Immunopharmacology and Anti-tumor Pharmacology

(5R)-5-hydroxytriptolide inhibits IFN-γ-related signaling

Abstract

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 transcription 1 (STAT1), T-box transcription factor, IL-12 receptor β2, 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)-1α, Mip-1β, regulated upon activation normally T-cell expressed and secreted, induc-ible 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.

References

  1. 1

    Grogan JL, Locksley RM . T helper cell differentiation: on again, off again. Curr Opin Immunol 2002; 14: 366–72.

  2. 2

    Schroder K, Hertzog PJ, Ravasi T, Hume DA . Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol 2004; 75: 163–89.

  3. 3

    Chang L, Karin M . Mammalian MAP kinase signalling cascades. Nature 2001; 410: 37–40.

  4. 4

    Ip YT, Davis RJ . Signal transduction by the c-Jun N-terminal kinase (JNK) — from inflammation to development. Curr Opin Cell Biol 1998; 10: 205–19.

  5. 5

    Dong C, Davis RJ, Flavell RA . MAP kinases in the immune response. Annu Rev Immunol 2002; 20: 55–72.

  6. 6

    Rincon M, Flavell RA, Davis RA . The JNK and p38 MAP kinase signaling pathways in T cell-mediated immune responses. Free Radic Biol Med 2000; 28: 1328–37.

  7. 7

    Tang W, Yang Y, Zhang F, Li YC, Zhou R, Wang JX, et al. Prevention of graft-versus-host disease by a novel immunosuppressant, (5R)-5-hydroxytriptolide (LLDT-8), through expansion of regulatory T cells. Int Immunopharmacol 2005; 5: 1904–13.

  8. 8

    Tang W, Zhou R, Yang Y, Li YC, Yang YF, Zuo JP . Suppression of (5R)-5-hydroxytriptolide (LLDT-8) on allograft rejection in full MHC-mismatched mouse cardiac transplantation. Transplantation 2006; 81: 927–33.

  9. 9

    Zhou R, Tang W, Ren YX, He PL, Zhang F, Shi LP, et al. (5R)-5-hydroxytriptolide (LLDT-8) attenuated collagen-induced arthritis in DBA/1 mice via suppressing IFN-γ production and its related signaling. J Pharmacol Exp Ther 2006; 318: 35–44.

  10. 10

    Zhou R, Tang W, Ren YX, He PL, Yang YF, Li YC, et al. Preventive effects of (5R)-5-hydroxytriptolide on concanavalin A-induced hepatitis. Eur J Pharmacol 2006; 537: 181–9.

  11. 11

    Zhou R, Zhang F, He PL, Zhou WL, Wu QL, Xu JY, et al. (5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide analog mediates immunosuppressive effects in vitro and in vivo. Int Immunopharmacol 2005; 5: 1895–903.

  12. 12

    Yang YF, Mukai T, Gao P, Yamaguchi N, Ono S, Iwaki H, et al. A non-peptide CCR5 antagonist inhibits collagen-induced arthritis by modulating T cell migration without affecting anti-collagen T cell responses. Eur J Immunol 2002; 32: 2124–32.

  13. 13

    Zhou R, Zheng SX, Tang W, He PL, Li XY, Yang YF, et al. Inhibition of inducible nitric-oxide synthase expression by (5R)-5-hydroxytriptolide (LLDT-8) in interferon-{gamma} and bacterial lipopolysaccharide stimulated macrophages. J Pharmacol Exp Ther 2006; 316: 121–8.

  14. 14

    Yang DD, Conze D, Whitmarsh AJ, Barrett T, Davis RJ, Rincon M, et al. Diferentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2. Immunity 1998; 9: 575–85.

  15. 15

    Dong C, Davis RJ, Flavell RA . Signaling by the JNK group of MAP kinases. c-jun N-terminal kinase. J Clin Immunol 2001; 21: 253–7.

  16. 16

    Lu HT, Yang DD, Wysk M, Gatti E, Mellman I, Davis RJ, et al. Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (Mkk3)-deficient mice. EMBO J 1999; 18: 1845–57.

  17. 17

    Rincon M, Enslen H, Raingeaud J, Recht M, Zapton T, Su MS, et al. Interferon-gamma expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway. Embo J 1998; 17: 2817–29.

  18. 18

    Gil MP, Bohn E, O'Guin AK, Ramana CV, Levine B, Stark GR, et al. Biologic consequences of Stat1-independent IFN signaling. Proc Natl Acad Sci USA 2001; 98: 6680–5.

  19. 19

    Hamilton NH, Banyer JL, Hapel AJ, Mahalingam S, Ramsay AJ, Ramshaw IA, et al. IFN-gamma regulates murine interferon-inducible T cell alpha chemokine (I-TAC) expression in dendritic cell lines and during experimental autoimmune encephalo-myelitis (EAE). Scand J Immunol 2002; 55: 171–7.

  20. 20

    Nazar AS, Cheng G, Shin HS, Brothers PN, Dhib-Jalbut S, Shin ML, et al. Induction of IP-10 chemokine promoter by measles virus: comparison with interferon-gamma shows the use of the same response element but with differential DNA-protein binding profiles. J Neuroimmunol 1997; 77: 116–27.

Download references

Author information

Correspondence to Jian-ping Zuo.

Additional information

Project supported by a grant from The Knowledge Innovation Program of the Chinese Academy of Sciences (No KSCX2-SW-202).

Rights and permissions

Reprints and Permissions

About this article

Keywords

  • (5R)-5-hydroxytriptolide
  • IFN-γ
  • MAPK
  • chemokine

Further reading