A novel anti-fibrotic agent pirfenidone suppresses tumor necrosis factor-α at the translational level
Introduction
In the primary immune response to infection or injury, macrophages synthesize proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6, and interferon-γ, which play important roles in promoting inflammatory processes Van Deuren et al., 1992, Bendtzen, 1988. The proinflammatory cytokines are highly expressed in the injured tissues and induced when macrophages are exposed to Gram-negative bacterial endotoxins Van Deuren et al., 1992, Bendtzen, 1988, Blackwell and Christman, 1996, Ozmen et al., 1994. Therefore, cytokine synthesis inhibitors prevent the immune response to invasive stimuli.
The proinflammatory cytokines are involved in the pathogenesis of fibrosis. The series of events can be divided into three stages: (1) the inflammatory process Piguet et al., 1989, Piguet et al., 1990, Piguet et al., 1993; (2) tissue injury; and (3) the subsequent restoration process of woundhealing Piguet et al., 1989, Khalil et al., 1996, Yoshida et al., 1995). The inflammatory process occurs with the production of proinflammatory cytokines, such as TNF-α. TNF-α was highly expressed in the lungs of mice, which had received bleomycin (Piguet et al., 1989). Therefore, the cytokine synthesis inhibitors have anti-fiborosis activity.
Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is currently being evaluated as an anti-fibrotic agent. Pirfenidone can prevent formation of experimentally induced fibrotic lesions, and reduce or remove excessive fibrotic lesions or scar tissue Iyer et al., 1995, Shimizu et al., 1996. In the progression of fibrosis, the proinflammatory cytokines continuously cause tissue inflammation and overexpression of collagen occurs due to an excessive response of repairing injured tissue Zhang and Phan, 1996, van den Blink et al., 2000, Selman et al., 2001. Previous studies showed that pirfenidone downregulates the proinflammatory cytokine expression, fibroblast proliferation, and collagen matrix synthesis Cain et al., 1998, Gurujeyalakshmi et al., 1999. Therefore, studies of the cytokine inhibition mechanism by pirfenidone are important for clarifying the effects of this drug on the mechanisms of immune system and fibrosis. However, the detailed mechanism remains to be elucidated.
In the present study, we show that the suppression of proinflamatory cytokines by lipopolysaccharide is specific, translational, and independent of mitogen-activated protein kinase (MAPK) 2, p38 MAP kinase and c-Jun N-terminal kinase (JNK). Because of the posttranscriptional inhibition, pirfenidone inhibits cytokine production even when cytokine transcription becomes fully activated by lipopolysaccharide. In the in vivo application of pirfenidone, cytokine inhibition is posttranscriptional and independent of MAPK2 as in vitro, and it can enhance the production of the protective interleukin-10.
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Animals and reagents
Female C57BL/6 mice (7–11 weeks) were purchased from Charles River Japan (Osaka, Japan). Lipopolysaccharide from Escherichia coli strain O55:B5 prepared by Boivon's method was obtained from Difco Laboratories (Detroit, MI). d-Galactosamine hydrochrolyde was purchased from Tokyo Chemicals (Tokyo, Japan). Actinomycin D was obtained from Sigma (St. Louis, MO). Multigel and immobilon polyvinylidene difluoride (PVDF) transfer membrane were purchased from Daiichi Pure Chemicals (Tokyo, Japan). RPMI
Pirfenidone suppression of TNF-α is translational
To investigate the mechanism of pirfenidone action on cytokine synthesis, we used the murine macrophage-like cell line RAW264.7. First, we determined whether pirfenidone inhibits TNF-α secretion or expression when RAW264.7 cells were exposed to lipopolysaccharide for 8 h. Exposure of RAW264.7 cells to pirfenidone significantly suppressed the levels of both cell-associated and secreted TNF-α by ELISA (Fig. 1A). But pirfenidone did not affect RAW264.7 cell viability (data not shown).
We then
Discussion
In the primary immune response, the inflammation is regulated by cytokine induction and function. The proinflammatory cytokines, such as TNF-α, interferon-γ, and interleukin-6, are highly expressed in the inflammatory tissue site, and induced when macrophages are exposed to Gram-negative bacterial endotoxins Van Deuren et al., 1992, Bendtzen, 1988, Blackwell and Christman, 1996, Ozmen et al., 1994. On the other hand, the anti-inflammatory cytokines, such as interleukin-10, suppress induction of
Acknowledgements
The authors thank Dr. K. Shirai and Dr. T. Sakata of Shionogi for their useful suggestions and expert technical assistance.
References (37)
- et al.
Number and location of AUUUA motifs: role in regulating transiently expressed RNAs
Blood
(1994) - et al.
Signalling to translational activation of tumour necrosis factor-alpha expression in human THP-1 cells
Cytokine
(2000) - et al.
Sepsis and cytokines: current status
Brit. J. Anesthesia
(1996) - et al.
Inhibition of tumor necrosis factor and subsequent endotoxin shock by pirfenidone
Int. J. Immunopharmacol.
(1998) - et al.
AUF1 binding affinity to A+U-rich elements correlates with rapid mRNA degradation
J. Biol. Chem.
(1996) New twists in gene regulation by glucocorticoid receptor: is DNA binding dispensable?
Cell
(1998)- et al.
Inhibition of cytokine activation processes in vitro by tenidap, a novel anti-inflammatory agent
Cytokine
(1995) - et al.
Introduction of the interleukin-10 gene into mice inhibited bleomycin-induced lung injury in vivo
Am. J. Physiol., Lung Cell. Mol. Physiol.
(2000) Interleukin 1, interleukin 6 and tumor necrosis factor in infection, inflammation and immunity
Immunol. Lett.
(1988)- et al.
A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promotor in macrophages
J. Immunol.
(2000)
CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency
Proc. Natl. Acad. Sci. USA
Interleukin-10 inhibits cytokine: production by activated macrophages
J. Immunol.
Pirfenidone inhibits PDGF isoforms in bleomycin hamster model of lung fibrosis at the translational level
Am. J. Physiol.
Inosine inhibits inflammatory cytokine production by a posttranscriptional mechanism and protects against endotoxin-induced shock
J. Immunol.
Spermine differentially regulates the production of interleukin-12 p40 and interleukin-10 and suppresses the release of the T helper 1 cytokine interferon-γ
Shock
Interleukin 10 protects mice from lethal endotoxemia
J. Exp. Med.
Dietary intake of pirfenidone ameliorates bleomycin induced lung fibrosis in hamsters
J. Lab. Clin. Med.
Effects of pirfenidone on transforming growth factor-beta gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis
J. Pharmacol. Exp. Ther.
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