Cross-reactivity of human and murine anti-DNA antibodies with heparan sulfate. The major glycosaminoglycan in glomerular basement membranes.

In 30 of 33 human systemic lupus erythematosus (SLE) sera and in 10 sera from MRL/l mice with spontaneous SLE, antibodies against heparan sulfate were detected. The anti-heparan sulfate titers showed a significant correlation with the anti-DNA antibody titers. By inhibition studies it was demonstrated that heparan sulfate could inhibit the binding of anti-DNA antibodies to DNA, whereas DNA could block the binding to heparan sulfate. That this reaction is due to crossreactivity of anti-DNA antibodies was further substantiated by the finding that two monoclonal anti-DNA antibodies also bound to heparan sulfate. Antibodies eluted from human and mouse kidneys with diffuse SLE glomerulonephritis showed a similar binding to DNA and heparan sulfate when these eluted antibodies were tested in vitro. Heparan sulfate is the major glycosaminoglycan constituent of the glomerular basement membrane. Our findings suggest that heparan sulfate might serve as a target antigen in vivo for cross-reactive anti-DNA antibodies.

Anti-(U1) small nuclear RNA antibodies in anti-small nuclear ribonucleoprotein sera from patients with connective tissue diseases.

Small nuclear ribonucleoprotein (snRNP) particles are a class of RNA-containing particles in the nucleus of eukaryotic cells. Sera from patients with connective tissue diseases often contain antibodies against the proteins present in these snRNPs. Antibodies against the RNA components of snRNPs, the U snRNAs, are thought to be rare. We tested 118 anti-snRNP sera for the presence of anti-snRNA antibodies and found them in 45 sera (38%). In all sera the antibodies (IgG and F(ab)2 fragments thereof) were exclusively directed against U1 snRNA. The anti-(U1) RNA antibodies were always accompanied by anti-(U1)RNP antibodies but were not found in sera which contain antibodies of the Sm serotype directed against all nucleoplasmic U snRNP particles. Like anti-RNP antibodies, anti-U1 RNA activity is confined to sera from patients with SLE or SLE overlap syndromes and is rarely found in patients with other connective tissue diseases. By analyzing binding to subfragments of U1 snRNA made in vitro, it was demonstrated that anti-(U1)RNA antibodies recognize epitopes distributed throughout the U1 RNA molecule. In most sera, however, either the second or the fourth hairpin loop is the main target of the antibody. The possible mechanisms that could lead to the production of this new type of autoantibody are discussed.

Electrical charge of the antigen determines intraarticular antigen handling and chronicity of arthritis in mice.

We studied the influence of antigenic charge on the handling of intraarticular antigen by the joint and on the ability of the antigen to induce chronic arthritis. Three different antigens were used: anionic native bovine serum albumin (BSA), and charge modified BSA made cationic (pI 8.5) either by methylation (mBSA), or amidation (aBSA). 125I-labeled antigen was injected into the knee joints of nonimmune mice and of mice immunized with antigen in Freund's complete adjuvant. Intraarticular antigen retention of the cationic antigens mBSA and aBSA was significantly increased compared with native BSA, both in immune and non-immune mice. In vitro studies indicated the electrostatic character of the binding of the cationic antigens to joint tissues and confirmed the large difference in antigen retention of the antigens found in vivo. A 100-fold amount of cationic antigen could be bound to non-cartilaginous collagenous tissue of the joint compared with antibody-mediated trapping of native BSA, and for hyaline articular cartilage, this difference was even greater. In immunized mice, chronic arthritis only developed after intraarticular injection of the cationic antigens. This phenomenon was apparently related to increased retention of mBSA and aBSA compared with BSA, since delayed hypersensitivity and humoral immunity were comparable for the three antigens used. Our data indicate that antigenic charge is an important determinant of antigen handling by the joint and, in addition, support the concept that the development of chronic arthritis depends on the amount of antigen retained in the joint.

Cationization of catalase, peroxidase, and superoxide dismutase. Effect of improved intraarticular retention on experimental arthritis in mice.

