Acta Pharmacologica Sinica 2006 December; 27 (12): 1616-1621; doi: 10.1111/j.1745-7254.2006.00457.x

Aim: (5R)-5-hydroxytriptolide (LLDT-8) displayed anti-arthritis and anti-allogenic transplantation rejection activities in our previous studies. Here, we aim to further clarify the effect of LLDT-8 on the pro-inflammatory cytokine IFN-γ.

Methods: T cells were activated with anti-CD3 antibody or concanavalin A (ConA). The expression of cell surface molecules was detected with flow cytometry. Cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) to test cell division. IFN-γ production was determined by enzyme-linked immunosorbent assay. Cell proliferation was evaluated by [³H]-thymidine uptake. Mice were immunized with ovalbumin to assess the in vivo immune response. RT-PCR and Real-time PCR were applied to determine the mRNA expression. The protein phosphorylation levels were detected by Western immunoblot assay.

Results: LLDT-8 at 100 nmol/L did not change the CD25, CD69, and CD154 expressions in anti-CD3-stimulated T cells. LLDT-8 markedly blocked the cell division of CD4 and CD8 T cells after ConA stimulation. LLDT-8 inhibited T cell-derived IFN-γ production. Moreover, LLDT-8 suppressed the ovalbumin-specific T cell proliferation and IFN-γ generation. In anti-CD3-activated T cells, LLDT-8 abrogated the mRNA expression of signal transducer and activator of transcription1 (STAT1), T-box transcription factor, IL-12 receptor b2, STAT4, and interferon regulatory factor 1 in the IFN-γ expression pathway. Western blot analysis showed that LLDT-8 blocked the phosphorylation levels of extracellular signal-regulated kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase in anti-CD3 plus anti-CD28-activated T cells. In addition, LLDT-8 reduced the transcripts of macrophage inflammatory protein (Mip)-1a, Mip-1b, regulated upon activation normally T-cell expressed and secreted, inducible protein-10, IFN-inducible T cell a chemoattractant, and monokine induced by IFN-γ in IFN-γ-stimulated murine macrophage cell line Raw 264.7 cells.

Conclusion: LLDT-8 was a potential inhibitor for IFN-γ-associated signaling.

Keywords: (5R)-5-hydroxytriptolide; IFN-γ; MAPK; chemokine