Elevated Nerve Growth Factor Levels in the Synovial Fluid of Patients with Inflammatory Joint Disease

A novel pH shock extraction procedure was used to measure nerve growth factor (NGF) levels in both normal and inflamed synovial fluids using a sensitive and specific two-site enzyme linked immunosorbant assay. To date no data is available on NGF levels in normal synovial fluids. Synovial fluids were taken from 5 normal volunteers, 12 patients with rheumatoid arthritis and 10 patients with other inflammatory arthropathies. The mean ± SEM NGF concentration in normal synovial fluids was 95 ± 33.2 pg/ml (range 39.1–143.1 pg/ml), whereas the mean NGF concentration in the synovial fluids taken from patients with rheumatoid arthritis was 532.5 ± 123.8 pg/ml (range 152–1686 pg/ml). The mean NGF concentration in patients with other inflammatory arthropathies was also raised (430.6 ± 90 pg/ml; range 89–1071 pg/ml). The NGF concentrations were significantly higher in the synovial fluids from both inflamed groups (ANOVA p < 0.05) compared to normals. Raised levels of NGF in synovial fluid may contribute directly to joint inflammation via activation of inflammatory cells.     

Chlorination and nitration of soy isoflavones.

Diets enriched in soy foods containing a high concentration of isoflavonoids are associated with a decrease in the incidence of several chronic inflammatory diseases. Studies with experimental models of diseases, such as atherosclerosis, suggest that these effects can be ascribed to the biological properties of the isoflavones. Since the isoflavones and tyrosine have structural similarities and modifications to tyrosine by inflammatory oxidants such as hypochlorous acid (HOCl) and peroxynitrite (ONOO(-)) have been recently recognized, we hypothesized that the isoflavones also react with HOCl and ONOO(-). Using an in vitro approach, we demonstrate in the present study that the isoflavones genistein, daidzein, and biochanin-A can be chlorinated and nitrated by these oxidants. These reactions were investigated using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. In the reaction with HOCl, both mono- and dichlorinated derivatives of genistein and biochanin-A are formed, whereas with daidzein only a monochlorinated derivative was detected. The reaction between genistein or daidzein and ONOO(-) yielded a mononitrated product. However, no nitrated product was detected with biochanin-A. Furthermore, the reaction between genistein and sodium nitrite and HOCl yielded a chloronitrogenistein derivative, as well as a dichloronitrogenistein derivative. These results indicate that the ability of the isoflavones to react with these oxidant species depends on their structure and suggest that they could be formed under conditions where these reactive species are generated under pathological conditions.     

High synovial expression of the inhibitory FcgammaRIIb in rheumatoid arthritis

Activating Fc gamma receptors (FcgammaRs) have been identified as having important roles in the inflammatory joint reaction in rheumatoid arthritis (RA) and murine models of arthritis. However, the role of the inhibitory FcgammaRIIb in the regulation of the synovial inflammation in RA is less known. Here we have investigated synovial tissue from RA patients using a novel monoclonal antibody (GB3) specific for the FcgammaRIIb isoform. FcgammaRIIb was abundantly expressed in synovia of RA patients, in sharp contrast to the absence or weak staining of FcgammaRIIb in synovial biopsies from healthy volunteers. In addition, the expression of FcgammaRI, FcgammaRII and FcgammaRIII was analyzed in synovia obtained from early and late stages of RA. Compared with healthy synovia, which expressed FcgammaRII, FcgammaRIII but not FcgammaRI, all activating FcgammaRs were expressed and significantly up-regulated in RA, regardless of disease duration. Macrophages were one of the major cell types in the RA synovium expressing FcgammaRIIb and the activating FcgammaRs. Anti-inflammatory treatment with glucocorticoids reduced FcgammaR expression in arthritic joints, particularly that of FcgammaRI. This study demonstrates for the first time that RA patients do not fail to up-regulate FcgammaRIIb upon synovial inflammation, but suggests that the balance between expression of the inhibitory FcgammaRIIb and activating FcgammaRs may be in favour of the latter throughout the disease course. Anti-inflammatory drugs that target activating FcgammaRs may represent valuable therapeutics in this disease.     

