Patients with inflammatory arthritic diseases harbor elevated serum and synovial fluid levels of free and immune-complexed glucose-6-phosphate isomerase (G6PI)

In K/BxN mice, anti-glucose-6-phosphate isomerase (G6PI) IgG antibodies (Abs) cause joint-specific inflammation and destruction. Anti-G6PI Abs are also present in humans with inflammatory arthritis, especially among patients with rheumatoid arthritis (RA). A contributing factor to the induction of such autoantibodies may be upregulated expression of the corresponding antigen G6PI in affected tissues and/or increased levels of G6PI in the circulation. To determine G6PI levels and the presence of free G6PI and/or G6PI-containing immune complexes in sera and synovial fluids (SF) of patients with different arthritides, serum and SF obtained concomitantly from 91 clinically well-defined arthritis patients were assessed in a blinded manner for G6PI enzymatic assay and for G6PI protein concentration by ELISA. Sera and SF from patients with immune-based inflammatory arthritis contained significantly higher levels of G6PI enzymatic activity compared to sera or SF from patients with non-immune-based inflammatory arthritis or healthy controls. In addition, significantly higher levels of total G6PI protein concentration (including both enzymatically active and inactive forms) were present in sera of RA patients vs. those with other immune-based or non-immune-based inflammatory arthritis.G6PI in sera and SF were present both as G6PI-containing immune complexes and as free G6PI, with the majority of free G6PI existing as tetramers with lesser amounts of dimers and monomers. Levels of G6PI enzymatic activity in the sera of most immune-based inflammatory arthritis patients are elevated and may reflect ongoing inflammation and cell destruction. The high serum levels of enzymatically inactive forms of G6PI in RA relative to those in other arthritic diseases are partially due to G6PI-containing immune complexes, a portion of which also contains C1q. Overall, our study supports the notion that elevated G6PI levels present in patients with immune-based inflammatory arthritis may contribute to elevated levels of anti-G6PI Abs and G6PI/anti-G6PI immune complexes. This, in turn, may trigger production of proinflammatory cytokines and perpetuate the inflammatory process.     

Human monoclonal IgG rheumatoid factor has structural homology with bacterial Fc receptor proteins

A cloned lymphoblast cell line, hRF-1, that secreted human monoclonal IgG4 rheumatoid factor autoantibody was produced by Epstein-Barr virus transformation of lymphocytes from rheumatoid arthritis synovium. The binding of hRF-1 rheumatoid factor to IgG globulins of different mammalian species was similar to the binding specificity of Staphylococcus aureus protein A (SpA) and to antibodies found in the sera from patients with rheumatoid arthritis. hRF-1 also had the same binding pattern to human IgG subclasses as SpA. Direct competition was observed between SpA and hRF-1 in binding IgG Fc. These results provide evidence for structural homology between a bacterial Fc receptor protein (SpA) and the monoclonal IgG rheumatoid factor.     

Reduced amyloid-A-degrading activity in serum in amyloidosis associated with rheumatoid arthritis.

The ability to degrade amyloid A fibrils was studied in the serum of 31 patients with amyloidosis associated with rheumatoid arthritis, 33 patients with rheumatoid arthritis without amyloidosis, and 47 healthy controls. Fibrillar amyloid A protein and the radial diffusion method were used. The mean degrading activity in serum was significantly lower in patients with rheumatoid arthritis complicated by amyloidosis (58 +/- 19% SD of the activity in a pooled sample of sera from 100 healthy blood donors used as standard) than in patients with rheumatoid arthritis alone (78 +/- 14%; p less than 0.001) or controls (99 +/- 19%; p less than 0.001). Alpha 1-antitrypsin, concentrations of which were raised in both groups of patients, inhibited the degrading activity in serum even in low concentrations. A negative correlation between degrading activity and alpha 1-antitrypsin concentrations was observed. These findings suggest that reduced amyloid-A-degrading activity is due to inhibition rather than to deficiency of enzyme.     

Low Incidence of Rheumatoid Factor and Autoantibodies in Nigerian Patients with Rheumatoid Arthritis

Sera from 53 Nigerian patients satisfying the American Rheumatism Association criteria for a diagnosis of definite or probable rheumatoid arthritis and sera from sick and healthy Nigerian controls were tested for rheumatoid factor, autoantibodies, and immunoglobulin levels. Rheumatoid factor and autoantibodies were found no more frequently in patients with rheumatoid arthritis than in controls. These findings confirm the clinical impression that Nigerian patients with polyarthritis satisfying the criteria for a diagnosis of rheumatoid arthritis differ from Caucasian patients with the disease in a number of important respects. They suggest that either these patients do not have rheumatoid arthritis but a distinct clinical syndrome or that in Nigeria the course of rheumatoid arthritis is modified by genetic or environmental factors.     

