1 The aim of this study was to elucidate the in vitro inhibitory potency of FK506 on production of the in¯ammatory cytokines, tumour necrosis factor (TNF)-a and interleukin (IL)-1b, with a view to assessing this immunosuppressive agent as a potential anti-rheumatic drug. 2 We employed an in vitro model which produces TNF-a and IL-1b through T cell activation. Human peripheral blood mononuclear cells (PBMC) were cultured with immobilized anti-CD3/ CD28 monoclonal antibody in this model. 3 FK506 inhibited anti-CD3/CD28 induced TNF-a and IL-1b production at concentrations less than 1 ng ml 71 . Flow cytometric analysis of intracellular TNF-a and IL-1b positive cells showed that FK506 potently suppresses in¯ammatory cytokine production from CD14+ monocytes as well as from T cells. 4 Cyclosporin A (CsA) and dexamethasone (DEX) also inhibited the anti-CD3/CD28 induced cytokine production, but were less potent than FK506. FK506 and CsA, but not DEX, speci®cally inhibited anti-CD3/CD28 induced in¯ammatory cytokine production without a ecting the lipopolysaccaride (LPS) induced e ect. Methotrexate (MTX) was completely inactive for suppressing cytokine production under either condition. 5 Anti-CD3/CD28 stimulated PBMC culture supernatants were found to enhance the expression of adhesion molecules in human vascular endothelial cells. FK506, CsA and DEX led to the suppression of adhesion molecule expression probably by inhibiting cytokine production from PBMC. 6 The inhibitory potency of agents on TNF-a and IL-1b production was compared with cytotoxicity and FK506 was not cytotoxic at concentrations several orders of magnitude greater than those required for cytokine inhibition. 7 These results strongly suggest that FK506 may be most e ective to speci®cally prevent T cell activation mediated in¯ammatory cytokine production in a clinical setting.
1 FK506 and cyclosporin A (CsA) are immunosuppressive drugs, that specifically inhibit T-cell activation via calcineurin inhibition. This study was undertaken to investigate whether calcineurin inhibitors exert analgesic actions in rat adjuvant-induced arthritis (AIA), an animal model of rheumatoid arthritis (RA). 2 AIA was induced in female Lewis rats. Single doses of FK506 and CsA were orally administered to arthritic rats 17 days after arthritis induction. Intensity of hyperalgesia was assessed by measuring the pain threshold of hind paws. Tumor necrosis factor (TNF)-a, IL-1b and PGE 2 levels in paw extracts were determined by ELISA. TNF activity was measured by L929 cell cytotoxicity assay. IL-1b and cyclooxygenase (COX) mRNA expression in arthritic paws were measured by RT -PCR. 3 Single doses of FK506 and CsA markedly reduced joint hyperalgesia 24 h after drug administration, without affecting inflammation in an advanced stage of AIA. 4 The calcineurin inhibitors partially reduced the elevated level of TNF-a in arthritic paws, however, the analgesic effects of these drugs were not associated with the reduction in TNF-a level. 5 Moreover, treatment with anti-rat TNF-a antibody did not affect the hyperalgesia, when TNF-a activity was suppressed in arthritic paws by that treatment. 6 Both calcineurin inhibitors reduced the elevated level of IL-1b in arthritic paws to a normal level, 24 h after drug administration. 7 FK506 reduced IL-1b and COX-2 mRNA expression and PGE 2 level in arthritic paws. 8 In conclusion, calcineurin inhibitors rapidly reduce joint hyperalgesia probably by downregulating IL-1b, but not TNF-a, in AIA. Our findings may provide a new strategy for the treatment of pain in RA.
These results show that FK506 is effective in suppressing inflammation, TNF-alpha expression in joint, and damage to bone and cartilage in rat CIA, and may be useful in the treatment of rheumatoid arthritis.
LEF inhibited anti-CD3/CD28 induced inflammatory cytokine production in human PBMC at concentrations showing deleterious effects on bone marrow cell proliferation. LEF is superior to MXT in improving arthritis and joint function in established AIA, but is inferior to FK506 in recovering joint function, probably due to its anti-proliferative actions.
Levels of IL-1beta and IL-6, but not TNF-alpha , in paw tissue were upregulated in association with the development of arthritis in rat CIA. These results suggest that IL-1beta and IL-6, rather than TNF-alpha , may play important roles at local inflammatory sites in producing joint destruction in rat CIA. FK506 may improve arthritis in established stages of CIA, by reducing the elevated level of IL-6.
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