Pathogenic antibody recognition of cartilage

Antibodies against cartilage proteins are highly prevalent in the sera and synovial fluids of rheumatoid arthritis (RA) patients and also precede disease induction in various spontaneous and induced animal models of arthritis. These antibodies play an important role in the induction and perpetuation of the clinical disease. Antibodies binding to cartilage protein(s), especially the major articular cartilage protein, collagen type II (CII) can induce, in naive mice, an acute form of arthritis that can substantially destroy the cartilage and bone architecture. More importantly, these anti-CII antibodies can also directly cause the destruction of the target tissue preceding and independently of disease development and in the absence of any other pathogenic inflammatory factors or the action of immune cells. Alternatively, antibodies to citrullinated protein antigens and rheumatoid factor are well-validated prognostic and diagnostic markers of severe erosive RA, although their arthritogenic potential is questioned. Recently, we have found that the monoclonal antibodies to citrulline-modified cartilage protein can bind cartilage and synovial tissue and mediate arthritis in mice. Similarly, one of the pathogenic anti-CII monoclonal antibodies has rheumatoid-factor-like activity, suggesting a disease-inducing role for these commonly prevalent antibodies in RA patients. Interestingly, recent findings have also shown that the enzymatic cleavage or modification of pathogenic IgG antibodies protects the cartilage surface, thereby opening up new therapeutic possibilities for protecting the cartilage from inflammatory damage.     

Oxidative damage to Ca2+-ATPase sarcoplasmic reticulum by HOCl and protective effect of some antioxidants

Injury of rabbit skeletal sarcoplasmic reticulum (SR) induced by hypochlorous acid (HOCl) was studied. HOCl inhibited Ca2+-ATPase activity in a concentration-dependent manner (IC50=100 micromol/l). The concentration of 13.5 micromol/l HOCl reduced the level of sulfhydryl (SH) groups by 50%, yet it did not influence the enzyme activity. In comparison with SH group oxidation and enzyme activity inhibition, a significantly longer time was necessary for the generation of protein carbonyls in SR injured by HOCl. Protective effects of some antioxidants (stobadine, trolox, EGb 761, Pycnogenol) were studied in SR oxidatively injured by HOCl. Trolox and EGb 761 exerted a protective effect on ATPase activity and on SH groups of SR oxidatively modified by HOCl. Stobadine and Pycnogenol inhibited markedly protein carbonyl formation. Stobadine was the only antioxidant able to scavenge HOCl. In conclusion, the protective effects of antioxidants against decrease of Ca2+-ATPase activity induced by HOCl might be caused by protection of SH groups. The compounds with both antioxidant and Ca2+-ATPase protecting effect offer dual defense against tissue damage occurring, e.g. in aging process.     

Gain-of-function mutations in FcgammaRI of NOD mice: implications for the evolution of the Ig superfamily.

It has been postulated that, during evolution of the Ig superfamily, modifications of the function of individual receptors might occur by acquisition of exons and their subsequent modification, though evidence of this is lacking. Here we have analysed the interaction of mouse IgG subclasses with high-affinity FcgammaRI (CD64) which contains three Ig-like domains and is important in innate and adaptive immunity. This analysis has identified a mechanism by which the postulated modification of newly acquired exons provides gains in function. Thus, the most widely distributed FcgammaRI allele in mice (e.g. BALB/c), bound only a single IgG subclass, IgG2a, with high affinity. However, non-obese diabetic (NOD) mice expressed a unique allele that exhibits broader specificity and, in addition to binding IgG2a, FcgammaRI-NOD bound monomeric IgG3 and bound IgG2b with high affinity, an IgG subclass not bound by FcgammaRI of other mouse strains, either as monomer or multivalent immune complexes. Analysis of mutants of FcgammaRI wherein segments of the interdomain junctions were exchanged between FcgammaRI-BALB and FcgammaRI-NOD identified these regions as having major influence in ''gain-of-function'' by the NOD form of FcgammaRI. Nucleotide sequence analysis of intron/exon boundaries encoding the interdomain junctions of the FcgammaRI alleles showed these to have arisen by mutation to alter existing or create new mRNA splice donor/acceptor sites, resulting in generation of modified junctions.     

Transforming growth factor-beta in synovial fluids modulates Fc gamma RII (CD16) expression on mononuclear phagocytes.

