Induction of inducible nitric oxide synthase, argininosuccinate synthase, and GTP cyclohydrolase I in arthritic joints of human tumor necrosis factor-alpha transgenic mice.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Hukkanen
Mika
M
Institute of Biomedicine/Anatomy, Biomedicum Helsinki, University of Helsinki and ORTON Research Institute, Orthopaedic Hospital of the Invalid Foundation, Finland. mika.hukkanen@helsinki.fi
Platts
Louise A M
LA
Haralambous
Sylva
S
Ainola
Mari
M
Konttinen
Yrjö T
YT
Kollias
George
G
Polak
Julia M
JM
D000818:Animals; D001124:Argininosuccinate Synthase / Q000235:genetics / Q000378:metabolism*; D001168:Arthritis / Q000378:metabolism* / Q000473:pathology; D006136:GTP Cyclohydrolase / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D009566:Nitrates / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D019683:Penetrance; D030421:Peroxynitrous Acid / Q000494:pharmacology; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis; D013583:Synovial Membrane / Q000201:enzymology / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D009566:Nitrates; D009573:Nitrites; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D006136:GTP Cyclohydrolase; D001124:Argininosuccinate Synthase