Immunoprecipitation of human adrenal microsomal antigen

Human adrenal microsomes have been labelled with 125I and immunoprecipitated with sera from patients with Addison's disease. The immunoprecipitates were then analysed by SDS-PAGE and autoradiography. 13 of the 23 sera from the Addison patients studied contained antibodies which reacted with a 55 kDa adrenal microsomal protein. The same 13 sera were also positive for adrenal antibodies as judged by immunofluorescence. The 55 kDa protein was not immunoprecipitated from placenta or thyroid microsomes by Addison sera. Furthermore, patients with Graves' disease or rheumatoid arthritis did not immunoprecipitate the 55 kDa protein from adrenal microsomes. Our studies suggest therefore that Addison sera contain antibodies to a 55 kDa adrenal specific protein which may well be the antigen observed on immunofluorescence.     

The level of IgM, IgG and IgA antibodies and total antibodies to the low-molecular, cell-wall protein of Streptococcus group A without type specificity in the serum of patients with a streptococcal infection

The levels of IgM, IgG and IgA antibodies, as well as total antibodies, to group A streptococcal low-molecular cell-wall protein without type specificity were studied in the sera of patients with primary erysipelas, rheumatism in the active and inactive phases, seronegative rheumatoid arthritis, as well as in the sera of healthy donors. The average level of antibodies to low-molecular protein in the sera of all groups of patients was significantly higher than the sera of healthy donors. The analysis of the distribution of antibodies in accordance with their isotypes revealed the specific features of response, characteristic of each group of patients. For rheumatism patients, the positive correlation between response to low-molecular protein and response to group-specific polysaccharide A was established. This correlation was most pronounced in patients with rheumatism in the inactive phase.     

Detection of disease-specific augmentation of abnormal immunoglobulin G in sera of patients with rheumatoid arthritis

Galactose-free immunoglobulin G (IgG), which is known to be higher in the sera of patients with rheumatoid arthritis, was prepared from IgG of healthy volunteers using enzymes. Its reactivity to lectins was analyzed. The galactose-free IgG showed no reactivity to Ricinus communis agglutinin 120 but displayed greater reactivity to concanavalin A and Lens culinaris lectin than did intact human IgG. Then, IgG in serum samples was bound to protein A immobilized on a nitrocellulose membrane, and its reactivity to biotinylated concanavalin A was measured with streptavidin-conjugated horseradish peroxidase. When the reactivity to concanavalin A of IgG in sera from healthy individuals and patients with rheumatoid arthritis (RA), osteoarthritis, systemic lupus erythematosus, or hepatic disease was compared, higher levels were shown in patients with RA, notably in 60% of the seronegative patients and 80% of the early phase patients. Therefore, it was suggested that augmentation of the abnormal IgG in sera was highly specific to patients with RA and that this novel serum test could be very useful for an accurate diagnosis of this disease.     

Measurement of Arginine Carboxypeptidase-Generating Activity of Adult Plasma

Arginine carboxypeptidase (CPR) is a novel carboxypeptidase which was first described by Campbell and Okada. CPR is generated from a stable precursor of CPR (proCPR) during coagulation or under other circumstances and is promptly inactivated at 37 C. Therefore, it is not easy to determine CPR in blood samples. Since proCPR can be separated from the other basic carboxypeptidase (carboxypeptidase N; CPN) by passing plasma through DEAE gel, we have established a method to determine the amount of proCPR after converting it to active CPR by trypsin treatment. We first separated the proCPR from CPN using a filter cup tube (FC tube) packed with DEAE Sephadex, and measured activity after conversion of the enzyme to its active form using trypsin. With this method, no significant decrease in proCPR was noted in the plasma of patients including those with rheumatoid arthritis (RA), although CPR activity in fresh sera has been reported to be decreased. This discrepancy suggests that proCPR is not depleted in most patient sera, but that the level of activity of the enzyme which converts proCPR into active CPR may be compromised in RA patients.     