Mononuclear phagocytes in the synovium of patients with arthritis, in contrast to blood monocytes, were found to express a third receptor for the constant region of Ig (Fc gamma RIII), in addition to Fc gamma RI and Fc gamma RII. Previously identified on mature mononuclear phagocytes or phagocytes exposed to transforming growth factor-beta (TGF-beta) in vitro, this study documents the presence of Fc gamma RIII (CD16) expressing cells at an inflammatory site. Furthermore, the presence of CD16 on the majority of the LeuM3 (CD14) positive synovial monocytic cells could be mimicked by exposing blood monocytes to synovial fluids from patients with rheumatoid arthritis (17 of 19) and synovial fluids from patients with osteoarthritis (4 of 4). In additional studies, the soluble factor in inflammatory synovial fluids responsible for regulating CD16 expression was found to be consistent with the presence of TGF-beta. Inhibition of the activity in synovial fluids with a neutralizing antibody to TGF-beta confirmed a role for this peptide in synovial phagocytic cell CD16 expression. Moreover, signal transduction through CD16 on synovial phagocytes resulted in augmented extracellular release of superoxide anion that may contribute to tissue damage and other inflammatory sequelae. Identification of TGF-beta and its association with upregulation of CD16 at sites of chronic inflammation may provide insight into the destructive lesions associated with inflammatory arthropathies.     

Hypochlorous acid modifies calcium release channel function from skeletal muscle sarcoplasmic reticulum.

We have previously demonstrated that H2O2 at millimolar concentrations induces Ca(2+) release from actively loaded sarcoplasmic reticulum (SR) vesicles and induces biphasic [(3)H]ryanodine binding behavior. Considering that hypochlorous acid (HOCl) is a related free radical and has been demonstrated to be a more effective oxidant of proteins, we evaluated the effects of HOCl on sarcoplasmic reticulum Ca(2+)-channel release mechanism. In a concentration-dependent manner, HOCl activates the SR Ca(2+) release channel and induces rapid release of Ca from actively loaded vesicles. HOCl-induced Ca(2+) release is inhibited in the presence of millimolar concentrations of DMSO. High-affinity [(3)H]ryanodine binding is also enhanced at concentrations from 10 to 100 microM. At HOCl concentrations of >100 microM, equilibrium binding is inhibited. HOCl stimulation of binding is inhibited by the addition of dithiothreitol. The direct interaction between HOCl and the Ca(2+) release mechanism was further demonstrated in single-channel reconstitution experiments. HOCl, at 20 microM, activated the Ca(2+) release channel after fusion of a SR vesicle to a bilayer lipid membrane. At 40 microM, Ca(2+)-channel activity was inhibited. Pretreatment of SR vesicles with HOCl inhibited the fluorescence development of a fluorogenic probe specific to thiol groups critical to channel function. These results suggest that HOCl at micromolar concentrations can modify SR Ca(2+) handling.     

Stimulation of eryptosis by anti-A IgG antibodies.

Anti-A IgG antibodies have previously been shown to stimulate Ca(2+) entry into red blood cells. Increased cytosolic free Ca(2+) concentration is known to trigger eryptosis, i.e. suicidal erythrocyte death, characterized by exposure of phosphatidylserine at the erythrocyte surface. As macrophages are equipped with phosphatidylserine receptors, they bind, engulf and degrade phosphatidylserine exposing cells. The present experiments have been performed to explore whether anti-A IgGs trigger phosphatidylserine exposure of erythrocytes. Phosphatidylserine exposure was estimated from annexin-V binding as determined in FACS analysis. Exposure to anti-A IgGs (0.5 microg/ml) indeed significantly increased annexin-V binding in erythrocytes with blood group A, but not in erythrocytes with blood group 0. According to Fluo3 fluorescence, anti-A IgGs increased cytosolic Ca(2+) concentration. Whole cell patch clamp recordings revealed the activation of a Ca(2+)-permeable cation channel following treatment with anti-A-IgGs. Annexin-V binding following anti-A IgG exposure was blunted by Ca(2+) removal while anti-A IgG-stimulated cation channel activity was not dependent on extracellular Ca(2+). Osmotic shock (exposure of erythrocytes to 850 mOsm) increased annexin binding, an effect further enhanced by exposure to anti-A IgGs. In conclusion, anti-A IgGs activate erythrocyte cation channels leading to Ca(2+) entry and subsequent erythrocyte cell membrane scrambling. The effect most likely contributes to the elimination of erythrocytes following an immune reaction against the A antigen.     

