Cyclosporin A and tacrolimus (FK506) suppress expression of inducible nitric oxide synthase in vitro by different mechanisms

1 The effects of the immunosuppressant drugs cyclosporin A and tacrolimus ( FK506) on nitric oxide synthesis were examined in a murine macrophage cell line (J774) and rat vascular smooth muscle cells (VSMC) in culture for 24 a nd 48 h, respectively. 2 Cyclosporin A (0.01-10 mM) inhibited by up to 90% accumulation of nitrite induced by lipopolysaccharide (LPS) in both cell lines: but FK506 (0.01-10 mu M) had a weaker effect on nitrite accumulation in these cells. Cyclospo rin A and FK506 (at 1 mu M) also significantly inhibited nitrite production induced by recombinant murine interferon-gamma (rIFN gamma) and recombinan t murine interleukin-1 beta (rIL-1 beta) in J774 and VSMC, respectively. 3 In J774 cells, cyclosporin A (but not FK506) at 1 mu M was inhibitory whe n co-incubated with the inducing agents but not when the cells were treated with the immunosuppressant before or after the inducer. In VSMC, nitrite p roduction was inhibited by co-incubation of cyclosporin A or FK506 with the inducer, or when the immunosuppressants were pre-incubated with cells. In contrast, N-monomethyl L-arginine (NMMA) abolished nitrite production when incubated with either cell type during or after addition of inducing agent, but not if cells were preincubated with NMMA. 4 RNA extracted from treated J774 and VSMC was subjected to reverse transcr iption-polymerase chain reaction (RT-PCR). Cyclosporin A, but not FK506, su ppressed expression of mRNA for NOS2 in a concentration-dependent manner wh en co-incubated with LPS. 5 The fact that the potency difference between cyclosporin A and FK506 for NO suppression is the opposite to that for inhibition of interleukin-2 gene ration suggests that the immunosuppressants act in J774 macrophages and VSM C through intracellular mechanisms that differ from those elucidated in T-c ells. Cyclosporin A suppresses NOS2 gene transcription, but FK506 acts post -transcriptionally to suppress NO generation in VSMC. 6 Taken together the present data suggest that therapeutic concentrations o f cyclosporin A, but not FK506, might well suppress NO production, but FK50 6 would not have this effect. Suppression of NO might contribute to the sid e effects of hypertension and nephrotoxicity associated with longterm use o f cyclosporin A to prevent transplant rejection.