Ralf Miesel

Elevated levels of xanthine oxidase in serum of patients with inflammatory and autoimmune rheumatic diseases

Sera of patients with various inflammatory and autoimmune rheumatic diseases were screened for the presence of xanthine oxidase (XOD) and compared to sera from healthy donors and patients with nonrheumatic diseases including AIDS, internal diseases, and different carcinomas. Up to 50-fold higher levels of XOD were detected in rheumatic sera (P < 0.001). In addition, serum sulfhydryls (SH) were determined as sensitive markers of oxidative stress. The SH status in rheumatic patients was diminished by 45–75% (P < 0.001) and inversely correlated to the concentration of serum XOD (R=0.73), suggesting a causal interrelation. The depletion of serum sulfhydryls by the oxyradical-producing XOD/acetaldehyde system was mimicked successfully ex vivo in human serum from healthy donors. Cortisone treatment of patients suffering from systemic lupus erythematosus and rheumatoid arthritis impressively normalized elevated XOD concentrations in rheumatic sera to those of healthy controls. The participation of xanthine oxidase in the depletion of serum antioxidants in rheumatic patients is discussed in the light of substrate availability andKm values.

Copper-dependent antioxidase defenses in inflammatory and autoimmune rheumatic diseases

Gel-filtered sera of patients with various inflammatory and autoimmune rheumatic diseases (N=354) were screened for the presence of the inflammation marker Cu-thionein. The concentrations of Cu-thionein were significantly diminished in patients with connective tissue diseases (P < 0.001). Sera of patients suffering from inflammatory rheumatic diseases were almost totally depleted of this low-molecular-weight copper protein that exerts pronounced Superoxide dismutase activity and scavenges effectively hydroxyl radicals and singlet oxygen. Cortisone treatment of patients with rheumatoid arthritis, systemic lupus erythematosus, and polymyalgia rheumatica replenished impressively the serum concentration of Cu-thionein. The partial oxidation of the EPR-silent Cu(I)-chromophore to Cu(II)/Cu(I)-thionein, which is essential for the caalytic dismutation of Superoxide, was monitored by electron paramagnetic resonance in the presence of activated neutrophils and monocytes. Release of Cu-thionein during the oxidative burst of peripheral blood monocytes was demonstrated in vitro. The role of prooxidant-antioxidant imbalances in the pathogenesis of rheumatic diseases is discussed.

Modulation of inflammatory arthritis by inhibition of poly(ADP ribose) polymerase

Poly(ADPR) polymerase (PARP; EC 2.4.2.30) is a nuclear enzyme, which, when activated by oxygen- and nitrogen-radical-induced DNA strand breaks, transfers ADP ribose units to nuclear proteins and initiates apoptosis by depletion of cellular NAD and ATP pools. The present study investigates whether the oxidative stressdependent activation of PARP plays a role in the etiopathogenesis of arthritis. The antiarthritic reactivity of the biogenic PARP inhibitor nicotinamide was tested in DBA/1 × B10A(4R) mice suffering from potassium peroxochromate-induced arthritis. Daily doses of 4 mmol/kg of NA suppressed the arthritis by 35% and inhibited the phagocytic generation of reactive oxygen species, which increases sixfold during the development of arthritis. The onset, progression, and remission of arthritis correlated positively to the phorbolester-activated respiratory burst of neutrophils and monocytes, and a dose-dependent inhibition of NADPH oxidase activity was determined with human phagocytes. Our data support the hypothesis that oxidative stressinduced alterations in cellular signal transduction pathways play a pivotal role in the development of arthritis, which can be suppressed by the simultaneous inhibition of poly(ADPR) polymerase and NADPH oxidase.

Synergistic effects of thalidomide and poly (ADP-ribose) polymerase inhibition on type II collagen-induced arthritis in mice.

The present study investigates synergistic effects of the TNF-α inhibitor thalidomide and the poly(ADP-ribose) polymerase (PARP)-inhibitor nicotinic acid amide (NAA) in male DBA/1 hybrid mice suffering from type II collagen-induced arthritis. Parameters including the arthritis index, chemiluminescence and anti-collagen antibody titers were used for the assessment of disease activity: The disease courses demonstrated clearly an inhibitory effect of thalidomide. NAA inhibited established collagen arthritis in a dose-dependent manner. The combined application of thalidomide and NAA caused a powerful synergistic inhibition of arthritis. Furthermore, thalidomide and NAA were tested ex vivo for their inhibition of the NADPH oxidase-dependent generation of reactive oxygen species by activated neutrophils and monocytes in unseparated human blood. Our data show that type II collagen-induced arthritis can be suppressed by the simultaneous inhibition of TNF-α, PARP, and NADPH oxidase.

