spectroscopy was employed to follow the temperature- and time-dependent conformational changes of human serum albumin in the model lubricant solution. Effects of protein conformation and polymer surface hydrophilicity on protein adsorption and the resulting friction in the boundary lubrication regime have been investigated. Unfolded proteins preferentially adsorb onto hydrophobic polymer surfaces, where they form a compact, passivating layer and increase sliding friction-an effect that can be largely suppressed by rendering the substrate more hydrophilic. A molecular model for protein-mediated boundary friction is proposed to consolidate the observations. The relevance of the results for in vivo performance and ex vivo hip-joint testing are discussed.     

An Experimental Model in Mink for Studying the Relation Between Amyloid Fibril Protein AA and the Related Serum Protein, SAA

Experimental amyloidosis was induced in mink by repeated injections with endotoxin. Amyloid fibrils extracted from liver and spleen were fractionated by gel filtration after treatment with guanidine-hydrochloride and a reducing agent, dithiothreitol. An elution profile very similar to that of human amyloid fibrils, having protein AA as a major component, was obtained. The mink amyloid protein eluted at a position similar to that of human protein AA was by amino acid composition and partial sequence studies shown to be very similar to the latter protein and was called mink protein AA. In addition, a protein AA-related component (protein SAA) was found in increased amounts in serum of amyloidotic mink, providing further evidence of the homology with human amy-loidosis. Experimental amyloidosis in mink represents a suitable model for studying amyloid proteins and related serum components.     

An Experimental Model in Mink for Studying the Relation Between Amyloid Fibril Protein AA and the Related Serum Protein, SAA

Experimental amyloidosis was induced in mink by repeated injections with endotoxin. Amyloid fibrils extracted from liver and spleen were fractionated by gel filtration after treatment with guanidine-hydrochloride and a reducing agent, dithiothreitol. An elution profile very similar to that of human amyloid fibrils, having protein AA as a major component, was obtained. The mink amyloid protein eluted at a position similar to that of human protein AA was by amino acid composition and partial sequence studies shown to be very similar to the latter protein and was called mink protein AA. In addition, a protein AA-related component (protein SAA) was found in increased amounts in serum of amyloidotic mink, providing further evidence of the homology with human amyloids. Experimental amyloidosis in mink represents a suitable model for studying amyloid proteins and related serum components.     

Early rheumatoid-like synovial lesions in rabbits drinking cow's milk. II. Antibody responses to bovine serum proteins

Thirty-six percent of Old English rabbits fed pasteurized cow's milk developed early rheumatoid-like synovial lesions. All rabbits drinking milk developed high titres of serum and synovial fluid Clq-binding activity. This has been shown to be due to specific antibody to Clq, produced in response to Clq in cow's milk. In addition, these rabbits responded to other bovine proteins present in cow's milk, including conglutinin, beta-lactoglobulin and IgG. There was no correlation between serum or synovial fluid titres of antibody to bovine milk proteins and synovial lesions.