Peroxynitrite modulates acidic fibroblast growth factor (FGF-1) activity

To establish peroxynitrite (ONOO(-)) as a mediator of acidic fibroblast growth factor (FGF-1) function, preparations of recombinant human FGF-1 were treated with the pro-oxidant in vitro and identified amino acid modifications were correlated with biologic activity. The sequence of FGF-1 amino acid modifications induced by increasing concentrations of ONOO(-) was from cysteine oxidation to dityrosine formation, and to tyrosine/tryptophan nitration. Low steady-state ONOO(-) concentrations (10-50 microM) induced formation of dityrosine, which involved less than 0.1% of the total tyrosines. Treatment of FGF-1 with ONOO(-) induced a dose-dependent (10-50 microM) loss of sulfhydryl groups that correlated with formation of reducible (dithiothreitol, arsenite) FGF-1 aggregates containing 50% latent biologic activity. Treatment with 0.1-0.5mM ONOO(-) induced increasing formation of non-reducible, inactivated FGF-1 structures. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that six residues (Y29, Y69, Y108, Y111, Y139, and W121) were nitrated by ONOO(-). ONOO(-) treatment (0.1mM) of an active FGF-1 mutant (cysteines converted to serines) induced dose-dependent, non-reversible inhibition of biologic activity that correlated with nitration of Y108 and Y111, both of which reside within a conserved domain encompassing the putative FGF-1 receptor binding site. Collectively, these observations predict a role for low levels of ONOO(-) during secretion of FGF-1 as an extracellular complex containing latent biologic activity. High steady-state levels of ONOO(-) may induce extensive cysteine oxidation, critical tyrosine nitration, and non-reversible inactivation of FGF-1, a potential inhibitory feedback mechanism restoring cellular homeostatis during the resolution of inflammation and repair.     

Potential role of methionine sulfoxide in the inactivation of the chaperone GroEL by hypochlorous acid (HOCl) and peroxynitrite (ONOO-)

GroEL is an Escherichia coli molecular chaperone that functions in vivo to fold newly synthesized polypeptides as well as to bind and refold denatured proteins during stress. This protein is a suitable model for its eukaryotic homolog, heat shock protein 60 (Hsp60), due to the high number of conserved amino acid sequences and similar function. Here, we will provide evidence that GroEL is rather insensitive to oxidants produced endogenously during metabolism, such as nitric oxide (.NO) or hydrogen peroxide (H(2)O(2)), but is modified and inactivated by efficiently reactive species generated by phagocytes, such as peroxynitrite (ONOO(-)) and hypochlorous acid (HOCl). For the exposure of 17.5 microm GroEL to 100-250 microm HOCl, the major pathway of inactivation was through the oxidation of methionine to methionine sulfoxide, established through mass spectrometric detection of methionine sulfoxide and the reactivation of a significant fraction of inactivated GroEL by the enzyme methionine sulfoxide reductase B/A (MsrB/A). In addition to the oxidation of methionine, HOCl caused the conversion of cysteine to cysteic acid and this product may account for the remainder of inactivated GroEL not recoverable through MsrB/A. In contrast, HOCl produced only negligible yields of 3-chlorotyrosine. A remarkable finding was the conversion of Met(111) and Met(114) to Met sulfone, which suggests a rather low reduction potential of these 2 residues in GroEL. The high sensitivity of GroEL toward HOCl and ONOO(-) suggests that this protein may be a target for bacterial killing by phagocytes.     

Loss of oxidized and chlorinated bases in DNA treated with reactive oxygen species: implications for assessment of oxidative damage in vivo