Exposure to Mimivirus Collagen Promotes Arthritis

Collagens, the most abundant proteins in animals, also occur in some recently described nucleocytoplasmic large DNA viruses such as Mimiviridae, which replicate in amoebae. To clarify the impact of viral collagens on the immune response of animals exposed to Mimiviridae, we have investigated the localization of collagens in Acanthamoeba polyphaga mimivirus particles and the response of mice to immunization with mimivirus particles. Using protein biotinylation, we have first shown that viral collagen encoded by open reading frame L71 is present at the surface of mimivirus particles. Exposure to mimivirus collagens elicited the production of anti-collagen antibodies in DBA/1 mice immunized intradermally with mimivirus protein extracts. This antibody response also targeted mouse collagen type II and was accompanied by T-cell reactivity to collagen and joint inflammation, as observed in collagen-induced arthritis following immunization of mice with bovine collagen type II. The broad distribution of nucleocytoplasmic large DNA viruses in the environment suggests that humans are constantly exposed to such large virus particles. A survey of blood sera from healthy human subjects and from rheumatoid arthritis patients indeed demonstrated that 30% of healthy-subject and 36% of rheumatoid arthritis sera recognized the major mimivirus capsid protein L425. Moreover, whereas 6% of healthy-subject sera recognized the mimivirus collagen protein L71, 22% of rheumatoid arthritis sera were positive for mimivirus L71. Accordingly, our study shows that environmental exposure to mimivirus represents a risk factor in triggering autoimmunity to collagens.     

Autoantibodies to the extracellular matrix microfibrillar protein, fibrillin-1, in patients with scleroderma and other connective tissue diseases.

A duplication in the fibrillin-1 gene has been implicated as the cause of the tight skin 1 (tsk1) phenotype, an animal model of scleroderma or systemic sclerosis (SSc). In addition to the production of abnormal fibrillin-1 protein, the tsk1 mouse also produces autoantibodies to fibrillin-1. Among a population of Choctaw Native Americans with the highest prevalence of SSc yet described, a chromosome 15q haplotype containing the fibrillin-1 gene has been strongly associated with SSc. With a recombinant human fibrillin-1 protein, autoantibodies to fibrillin-1 were detected in the sera of Native American SSc patients that correlated significantly with disease. Abs to fibrillin-1 also were detected in sera from Japanese, Caucasian, and African-American SSc patients. Compared with other ethnic groups, Japanese and Native American SSc patients had significantly higher frequencies of anti-fibrillin-1 Abs. Sera from patients with diffuse SSc, calcinosis, Raynaud's, esophageal dysmotility, sclerodactyly, and telangiectasias syndrome and mixed connective tissue disease also had significantly higher frequencies of anti-fibrillin-1 Abs than sera from controls or patients with other non-SSc connective tissue diseases (lupus, rheumatoid arthritis, and Sj?gren's syndrome). Ab specificity for fibrillin-1 was demonstrated by the lack of binding to a panel of other purified autoantigens. The results presented demonstrate for the first time the presence of high levels of anti-fibrillin-1 Abs in a significant portion of patients with SSc.     

Structural similarities in the kappa light chains of human rheumatoid factor paraproteins and serum immunoglobulins bearing a cross-reactive idiotype

The monoclonal antibody (MoAb) 17.109 recognizes a cross-reactive idiotype (CRI) associated with the light chains of Waldenstrom's macroglobulins with rheumatoid factor (RF) activity. The MoAb also reacts with a proportion of IgM-RF molecules from the sera of rheumatoid arthritis and primary Sjogren's syndrome patients, and from the sera of seropositive normal human subjects. In the present experiments, we used affinity chromatography to purify the 17.109 CRI-positive immunoglobulin from serum and have analyzed the isolated material by Western blotting. The purified 17.109 CRI-positive material from the sera of rheumatoid arthritis patients, Sjogren's syndrome patients, and normal subjects contained exclusively kappa light chains, and had demonstrated RF activity. In every case the 17.109 CRI-positive isolates reacted with antibodies against synthetic peptides corresponding to both the conserved second and third complementarity-determining regions (CDR) of the monoclonal kappa IgM-RF paraprotein Sie. The binding was inhibited specifically by the free peptides in solution. The antipeptide antibodies did not react appreciably with unfractionated human immunoglobulin. The data establish that the 17.109 CRI-positive immunoglobulin from diverse human sera have similar or identical second and third light chain CDR. These results suggest i) that the MoAb 17.109 identifies the protein product of a single or a very few V kappa genes, ii) that the ability to make kappa light chains with the 17.109-associated variable region is widespread in the human population, and iii) that the 17.109-defined kappa variable region segment is associated with IgM-RF autoantibodies.     

C1q-containing immune complexes purified from sera of juvenile rheumatoid arthritis patients mediate IL-8 production by human synoviocytes: role of C1q receptors.

