3-nitrotyrosine and Arthritis

Transgenic mice that express human tumor necrosis factor-alpha (Tg197 h-TNF-alpha) develop polyarthritis at 3 to 4 weeks of age leading to severe joint destruction at 8 to 10 weeks of age. Studies have suggested that inducible nitric oxide synthase (iNOS) activity can modulate the progression of arthritis. We investigated the induction of iNOS together with argininosuccinate synthase (AS) and GTP cyclohydrolase I (GTPCH), 2 of the rate-limiting enzymes for high output NO generation, in the Tg197 h-TNF-alpha transgenic model of arthritis.. We used 4 and 8-week-old Tg197 h-TNF-alpha transgenic mice and wild-type CBA C57B1/6 control mice to investigate the expression of iNOS with respect to that of AS, GTPCH, and 3-nitrotyrosine by quantitative RT-PCR and immunocytochemistry. Urinary NO metabolites were analyzed using a chemiluminescence assay.. Inducible NOS, AS, and GTPCH mRNA was found in all study groups; however, only iNOS mRNA showed a clear increase in 4-week-old Tg197 h-TNF-alpha transgenics in comparison to age matched wild-type controls. Abundant iNOS protein expression was found in macrophages and vascular smooth muscle cells in hyperplastic synovium and pannus. AS expression was found in vascular endothelium and fibroblasts of the inflammatory synovium and pannus. GTPCH immunoreactivity was mostly restricted to macrophages in inflammatory synovium. Localization of 3-nitrotyrosine overlapped with that of iNOS, indicating formation of reactive nitrogen species. Consistent with the high output NO generation, there was a 5-fold increase in urinary NO metabolites in 8-week-old Tg197 h-TNF-alpha transgenic mice.. We characterized the Tg197 h-TNF-alpha transgenic model of inflammatory arthritis in terms of high output NO-generating pathway, and showed that both AS and GTPCH are intimately associated with inflammatory arthritis. The concomitant induction of AS and GTPCH with that of iNOS suggests that they may be important modulators of arthritis, and that they may represent novel targets for modulation of disease activity.

Topics: Animals; Argininosuccinate Synthase; Arthritis; Gene Expression Regulation, Enzymologic; GTP Cyclohydrolase; Humans; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Oxidative Stress; Penetrance; Peroxynitrous Acid; Phenotype; RNA, Messenger; Synovial Membrane; Tumor Necrosis Factor-alpha; Tyrosine

1. The aim of the present study was to investigate the effects of cloricromene, a coumarine derivative, in rats subjected to collagen-induced arthritis. 2. Collagen-induced arthritis (CIA) was induced in Lewis rats by an intradermal injection of 100 microl of the emulsion (containing 100 microg of bovine type II collagen) (CII) and complete Freund's adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. 3. Lewis rats developed an erosive hind paw arthritis when immunized with CII in CFA. Macroscopic clinical evidence of CIA first appeared as peri-articular erythema and oedema in the hind paws. The incidence of CIA was 100% by day 27 in the CII challenged rats and the severity of CIA progressed over a 35-day period with radiographic evaluation revealing focal resorption of bone together with osteophyte formation in the tibiotarsal joint and soft tissue swelling. 4. The histopathology of CIA included erosion of the cartilage at the joint margins. Treatment of rats with cloricromene (10 mg kg(-1) i.p. daily) starting at the onset of arthritis (day 23), delayed the development of the clinical signs at days 24 - 35 and improved histological status in the knee and paw. 5. Immunohistochemical analysis for iNOS, COX-2, nitrotyrosine and for poly (ADP-ribose) synthetase (PARS) revealed a positive staining in inflamed joints from collagen-treated rats. The degree of staining for iNOS, COX-2, nitrotyrosine and PARS were markedly reduced in tissue sections obtained from collagen-treated rats, which had received cloricromene. 6. Radiographic signs of protection against bone resorption and osteophyte formation were present in the joints of cloricromene-treated rat. 7. This study provides the first evidence that cloricromene, a coumarine derivative, attenuates the degree of chronic inflammation and tissue damage associated with collagen-induced arthritis in the rat.

Topics: Animals; Arthritis; Body Weight; Cattle; Chromonar; Collagen; Cyclooxygenase 2; Disease Progression; Enzyme Activation; Hindlimb; Interleukin-1; Isoenzymes; Male; Malondialdehyde; Nitric Oxide; Poly(ADP-ribose) Polymerases; Prostaglandin-Endoperoxide Synthases; Radiography; Rats; Rats, Inbred Lew; Severity of Illness Index; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine

There is limited evidence that inhibition of the activity of the protease calpain I reduces inflammation. Here we investigate the effects of calpain inhibitor I in animal models of acute and chronic inflammation (carrageenan-induced pleurisy and collagen-induced arthritis). We report here for the first time that calpain inhibitor I (given at 5, 10, or 20 mg/kg i.p. in the pleurisy model or at 5 mg/kg i.p every 48 hours in the arthritis model) exerts potent anti-inflammatory effects (eg, inhibition of pleural exudate formation, mononuclear cell infiltration, delayed the development of the clinical signs and histological injury) in vivo. Furthermore, calpain inhibitor I reduced (1) the staining for nitrotyrosine and poly (ADP-ribose) polymerase (immunohistochemistry) and (2) the expression of inducible nitric oxide synthase and cyclooxygenase-2 in the lungs of carrageenan-treated rats and in joints from collagen-treated rats. Thus, prevention of the activation of calpain I reduces the development of acute and chronic inflammation. Inhibition of calpain I activity may represent a novel therapeutic approach for the therapy of inflammation.

Topics: Acute Disease; Animals; Arthritis; Carrageenan; Chronic Disease; Collagen; Cyclooxygenase 2; Cysteine Proteinase Inhibitors; Glycoproteins; Isoenzymes; Lung; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pleurisy; Poly Adenosine Diphosphate Ribose; Prostaglandin-Endoperoxide Synthases; Radiography; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Tarsus, Animal; Tyrosine