Several enzymes and other proteins were made cationic either by coupling to polylysine or by shielding of anionic sites. These cationic proteins, all having an isoelectric point greater than 8.5 exhibited excellent retention in articular structures when injected in mouse knee joints. Autoradiography and histochemistry showed that cationic forms of catalase, superoxide dismutase, and horseradish peroxidase were firmly retained by synovial and cartilaginous tissues. The half-life of these enzymes in the joint is thus significantly extended compared with native enzymes. The native enzymes and their cationic derivatives were tested for antiinflammatory properties in mice, using antigen-induced arthritis and zymosan-induced arthritis. It was found that injection of cationic catalase or peroxidase induced a marked suppression of some parameters of the inflammatory response in both types of arthritis, as measured by 99m technetium pertechnetate uptake and leakage of 125I-labeled albumin. Native catalase and peroxidase were less, or not at all effective. Cationic superoxide dismutase or cationic nonenzyme proteins did not suppress inflammation. The observed suppression of two different types of inflammation (an immune and a nonimmune arthritis) by catalase and peroxidase suggests that elimination of peroxides contributes to the suppression of an inflammatory response. We would hypothesize that cationic enzymes offer the possibility for investigating the mechanisms of inflammation and, in addition, might be interesting from a therapeutical point of view.

Scl-86, a marker antigen for diffuse scleroderma.

More than 300 sera from patients with a connective tissue disease were analyzed with the immunoblotting technique. The presence of autoantibodies against an 86,000-mol wt marker antigen for diffuse scleroderma (Scl-86) was found in 14 out of 33 patients with scleroderma. The presence of anti-Scl-86 antibodies seemed to correlate with the diagnosis of diffuse scleroderma since they were found in 13 out of 22 diffuse scleroderma patients and in only one out of 11 patients with limited scleroderma. All scleroderma sera (33 patients' sera and 13 reference sera) were also tested for the presence of anti-Scl-70 antibodies. It was found that all anti-Scl-70 positive sera (n = 25) contained anti-Scl-86 antibody as well, suggesting a relationship between these two antigens. However, the Scl-86 antigen was shown to be an extremely insoluble nonchromosomal protein, resistant to boiling in sodium dodecyl sulfate. This contrasts with the Scl-70 antigen, which has been described as a thermolabile, soluble antigen present in the chromatin fraction. Together, our results are consistent with the idea that Scl-70 is a degradation product of Scl-86. The Scl-86 antigen is present in freshly prepared rabbit thymus, spleen, and liver nuclei as well as in nuclei from various cultured cell lines, but is not detectable in extractable nuclear antigen from rabbit thymus. In a limited retrospective study, the anti-Scl-86 antibodies were found in two sera from patients with Raynaud's phenomenon before the development of diffuse scleroderma. Therefore, it is possible that screening of patients' serum for this antibody might predict the development of diffuse scleroderma.

Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies.

Only a few autoantibodies that are more or less specific for RA have been described so far. The rheumatoid factor most often tested for is not very specific for RA, while the more specific antiperinuclear factor for several reasons is not routinely used as a serological parameter. Here we show that autoantibodies reactive with synthetic peptides containing the unusual amino acid citrulline, a posttranslationally modified arginine residue, are specifically present in the sera of RA patients. Using several citrulline-containing peptide variants in ELISA, antibodies could be detected in 76% of RA sera with a specificity of 96%. Sera showed a remarkable variety in the reactivity pattern towards different citrulline-containing peptides. Affinity-purified antibodies were shown to be positive in the immunofluorescence-based antiperinuclear factor test, and in the so-called antikeratin antibody test, and were reactive towards filaggrin extracted from human epidermis. The specific nature of these antibodies and the presence of these antibodies early in disease, even before other disease manifestations occur, are indicative for a possible role of citrulline-containing epitopes in the pathogenesis of RA.

Hypothalamic-pituitary-adrenal axis activity in patients with rheumatoid arthritis.