Immune complexes that vary in size and composition are present in the sera and synovial fluid of juvenile rheumatoid arthritis (JRA) patients. They are believed to be potent inducers of the ongoing inflammatory process in JRA. However, the precise composition and role of these complexes in the pathophysiology of JRA remain unclear. We hypothesized that circulating ICs have the potential to interact with resident joint synovial fibroblasts (synoviocytes) and induce the expression of inflammatory cytokines. To test this hypothesis, cultures of synoviocytes from healthy individuals were treated with ICs isolated from the sera of JRA patients. Studies reported in this work demonstrate that IgM affinity-purified ICs from the sera of JRA patients contain IgM, C1q, IgG, and C3 to a variable extent. These ICs induce IL-8 mRNA and protein production in normal synoviocytes. Our data indicate that C1q in these ICs mediates, in part, IL-8 induction in synoviocytes. This is based on our findings of C1q-binding proteins for collagen stalks (cC1qR) and globular heads (gC1q-binding protein) of C1q in synoviocytes. In addition, collagen stalk and to some extent globular head fragments of C1q inhibit IC-mediated IL-8 induction in synoviocytes. Together, these findings provide evidence for a novel mechanism of IL-8 production by synoviocytes, which could play a key role in inflammation by recruiting leukocytes to synovial tissue and fluid-and subsequently contributing to joint disease.     

Primary biliary cirrhosis sera recognize not only gp210 but also proteins of the p62 complex bearing N-acetylglucosamine residues from rat liver nuclear envelope

We have recently observed reactivity of primary biliary cirrhosis (PBC) sera with several proteins bearing N-acetylglucosamine residues from rat liver nuclear envelopes. The aim of this study was to characterize the reactive antigens. Sera from 31 patients with PBC, 30 with rheumatoid arthritis (RA) and 30 with Sjögren's syndrome (SS) were examined. Rim-like immunofluorescence staining was observed in 15 of 31 (48%) sera from patients with PBC, in 1 of 30 with RA and in 1 of 30 with SS. Upon immunoblotting using preparations of whole rat liver nuclear envelopes and their Triton X 100-KCl extract as antigen souces, a 200 kDa protein band was observed in 9 of sera with PBC. Furthermore, upon immunoblotting using the wheat germ aggulutinin-bound fraction of rat liver envelope as antigen, 62, 60 and 54 kDa protein bands corresponding to components of the p62 complex in the nuclear pore complex (Kita et al. Biochem. 113, 377–382) were observed in 7, 5 and 6 samples respectively, of the 31 PBC sera. Our data suggest that PBC sera recognize not only the 210 kDa protein but also the p62 complex proteins.Abbreviations ANA antinuclear antibody - AMA anti-mitochondrial antibodies - IF immunofluorescence - LAP2 lamina-associated polypeptide 2 - LBR lamin B receptor - anti-NBP 60 anti-nuclear localization signal binding protein 60 - NE nuclear envelope - NPC nuclear pore complex - PBC primary biliary cirrhosis - RA rheumatoid arthritis - SLE systemic lupus erythematosus - SS Sjögren's syndrome - WGA wheat germ agglutinin     

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.     

Evidence for defect of complement-mediated phagocytosis by monocytes from patients with rheumatoid arthritis and cutaneous vasculitis.

In-vitro measurements of the rate of monocyte phagocytosis of heat-killed yeast preopsonised in human AB serum from 14 patients with rheumatoid arthritis and 14 normal controls showed a significant reduction in five patients with active vasculitis but no change in nine with active arthritis alone. Further studies of complement- and Fc-mediated monocyte phagocytosis in which the rate constants (Kc and KFc respectively) were determined using complement-coated Saccharomyces cerevisiae and Candida albicans opsonised with IgG in monocytes from nine patients with rheumatoid vasculitis and 12 controls showed a significant reduction in Kc (p less than 0.01) but normal KFc. Kc was normal in three patients with inactive vasculitis. Low Kc was correlated with low serum C3 concentrations but not with Clq binding or anticomplementary activity, and no evidence of intracytoplasmic or membrane-bound immune complexes was detected in monocytes from patients with active vasculitis. These results show that cutaneous vasculitis in rheumatoid arthritis is associated with selective impairment of complement-mediated monocyte phagocytosis, which does not appear to result from receptor blockade by immune complexes.     

A monoclonal anti-idiotype specific for human polyclonal IgM rheumatoid factor.