Antiinflammatory effects of nadph oxidase inhibitors

Proinflammatory cytokines prime the membrane-bound NADPH oxidase of neutrophils and monocytes of mice suffering from experimental arthritis so as to attain an activated state, which, upon a second stimulus, releases 6-fold increased levels of reactive oxygen species (ROS) than do unprimed phagocytes. Enhanced NADPH oxidase activity deregulates ROS-dependent signal transduction pathways of inflammation, which play a crucial role in the pathogenesis of arthritis. The antiarthritic reactivity of two inhibitors of NADPH oxidase, diphenylene iodoniumchloride (DPI) and stauroporine, was tested in male DBA/1 × B10A(4R) hybrid mice suffering from potassium peroxochromate arthritis. Daily doses of 2.8 μmol/kg of DPI or 30 nmol/kg of staurosporine sufficed to inhibit the arthritis by 50%. A complete inhibition was obtained with 10 μmol/kg of DPI, and 100 nmol/kg of stauroporine suppressed the arthritis by 85%. The onset, progression, and remission of arthritis correlated to both the activity of phagocytic NADPH oxidase (r=0.750) and to overt disease symptoms as judged by the arthritis index. Our data support the hypothesis that oxidative stress plays a pivotal role in the pathology of arthritis, which can be therapeutically targeted by NADPH oxidase inhibitors.

Reactivity of active centre analogues of Cu2Zn2-superoxide dismutase during the aqueous decay of K3CrO8

Abstract The aqueous decay of K3CrO8 was used to compare the reactivity of Cu2Zn2-superoxide dismutase and two active centre analogues where the first shell atoms around the copper are four unsaturated nitrogens. Unlike the acetate- or biuret-type Cu(II) chelates these di-Schiff base complexes had an identical reactivity compared to that of the intact enzyme. Nanomolar concentrations of copper coordinated in these complexes were sufficient to inhibit the K3CrO8 induced chemiluminescence by 50%. They were also able to inhibit the hyaluronic acid degradation successfully by 27%.

Effects of allopurinol on in vivo suppression of arthritis in mice and ex vivo modulation of phagocytic production of oxygen radicals in whole human blood

Recently, we demonstrated elevated levels of xanthine oxidase in serum of patients with various inflammatory and autoimmune rheumatic diseases. The present study reports the antiarthritic efficacy of the xanthine oxidase inhibitor and immunosuppressant allopurinol in DBA/1xB10A(4r) mice suffering from peroxochromateinduced arthritis. A profound dose-dependent suppression of arthritis was noted (P <0.001). The ED50 was 80±14μmol/kg/day. The arthritis index correlated positively to the phagocytic production of oxygen radicals (r2>0.672) and negatively to the concentration of allopurinol (r2=0.915). Ex vivo, allopurinol and various conventional antirheumatic drugs were screened for the inhibition of 12-otetradecanoylphorbol-13-acetate-stimulated whole human blood chemiluminescence. The concentrations of antirheumatic drugs required to inhibit the chemiluminescence by 50% were compared to the therapeutic doses administered to rheumatic patients. Whiled-penicillamine andcis-platinum(II) increased the phagocytic generation of superoxide, nonsteroidal antiinflammatory drugs (NSAIDs), steroids, and slow-acting antirheumatic drugs (SAARDs) inhibited the whole blood chemiluminescence in a dose-dependent manner. Therapeutic doses of NSAIDs, SAARDs, or steroids inhibited the phagocytic generation of reactive oxygen species by 10–50%. In addition to well-known mechanisms of action of NSAIDs and SAARDs, our results support the hypothesis that most common anti-rheumatic drugs act also by modulating the levels of reactive oxygen species, which serve important mediator and signal transduction functions in inflammatory and autoimmune diseases. Pharmacologically safe antioxidants like allopurinol, which simultaneously modify the oxidative burst of phagocytes, inhibit xanthine oxidase, and display immunosuppressive effects may well be suited to control the consequences of chronic phagocytic hyperreactivity in rheumatic patients.