OBJECTIVE: To study the hypothalamic-pituitary-adrenal (HPA) axis in patients with rheumatoid arthritis (RA). METHODS: Fifty patients with RA participated in 3 groups: recent onset active RA (n = 20), longstanding active RA (n = 20) and long-standing RA in remission (n = 10), and were compared with 20 healthy controls. The activity of the HPA-axis was assessed under basal conditions and in response to stress (insulin tolerance test, ITT). In addition, patients with recent onset RA underwent a corticotropin releasing hormone (CRH) test and a dexamethasone suppression test. Plasma levels of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha) and IL-6 were also measured. RESULTS: Basal plasma, salivary and urinary cortisol levels and plasma adrenocorticotropic hormone (ACTH) levels were not different between patients with RA and healthy controls. During the ITT, cortisol levels were consistently lower in RA patients than in healthy controls. ACTH levels during the ITT were not different between patients with RA and healthy controls. ACTH and cortisol responses to CRH were assessed only in patients with recent onset RA and were found to be within normal limits. Basal circulating plasma IL-6 levels were significantly higher in patients with active RA than in the other groups. CONCLUSION: Under the standardized conditions of the ITT, patients with RA have decreased plasma cortisol levels compared to healthy controls, despite elevated levels of IL-6. The defect is probably located at the adrenal level and may be of pathogenetic significance for the development of chronic arthritis.

Monocytes/macrophages rather than PMN are involved in early cartilage degradation in cationic immune complex arthritis in mice.

We investigated the involvement of polymorphonuclear granulocytes (PMN) and monocytes in cartilage degradation in immune complex-mediated arthritis (ICA). ICA induced with lysozyme-antilysozyme in the murine knee joint is characterized by a major influx of PMNs followed by monocytes and marked cartilage proteoglycan (PG) depletion develops within 2 days. Around 60% of 35S-prelabeled PG is lost at day 2. Influx of cells was manipulated using interleukin-1 (IL-1) receptor antagonist (IL-1ra) or antibodies to adhesion molecules. Cellular infiltrate was analyzed on hematoxylin-stained joint sections. Early systemic treatment with IL-1ra highly reduced PMN influx, whereas monocyte influx was hardly diminished. PG loss was not significantly reduced, declining from 62% in controls to 47% in IL-1ra-treated mice. Total blockade of cell influx was found after intravenous treatment with monoclonal antibodies 5C6 (anti-CD11b/CD18:anti-CR3) or NIMP.R14 (25-30 kDa protein mainly present on PMN) and PG loss was reduced to 5-10%. A similar reduction was observed after prior depletion of circulating PMNs with total body irradiation. Because amounts of IL-1 produced in leukopenic and control arthritic joints are comparable, this suggests that IL-1 is only marginally involved in PG loss in the first phase of ICA. This study indicates that monocytes rather than PMN might be involved in PG loss in this form of arthritis, either directly or by local activation of synovial layer cells of the joint.

Expression of macrophage-derived chemokine (MDC) mRNA in macrophages is enhanced by interleukin-1beta, tumor necrosis factor alpha, and lipopolysaccharide.

A cDNA encoding the C-C chemokine MDC was isolated from a human macrophage cDNA library by differential hybridization using monocyte- and macrophage-specific cDNA probes. During monocyte to macrophage differentiation in vitro, MDC expression is first detected after 1 day of culturing and reaches maximum levels after 6 days when macrophages have fully matured, as judged from the expression of known macrophage marker genes. Exposure of macrophages to lipopolysaccharide (LPS) results in a dose-dependent increase in MDC mRNA levels, with maximum induction occurring after 6-8 h, whereas expression levels of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-2, interleukin-1beta (IL-1beta), and tumor necrosis factor alpha (TNF-alpha) respond much faster to LPS. Furthermore, MDC expression in macrophages is enhanced by the inflammatory mediators TNF-alpha and IL-1beta. Similar to other TNF-alpha/IL-1beta-inducible genes, costimulation of macrophages with both cytokines leads to higher MDC expression levels than stimulation with a single cytokine. By contrast, both resting and activated monocytes do not express MDC mRNA.

