A novel anti-fibrotic agent pirfenidone suppresses tumor necrosis factor-α at the translational level

Abstract

A new experimental drug pirfenidone (5-methyl-1-phenyl-2-1H-pyridine-2-one) has been reported to have beneficial effects for the treatment of certain fibrotic diseases. Here, we studied the anti-inflammatory activities of pirfenidone by investigating the mechanism of its inhibitory effect on cytokine production. In RAW264.7 cells, a murine macrophage-like cell line, pirfenidone suppressed the proinflammatory cytokine tumor necrosis factor-α (TNF-α) by a translational mechanism, which was independent of activation of the mitogen-activated protain kinase (MAPK) 2, p38 MAP kinase, and c-Jun N-terminal kinase (JNK). In the murine endotoxin shock model, pirfenidone potently inhibited the production of the proinflammatory cytokines, TNF-α, interferon-γ, and interleukin-6, but enhanced the production of the anti-inflammatory cytokine, interleukin-10. The in vivo model also showed that pirfenidone suppressed the cytokine production by a translational mechanism, though interleukin-10 transcription was activated by pirfenidone. These findings show that pirfenidone inhibits the production of the proinflammatory cytokine selectively at the translational level. Therefore, cytokine inhibitory activities play an important role in the anti-inflammatory activities of pirfenidone. Coupled with the fact that this inhibitory effect is selective, translational, and not for total protein synthesis, this drug may have a clinical effect on inflammation and fibrosis with very low toxicity.

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.