Antiinflammatory reactivity of copper(I)-thionein

In unseparated human blood the reactivity of yeast copper (I)-thionein on TPA-activated polymorphonuclear leukocytes was evaluated and compared with low Mr copper chelates exerting Cu2Zn2 superoxide dismutase mimetic activity. Cu, 18ΜM, in the form of Cu-thionein was sufficient to inhibit the Superoxide production of activated human blood phagocytes by 50%. Furthermore, the scavenging of hydroxyl radicals and singlet oxygen by Cu(I)-thionein was determined, using the 2-deoxyribose fragmentation assay induced by decaying K3CrO8 and the NADPH oxidation caused by UVA illuminated psoralen, respectively. The inhibitory reactivity of Cu-thionein in both assays was compared with that of serum proteins including albumin, ceruloplasmin, transferrin, and ferritin. The galactosamine/endotoxininduced hepatitis in male NMRI mice was used to evaluate the antiinflammatory reactivity of Cu-thionein in vivo. The serum copper, superoxide dismutase, and sorbitol dehydrogenase concentrations, as well as the activity of polymorphonuclear leukocytes in unseparated blood seemed most appropriate to quantify the protective capacity of Cu-thionein in the course of an oxidative stress-dependent liver injury. The intraperitoneal application of 32.5 Μmol/kg thionein-Cu limited this damage to 45%.

Assessment of Collagen Type II Induced Arthritis in Mice by Whole Blood Chemiluminescence

Lucigenin-enhanced chemiluminescence can be measured in 100 microliters samples of whole, unseparated mouse blood. A procedure for doing so is here described in detail, using a standard clinical luminometer. The assay measures the TPA-induced oxidative burst from granulocytes and macrophages, which is believed to depend on the overall level of inflammation in the body. It is here applied to mice suffering from type II collagen-induced arthritis, and its relation to overt disease symptoms (the arthritis score) is characterised during the course of the disease. A correlation between the assay and the arthritis score is found at the height of the disease (r = 0.42, p = .039), but not at early or very late time points, although there is a strong hint that the results of an early assay may predict the subsequent disease course. The assay provides a rapid, convenient, quantitative and economical method of assessing disease activity, which can be carried out repeatedly on the same individual. It should be applicable in other mouse models of chronic inflammatory disease. It may find application for rapid screening of novel anti-rheumatic drugs and treatments.

Induction of arthritis in mice and rats by potassium peroxochromate and assessment of disease activity by whole blood chemiluminescence and 99mpertechnetate-imaging.

Arthritis develops in DBA/1xB10A(4R) mice and Wistar rats upon intraplantar injection of potassium peroxochromate (K3CrO8), and is here quantified by whole blood chemiluminescence (CL) and 99mpertechnetate-imaging (99mTcO4-), and related to overt disease symptoms (the arthritis index). During the aqueous decay of K3CrO8 to chromate (VI), the chromium(V)-bound oxygen is released as superoxide, hydroxyl radicals, singlet oxygen and hydrogen peroxide, the same reactants, which are produced by activated phagocytes during inflammation. Reactive oxygen species (ROS) trigger the breakdown of the sulfhydryl-dependent antioxidant defence system and induce the nuclear factor kappa B-dependent expression of pro-inflammatory cytokines, which prime phagocytic NADPH oxidases to the enhanced production of ROS. During both the acute inflammatory response and the onset of the secondary response in non-injected paws, the phorbolester-stimulated ROS production of phagocytes was significantly enhanced (p < 0.001) and correlated well to the arthritis index (r = 0.797) and the uptake of 99mTcO4- into inflamed joints. Chromate(VI), formed during the decay of K3CrO8, contributes to the progression of arthritis by inhibition of glutathione reductase, thereby increasing intracellular H2O2 concentrations. In addition, Cr(VI) reduced to Cr(V) by ascorbate, catalyzes hydroxyl radical production in the presence of hydrogen peroxide. A stable loop forms, in which ROS, continuously produced by Cr(VI)/Cr(V) redox-cycling, drive the primary response into chronic self-perpetuating inflammation. We see the main application of K3CrO8-induced arthritis and its assessment by both 99mTcO4- imaging and chemiluminescent immunosensoring of phagocytic activity in unseparated blood as for the rapid screening of novel anti-rheumatic drugs and treatments.