Serum IgA and gold-induced toxic effects in patients with rheumatoid arthritis.

Serum Immunoglobulin concentrations were measured prospectively in 25 patients with rheumatoid arthritis at months 0, 1, 3, 6, and 12 of aurothloglucose treatment. Substantial lowering of IgA and IgM levels was found at month 3 and thereafter, and of IgG at month 12 only. When patients in whom drug-induced toxic effects developed at any time during treatment (toxic group) were compared with those who did not (nontoxic group), serum levels of IgA and to a lesser degree of IgG, but not of IgM, were found to be substantially lower in the toxic than in the nontoxic group, both at the onset and during treatment, except for IgG at month 12. When measured at the moment of toxic effect, only igA, but not IgG and IgM, was substantially lower than in serum samples of patients without toxic effects at that moment. The serum IgA concentration in patients with rheumatoid arthritis seems to be related to whether or not aurothioglucose-induced toxic effects occur.

Unresolved issues in the role of cyclooxygenase-2 in normal physiologic processes and disease.

Originally suggested to function mainly in inflammatory situations, recent data have implied important roles for the cyclooxygenase-2 isoenzyme in reproductive biologic processes, renal and neurologic function, and the antithrombotic activities of endothelial cells. As cyclooxygenase-2-specific inhibitors have recently become available as analgesic and anti-inflammatory drugs, a comprehensive view of this rapidly evolving field is necessary to anticipate both the potential therapeutic benefits and toxic effects associated with these agents.

Naproxen kinetics and disease activity in rheumatoid arthritis: a within-patient study.

The effects of rheumatoid arthritis disease activity on the pharmacokinetics of the highly albumin-bound nonsteroidal anti-inflammatory drug naproxen were studied in six patients during chronic therapy. In the same patients, kinetics during active disease were compared with those in improvement. Active disease is commonly associated with hypoalbuminemia: 30 +/- 4 gm/L vs. 41 +/- 2 gm/L (mean +/- SD) at the time of improvement. Total naproxen concentrations were significantly lower in active disease, together with a larger apparent volume of distribution (10.6 +/- 1.8 L vs. 8.4 +/- 1.3 L; P less than 0.05) and total body clearance (0.79 +/- 1.8 L/hr vs. 0.59 +/- 0.14 L/hr; P less than 0.001). Peak unbound naproxen concentrations were 29% +/- 19% (P less than 0.05) lower at the time of improvement. The unbound clearance was found diminished during active disease (390 +/- 277 L/hr) in comparison with improvement (488 +/- 343 L/hr; P less than 0.05). Clinical implications of the alterations in naproxen kinetics induced by polyarticular inflammation in patients with rheumatoid arthritis are discussed.

Lymphocyte isolation from chronically inflamed synovial membranes.

A simple two-step procedure was developed for isolation of lymphocytes from chronically inflamed human synovial membranes. In the first step minced inflamed synovial tissues are disrupted enzymatically by deoxyribonuclease and collagenase. The second step consists of nylon-wool column filtration of the isolated cells. 7 min of preincubation of up to 37.4 X 10(6) cells in a column packed with 600 mg nylon-wool in 6 ml prior to filtration did not result in significant selective losses of either T or B cells, whereas 45 min of preincubation did. Recovery of lymphocytes after nylon-wool column filtration ranged from 68 to 95% (mean 80%) and viability was always higher than 90%. Nylon-wool column filtration increased the proportion of lymphocytes by a mean 73%. The method allows rapid identification of synovial tissue lymphocyte subpopulations as well as characterization of their function.

Cryosectioning of undecalcified tissues for immunofluorescence.

The present report describes a procedure for preparing 4--6 micrometers cryostat sections of undecalcified fresh frozen tissue which contain hard tissue, for immunofluorescence. The apparatus used is a cryomicrotome originally designed for cutting sections for whole body autoradiography. To obtain cryostat sections suitable for tissue immunofluorescence the standard procedure was modified with respect to the hardness and edges of the microtome knife, the temperature of the cryostat and the carboxymethyl cellulose concentration of the embedding material.

