FK506 enhances triptolide-induced down-regulation of cyclooxygenase-2, inducible nitric oxide synthase as well as their products PGE2 and NO in TNF-alpha-stimulated synovial fibroblasts from rheumatoid arthritic patients.
OBJECTIVE
To explore the effects of FK506 on the inhibition of cell proliferation and the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and their products PGE subset2 and NO in TNF-alpha-stimulated human rheumatoid arthritis synovial fibroblasts (RASF) treated with triptolide (TP), and to study the mechanisms involved when combining FK506 and TP in RA therapy.


METHODS
RASF used in the experiments were obtained from the synovial tissue of patients with RA before being cultured. RASF were pretreated with FK506 (10 approximately 1000 nM) for 2 hours before being stimulated with TNF-alpha (20 ng/ml) in the presence or absence of TP (10 ng/ml) . RASF proliferation was determined by [3 supersetH]-TdR incorporation. Production of PGE subset2 and NO in culture supernatants of RASF was detected by competitive ELISA and enzymatic reduction of nitrate, respectively. Expressions of COX-2 and iNOS mRNA in RASF were analyzed by semi-quantitative RT-PCR. Expressions of COX-2 and iNOS protein were estimated by Western-blot and a cellular enzyme immunoassay. NFkappaB activity in whole-cell extract of treated RASF was also measured using an ELISA-based method.


RESULTS
Neither FK506 nor TP at a lower concentration (10 ng/ml) affected TNF-alpha-induced COX-2 and iNOS expressions or PGE subset2 and NO productions in synovial cells. Combined treatment of FK506 and a lower concentration of TP (10 ng/ml) reduced both COX-2 and iNOS mRNA and protein expression, and correspondingly reduced PGE subset2 and NO produced by synovial fibroblasts. This effect was highly correlated with FK506 concentration (10 approximately 1000 nM). NFkappaB activity in TNF-alpha-stimulated synovial cells was suppressed more profoundly by FK506 plus TP (10 ng/ml) than by TP (10 ng/ml) alone. However, no change was observed regarding the inhibition of synovial cell proliferation after combined treatment of FK506 and TP.


CONCLUSIONS
FK506 enhanced TP-mediated down-regulation of COX-2 and iNOS as well as their products PGE subset2 and NO in human TNF-alpha-stimulated RASF by more profoundly suppressing the activity of NFkappaB.
Yao
H
H
Department of Dermatology and Allergology, Ruhr-University Bochum, St. Josef-Hospital, Gudrunstr. 56, D-44791 Bochum, Germany.
Zhou
J
J
Li
D
D
Wu
N
N
Bader
A
A
Höxtermann
S
S
Altmeyer
P
P
Brockmeyer
Norbert H
NH
D001172:Arthritis, Rheumatoid / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D001483:Base Sequence; D002460:Cell Line; D051546:Cyclooxygenase 2; D017931:DNA Primers; D015232:Dinoprostone / Q000378:metabolism*; D004224:Diterpenes / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects*; D004357:Drug Synergism; D004852:Epoxy Compounds; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008565:Membrane Proteins; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010616:Phenanthrenes / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D017931:DNA Primers; D004224:Diterpenes; D004852:Epoxy Compounds; D007166:Immunosuppressive Agents; D008565:Membrane Proteins; D010616:Phenanthrenes; D014409:Tumor Necrosis Factor-alpha; C001899:triptolide; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; D016559:Tacrolimus