Oxidative damage to DNA has been reported to occur in a wide variety of disease states. The most widely used "marker" for oxidative DNA damage is 8-hydroxyguanine. However, the use of only one marker has limitations. Exposure of calf thymus DNA to an .OH-generating system (CuCl(2), ascorbate, H(2)O(2)) or to hypochlorous acid (HOCl), led to the extensive production of multiple oxidized or chlorinated DNA base products, as measured by gas chromatography-mass spectrometry. The addition of peroxynitrite (ONOO(-)) (<200 microM) or SIN-1 (1mM) to oxidized DNA led to the extensive loss of 8-hydroxyguanine, 5-hydroxycytosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2-hydroxyadenine, 8-hydroxyadenine, and 4,6-diamino-5-formamidopyrimidine were lost at higher ONOO(-) concentrations (>200 microM). Exposure of DNA to HOCl led to the generation of 5-Cl uracil and 8-Cl adenine and addition of ONOO(-) (<200 microM) or SIN-1 (1mM) led to an extensive loss of 8-Cl adenine and a small loss of 5-Cl uracil at higher concentrations (>500 microM). An .OH-generating system (CuCl(2)/ascorbate/H(2)O(2)) could also destroy these chlorinated species. Treatment of oxidized or chlorinated DNA with acidified nitrite (NO(2)(-), pH 3) led to substantial loss of various base lesions, in particular 8-OH guanine, 5-OH cytosine, thymine glycol, and 8-Cl adenine. Our data indicate the possibility that when ONOO(-), nitrite in regions of low pH or .OH are produced at sites of inflammation, levels of certain damaged DNA bases could represent an underestimate of ongoing DNA damage. This study emphasizes the need to examine more than one modified DNA base when assessing the role of reactive species in human disease.     

Salvia plebeia extract inhibits the inflammatory response in human rheumatoid synovial fibroblasts and a murine model of arthritis

Salvia plebeia R. Br. has been used to treat a variety of inflammatory diseases and as an antioxidant in many countries, including Korea and China. In this study, we investigated the effects of S. plebeia extract (SPE) on inflammatory arthritis and the underlying mechanisms of action. We used a collagen-induced arthritis (CIA) mouse model. TNF-α-stimulated rheumatoid arthritis (RA) synovial fibroblasts were used to elucidate the underlying mechanisms of action. Oral administration of SPE improved the clinical arthritis score, footpad thickness, and histologic changes, as well as serum IgG1 and IgG2a levels. SPE administration inhibited Th1/Th2/Th17 phenotype CD4⁺ T lymphocyte expansion in inguinal lymph node and expression of inflammatory mediators such as cytokines, MMP-1, and MMP-3 in the ankle joint tissue. SPE significantly suppressed the expression of cytokines and MMP-1 by down-regulating NF-κB, Akt, and mitogen-activated protein kinases in RA synovial fibroblasts. Taken together, these results indicate that SPE is therapeutically efficacious against chronic inflammatory arthritis, suggesting that SPE is a candidate for treating RA.     

Aggregation-Prone Motifs in Human Immunoglobulin G

Therapeutic antibodies of many different IgG subclasses (IgG1, IgG2 and IgG4) are used in the treatment of various cancers, rheumatoid arthritis and other inflammatory and infectious diseases. These antibodies are stored for long durations under high concentrations as required in the disease treatment. Unfortunately, these antibodies aggregate under these storage conditions, leading to a decrease in antibody activity and raising concerns about causing an immunological response. Thus, there is a tremendous need to identify the aggregation-prone regions in different classes of antibodies. We use the SAP (spatial-aggregation-propensity) technology based on molecular simulations to determine the aggregation-prone motifs in the constant regions of IgG1 classes of antibodies. Mutations engineered on these aggregation-prone motif regions led to antibodies of enhanced stability. Fourteen aggregation-prone motifs are identified, with each motif containing one to seven residues. While some of these motifs contain residues that are neighbors in primary sequence, others contain residues that are far apart in primary sequence but are close together in the tertiary structure. Comparison of the IgG1 sequence with those of other subclasses (IgG2, IgG3 and IgG4) showed that these aggregation-prone motifs are largely preserved among all IgG subclasses. Other broader classes of antibodies (IgA1, IgD, IgE and IgM), however, differed in these motif regions. The aggregation-prone motifs identified were therefore common to all IgG subclasses, but differ from those of non-IgG classes. Moreover, since the motifs identified are in the constant regions, they are applicable for all antibodies within the IgG class irrespective of the variable region. Thus, the motif regions identified could be modified on all IgGs to yield antibodies of enhanced stability.     

Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells

Intact rabbit immunoglobulin G molecules (IgGs) and their papain or pepsin fragments were radio-iodinated and injected into HeLa cells. Whole IgGs, Fab2, and Fc fragments were degraded with half-lives of 60- 90 h, whereas half-lives of Fab fragments were 110 h. These results indicate that proteolytic cleavage in the hinge region of the IgG molecule is not the rate-limiting step in its intracellular degradation. The hingeless human myeloma protein, Mcg, was degraded at the same rate as bulk human IgG, providing further evidence that the proteolytically susceptible hinge region is not important for intracellular degradation of IgG molecules. SDS acrylamide gel analysis of injected rabbit IgG molecules revealed that heavy and light chains were degraded at the same rate. Injected rabbit IgGs and rabbit IgG fragments were also examined on isoelectric focusing gels. Fab, Fab2, and Fc fragments were degraded without any correlation with respect to isoelectric point. Positively charged rabbit IgGs disappeared more rapidly than their negative counterparts, contrary to the trend reported for normal intracellular proteins. The isoelectric points of two mouse monoclonal antibodies were essentially unchanged after injection into HeLa cells, suggesting that the altered isoelectric profile observed for intact rabbit IgG resulted from degradation and not protein modification. The intracellular distributions of IgG fragments and intact rabbit IgG molecules were determined by autoradiography of thin sections through injected cells. Intact IgG molecules were excluded from HeLa nuclei whereas both Fab and Fc fragments readily entered them. Thus, for some proteins, entry into the nuclear compartment is determined primarily by size.     

Scavenging of reactive oxygen and nitrogen species by the prodrug sulfasalazine and its metabolites 5-aminosalicylic acid and sulfapyridine

Sulfasalazine is a prodrug composed by a molecule of 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP), linked by an azo bond, which has been shown to be effective in the therapy of inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease, as well as of rheumatic diseases, such as rheumatoid arthritis and ankylosing spondylitis. The precise mechanism of action of sulfasalazine and/or its metabolites has not been completely elucidated, though its antioxidant effects are well established and are probably due to its scavenging effects against reactive oxygen and nitrogen species (ROS and RNS), as well as metal chelating properties, in association to its inhibitory effects over neutrophil oxidative burst. The present work was focused on screening and comparing the potential scavenging activity for an array of ROS (O(2)(?-), H(2)O(2), (1)O(2), ROO(?) and HOCl) and RNS ((?)NO and ONOO(-)), mediated by sulfasalazine and its metabolites 5-ASA and SP, using validated in vitro screening systems. The results showed that both 5-ASA and sulfasalazine were able to scavenge all the tested ROS while SP was practically ineffective in all the assays. For HOCl, (1)O(2), and ROO(?), 5-ASA showed the best scavenging effects. A new and important finding of the present study was the strong scavenging effect of 5-ASA against (1)O(2). 5-ASA was shown to be a strong scavenger of (?)NO and ONOO(-). Sulfasalazine was also able to scavenge these RNS, although with a much lower potency than 5-ASA. SP was unable to scavenge (?)NO in the tested concentrations but was shown to scavenge ONOO(-), with a higher strength when the assay was performed in the presence of 25 mM bicarbonate, suggesting further scavenging of oxidizing carbonate radical. In conclusion, the ROS- and RNS-scavenging effects of sulfasalazine and its metabolites shown in this study may contribute to the anti-inflammatory effects mediated by sulfasalazine through the prevention of the oxidative/nitrative/nitrosative damages caused by these species.     

Radioimmunoassay of IgG and IgM rheumatoid factors reacting with human IgG.

Although IgG rheumatoid factor may play a central role in the pathogenesis of rheumatoid arthritis, previously there have been no precise methods for its specific measurement in serum and synovial fluid. This paper describes a solid phase radioimmunoassay for the independent quantification of IgM and IgG rheumatoid factor reacting with the Fc fragment of human IgG. As measured by this assay, serum IgG rheumatoid factor levels differed significantly between patients with seropositive and seronegative rheumatoid arthritis and normal control subjects. In addition, several sera and joint fluids from patients with seropositive rheumatoid arthritis, even without vasculitis, were shown by gel chromatography to have acid-dissociable complexes of IgG rheumatoid factor suggestive of IgG-IgG dimer or trimer formation.     