Peripheral blood monocytes of rheumatoid arthritis patients do not express elevated TNF alpha, IL-1beta, and IL-8 mRNA levels. A comparison of monocyte isolation procedures.

We have compared three monocyte isolation procedures for their suitability in the analysis of cytokine mRNA expression in circulating monocytes. Monocytes were isolated from peripheral blood (1) using antiCD14 coated magnetic beads, (2) by Ficoll centrifugation, or (3) by Ficoll centrifugation followed by plastic adherence. The effect of the isolation procedure on the cytokine mRNA expression levels of the isolated monocytes was evaluated by RT-PCR. Results show that the expression of cytokine mRNAs determined in monocytes isolated using antiCD14 coated magnetized beads reflects best the cytokine mRNA levels in circulating monocytes. We subsequently applied this method to the analysis of cytokine mRNA expression levels in rheumatoid arthritis and control monocytes, which revealed that RA and control monocytes isolated by antiCD14 beads produce similar, very low TNF alpha, IL-1beta, and IL-8 mRNA levels.

Effects of NSAIDs on the metabolism of sulphated glycosaminoglycans in healthy and (post) arthritic murine articular cartilage.

Several non-steroidal anti-inflammatory drugs (NSAIDs) were studied for their effects on normal and damaged murine articular cartilage, both in vitro and in vivo. In vitro, in the absence of serum, sodium salicylate caused significant suppression of 35S-glycosaminoglycan (GAG) synthesis, whereas tiaprofenic acid, piroxicam, prednisolone sodium phosphate and several other NSAIDs were without effect. Trypsin-mediated proteoglycan depletion did not change the susceptibility of the articular chondrocyte to these drugs. Similarly, no enhancement of drug effect was seen when arthritic cartilage was taken from an acutely inflamed joint, and prenisolone sodium phosphate even seemed to diminish inflammation-mediated suppression of 35S-GAG synthesis. The short term in vivo effects of some of the drugs were tested in mice with unilateral zymosan-induced arthritis. At day 1 after arthritis induction, in vivo 35S-GAG synthesis by the cartilage of the arthritic joint was decreased to 63%. Only sodium salicylate suppressed in vivo 35S-GAG synthesis in the healthy and arthritic joint to the same extent in both. At day 28, GAG synthesis in the postarthritic joint was enhanced to 160%. This type of cartilage appeared to be more susceptible to drug effects, since all drugs tested showed clear suppression of the augmented GAG production in vivo. Finally, in vivo drug effects were tested on normal and enhanced 35S-GAG degradation, the latter in the zymosan-induced arthritic joint. Both tiaprofenic acid and prednisolone sodium phosphate appeared to suppress degradation in healthy and, to some extent, in arthritic cartilage.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of glycosaminoglycan synthesis in anatomically intact rat patellar cartilage by paracetamol-induced serum sulfate depletion.

We have studied the effect of low sulfate concentrations on the glycosaminoglycan synthesis in rat patellar cartilage in vivo as well as in vitro. The oral administration of 200 mg/kg paracetamol to male Wistar rats resulted in a significant reduction of the serum sulfate concentration. Reduced serum sulfate availability resulted in a 34% decrease of glycosaminoglycan synthesis in patellar cartilage. This is due to sulfate depletion since paracetamol had no direct effects on glycosaminoglycan synthesis and a slight but significant inhibitory effect on the catabolism of radiolabeled glycosaminoglycans in vitro. The glycosaminoglycans synthesized at low sulfate concentrations in vivo were similar to the glycosaminoglycans synthesized at physiological sulfate concentrations. Studying the effect of sulfate availability in vitro on glycosaminoglycan synthesis in patellar cartilage we found that incubation of rat patellae in medium containing less than 0.5 mM inorganic sulfate led to a decreased sulfate incorporation. The use of potential sulfate decreasing drugs can lead to inhibition of glycosaminoglycan synthesis. This argues for a reconsideration of the use of these drugs in patients with already dysfunctioning cartilage metabolism as in rheumatoid arthritis and osteoarthrosis.