One of the hallmarks of rheumatoid arthritis (RA) is the production of high titers of rheumatoid factor (RF) antibody directed against the Fc portion of IgG. Anti-Id that recognize the majority of monoclonal RF from patients with B cell dyscrasias are reactive with only 1 to 2% of these polyclonal RF from RA patients. We describe a new monoclonal anti-Id, 4C9, that recognizes a L chain determinant on polyclonal IgM RF from patients with RA but does not recognize a panel of monoclonal RF from patients with B cell malignancies. 4C9 reactivity is found in the serum of 34/43 RF-positive RA patients and in 12/12 RF-positive synovial fluids, but in only 1/14 RF-negative sera from RA patients and 1/22 sera containing monoclonal IgM RF. 4C9 reactivity is highly enriched in purified IgM RF from nine RA patients and represents a variable percentage of total IgM RF up to a maximum of 23%. Furthermore, 4C9 reactivity is enriched in the synovial fluid of three of five RA patients compared with serum, suggesting that 4C9-reactive IgM RF are synthesized within the joint. IgG RF from RA synovial fluids are not 4C9 reactive, indicating either that different genes are used to encode IgM and IgG RF in RA patients, or that IgG RF have somatically mutated away from idiotypic reactivity.     

Comparison between penicillamine and sulphasalazine in rheumatoid arthritis: Leeds-Birmingham trial.

Sulphasalazine was first formulated by Svartz in the early 1940s, specifically for use as a remission inducing drug in rheumatoid arthritis. After the publication of an unfavourable trial, however, the drug was restricted to patients with ulcerative colitis. In the late 1970s sulphasalazine was re-examined in rheumatoid arthritis and favourable results reported in "open" trials. A double blind controlled trial was therefore conducted comparing enteric coated sulphasalazine and D-penicillamine in patients with active rheumatoid arthritis. A total of 63 patients were recruited in two centres; 31 were treated with sulphasalazine and 32 received penicillamine. After 16 weeks'' treatment both drugs had produced significant improvements in clinical score, pain score measured on a visual analogue scale, grip strength, Ritchie articular index, erythrocyte sedimentation rate, and serum C reactive protein concentration. Nausea was the major side effect in the sulphasalazine treated group. No potentially dangerous effects of sulphasalazine were encountered in contrast with those seen in the penicillamine group. The results suggest that sulphasalazine is an effective and safe drug capable of producing remissions in active rheumatoid arthritis. They also lend confidence to the use of preliminary "open" trials as a means of screening for remission inducing drugs in rheumatoid arthritis.     

Hanganutziu-Deicher antigen and antibody in pathologic sera and tissues.

Heterophile, Hanganutziu-Deicher (H-D) antigen was studied in pathologic sera by means of inhibition of agglutination of bovine erythrocytes by H-D antibodies. H-D antigen was demonstrated in 38% of random cancer sera, 25% of lymphoma or leukemia sera, 25% of leprosy sera, 8% of infectious mononucleosis sera, 6% of rheumatoid arthritis sera, and 27% of synovial fluids of rheumatoid arthritis patients. None of 134 normal human sera gave positive results. Some of the inhibition-positive cancer sera formed precipitation lines with H-D antibody-containing sera. Over 50% of various extracts of cancer tissues as well as spleens of lymphoma or leukemia patients were shown to contain H-D antigen by means of the inhibition test.     

A comparison of euglobulin fractions and whole serum in the hemagglutination and latex fixation tests

One hundred and thirty-two sera were investigated with the Waaler-Rose and latex fixation reactions. The reactions were performed with serum, with acid-precipitated euglobulin, and with cold-precipitated euglobulin. The material consisted of 35 sera from healthy persons, 23 from patients with various diseases, 28 from patients with joint symptoms not due to rheumatoid arthritis, and 46 from patients with classical rheumatoid arthritis.In rheumatoid arthritis sera, an increase in positive reactions was obtained in the Waaler-Rose test from 70 per cent in serum to 83 per cent in acid-precipitated euglobulin. This increase was due to a greater specificity of reactions with low titers. The cold-precipitated euglobulin gave less positive Waaler-Rose reactions than the acid-precipitated euglobulin. With the latex fixation test an increase from 65 per cent positive reactions in serum to about 71 per cent with both cold- and acid-precipitated euglobulin fractions was obtained. Here, the increase consisted of reactions negative in serum but positive in the euglobulin fractions, but again with low titers. Because the increase in positive reactions consists merely of low titer values, fractionation of sera only slightly enhances the reliability of the serological tests.Negatively reacting rheumatoid arthritis sera often had low values of the _2A globulin.     

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.