Destructive arthritis in the absence of both FcgammaRI and FcgammaRIII

Fc receptors for IgG (FcgammaR) have been implicated in the development of arthritis. However, the precise contribution of the individual FcgammaR to joint pathology is unclear. In this study, the role of the different FcgammaR was assessed both in an active and in a passive mouse model of arthritis by analyzing disease development in double and triple knockout (KO) offspring from crosses of FcgammaRI KO, FcgammaRIII KO, FcgammaRI/III double KO, or FcR gamma-chain KO with the FcgammaRII KO on C57BL6 background, which is susceptible for collagen-induced arthritis (CIA). In the active CIA model, onset was significantly delayed in the absence of FcgammaRIII, whereas incidence and maximum severity were significantly decreased in FcgammaRI/II/III triple KO but not in FcgammaRII/III double KO and FcgammaRI/II double KO mice as compared with FcgammaRII KO animals. Remarkably, fully destructive CIA developed in FcgammaRI/II/III triple KO mice. In contrast, FcR gamma/FcgammaRII double KO mice were resistant to CIA. These findings were confirmed with the passive KRN serum-induced arthritis model. These results indicate that all activating FcgammaR play a role in the development of arthritis, mainly in the downstream effector phase. FcgammaRIII is critically required for early arthritis onset, and FcgammaRI can substantially contribute to arthritis pathology. Importantly, FcgammaRI and FcgammaRIII were together dispensable for the development of destructive arthritis but the FcR gamma-chain was not, suggesting a role for another FcR gamma-chain associated receptor, most likely FcgammaRIV. In addition, FcgammaRII plays a negative regulatory role in both the central and effector phase of arthritis.     

Role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis. II. Antibodies to Salmonella and Yersinia in sera and synovial fluids

IgA, IgG and IgM antibodies against Yersinia Yop proteins, Yersinia LPS and Salmonella LPS from different serogroups were determined by enzyme-linked immunosorbent assay (ELISA) in a 885 serum samples and 92 synovial fluids. The control group consisted of 200 healthy blood donors. Compared with control subjects, patients with arthritis showed significantly increased titres of antibodies against Yersinia Yop, Yersinia LPS and Salmonella LPS appropriately in 21.7%, 44.0% and 56.0% serum samples. The prevalence of positive antibody levels was highest in Yersinia serogroup O3 and Salmonella serogroup B and D antibodies. The IgA titres were found to be much higher in adults than in children and youngsters but IgM titres consequently decreased with age. Investigation of synovial fluids obtained from patients with arthritis showed that Yersinia and Salmonella antibodies in synovial fluid mirror those in serum by concentration, by specificity and by distribution in classes.     

Anti-inflammatory effect of a PAF receptor antagonist and a new molecule with antiproteinase activity in an experimental model of acute urate crystal arthritis

Platelet activating factor (PAF) is a potent mediator of allergic and inflammatory reactions in different pathological conditions. During recent years there has been increasing evidence that PAF can play an important role in the pathogenesis of arthritis. The PMN proteinases make an important contribution to the final tissue joint destruction in arthritis. In a rabbit model of acute crystal arthritis, we have compared the anti-inflammatory effect of two new molecules: BN 50727 with anti-PAF activity, and BN 50548 an inhibitor of PMN proteinases. These molecules were administered dissolved in DMSO at doses of 6 mg/kg three times daily i.p., beginning 24 h before the induction of arthritis. Compared with the untreated animals those receiving the drugs, presented a significant diminution in: (1) the synovial fluid volume; (2) the amount of cells infiltrating the joint cavity and the synovial membrane; and (3) the PGE₂ concentration. Furthermore, in both groups of treated rabbits there was a significant decrease in synovial IL-6 concentration and in C-reactive protein serum levels and an important decline of histopathological score. The treatment with BN 50548 induced a significant reduction of TNF levels in the synovial fluid vs DMSO-treated and untreated rabbits. These results further strengthen that in an acute experimental arthritis model, molecules with capacity to antagonize the in vivo action of PAF have an anti-inflammatory effect reflecting an important role for this mediator in the pathogenesis of arthritis. We have also seen that an inhibitor of proteinases is capable of improving the joint inflammation apparently through a decrease in tumor necrosis factor (TNF) and interleukin-6 (IL-6) synovial levels. Furthermore, the proteinase inhibitor treatment preserves the loss of articular proteoglycan content, in an acute arthritis model. In conclusion, BN 50727 and BN 50548, two compounds with PAF antagonist and antiproteinase activity, respectively exert an anti-inflammatory effect in an experimental model of acute urate crystal arthritis, probably due to a decrease in TNFa and IL-6 synthesis.