Effects of antirheumatic agents on cytokines.

A review of the literature concerning the effects of traditional antirheumatic drugs on cytokines and the cytokine and anticytokine approaches already used in the therapy of rheumatoid arthritis (RA) is presented. Many antirheumatic drugs are capable of cytokine modulation in vitro. Corticosteroids inhibit the transcription of a broad spectrum of genes including those encoding monocyte, T cell-derived cytokines and several hemopoietic growth factors, whereas drugs such as cyclosporin A and D-penicillamine interfere with T cell activation more specifically by suppressing interleukin 2 (IL-2) production. The in vivo effects of drug therapy on cytokines in RA patients are less well established. Gold compounds reduce circulating IL-6 levels and the expression of monocyte-derived cytokines, such as IL-1, tumor necrosis factor (TNF), and IL-6, in the rheumatoid synovium. Decreases in circulating IL-6, soluble IL-2 (sIL-2R), and TNF receptors and in synovial fluid IL-1 levels have been reported with methotrexate. Reductions in circulating IL-6 and sIL-2R concentrations have also been observed with cyclosporin and corticosteroids, whereas azathioprine reduces IL-6 but not sIL-2R. Studies on sulfasalazine are conflicting and the in vivo effects of D-penicillamine and antimalarials have not been studied yet. Interferon gamma therapy is not effective in RA but may prove a useful antifibrotic for systemic sclerosis. Colony stimulating factors improve the granulocytopenia associated with Felty's syndrome or drug toxicities but can induce arthritis flares and should be reserved to treat infectious complications. Promising results are being obtained with selective antagonism of TNF and IL-1 in RA, and combinations of anticytokine strategies with traditional antirheumatic drugs have been already envisaged. These should preferably be based in a broader knowledge of the effects of antirheumatic agents on the cytokine network.

Infections during low-dose methotrexate treatment in rheumatoid arthritis.

We studied the infection rate in patients with rheumatoid arthritis (RA) treated with low-dose methotrexate (MTX) in a 6-year open prospective study and in a 12-month randomized double blind trial comparing MTX with azathioprine (AZA) that was followed by a 3-year open prospective study. The literature on infections during low dose MTX in RA was reviewed. We also did a search for therapy-related opportunistic infections in RA and in MTX-treated psoriasis and psoriatic arthropathy patients. In our studies the infection rate during MTX treatment was higher in severe RA than in moderate RA. In severe RA there were often 2 infections simultaneously. The majority of the infections occurred in the first 1.5 years of treatment. There was no difference in the infection rate of MTX and AZA in the comparative trial. In the literature the infection rate was highest in short-term double-blind studies. Opportunistic infections are increasingly reported in RA treated with MTX and rarely with AZA, cyclosporine A, and cyclophosphamide or in MTX treated psoriasis and psoriatic arthropathy. In RA it appears that the initial period of treatment with MTX is the most vulnerable phase for infections, with the exception of opportunistic infections, which are not limited to a certain treatment period. Probably there are more MTX-associated infections in severe RA than in moderate RA.

Second-line treatment in seronegative spondylarthropathies.

The literature concerning second-line treatment of seronegative spondylarethropathies from 1940 to August 1993 was reviewed. Sulfasalazine appeared to be effective in the treatment of ankylosing spondylitis (AS) and promising in reactive arthritis (ReA) and Reiters' syndrome (RS). Methotrexate and azathioprine were associated with a remarkable improvement in some cases of AS and RS. Methylprednisolone and levamisole were both efficacious in AS, but levamisole was associated with occasional severe side effects. Radiation therapy led to short-term improvement in AS, but was abandoned because of severe long-term side effects. Only sulfasalazine has been studied in sufficient detail to allow definitive conclusions, but methotrexate and azathioprine may be promising drugs.