Terbinafine stimulates the pro-inflammatory responses in human monocytic THP-1 cells through an ERK signaling pathway

doi:10.1016/j.lfs.2010.08.010

Life Sciences

Volume 87, Issues 17–18, 23 October 2010, Pages 537-544

https://doi.org/10.1016/j.lfs.2010.08.010 Get rights and content

Abstract

Aims

Oral antifungal terbinafine has been reported to cause liver injury with inflammatory responses in a small percentage of patients. However the underlying mechanism remains unknown. To examine the inflammatory reactions, we investigated whether terbinafine and other antifungal drugs increase the release of pro-inflammatory cytokines using human monocytic cells.

Main methods

Dose- and time-dependent changes in the mRNA expression levels and the release of interleukin (IL)-8 and tumor necrosis factor (TNF)α from human monocytic THP-1 and HL-60 cells with antifungal drugs were measured. Effects of terbinafine on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK)1/2 were investigated.

Key findings

The release of IL-8 and TNFα from THP-1 and HL-60 cells was significantly increased by treatment with terbinafine but not by fluconazole, suggesting that terbinafine can stimulate monocytes and increase the pro-inflammatory cytokine release. Terbinafine also significantly increased the phosphorylation of ERK1/2 and p38 MAP kinase in THP-1 cells. Pretreatment with a MAP kinase/ERK kinase (MEK)1/2 inhibitor U0126 significantly suppressed the increase of IL-8 and TNFα levels by terbinafine treatment in THP-1 cells, but p38 MAPK inhibitor SB203580 did not. These results suggested that an ERK1/2 pathway plays an important role in the release of IL-8 and TNFα in THP-1 cells treated with terbinafine.

Significance

The release of inflammatory mediators by terbinafine might be one of the mechanisms underlying immune-mediated liver injury. This in vitro method may be useful to predict adverse inflammatory reactions that lead to drug-induced liver injury.

Introduction

Drug-induced hepatotoxicity is one of the major causes of liver injury and is classified into intrinsic and idiosyncratic types. Idiosyncratic drug reactions do not occur in most patients at any dose and they are often referred to as rare, with a typical incidence of from 1/100 to 1/100,000 (Uetrecht 1999). It has been hypothesized that inflammatory stress might be caused by some xenobiotics leading to an adverse drug reaction. The sporadic occurrence of acute inflammatory episodes could explain the onset of some idiosyncratic reactions during clinical drug therapy (Ganey et al., 2004, Roth et al., 2003, Tafazoli et al., 2005). Inflammatory reactions in liver are induced by the activation of immune cells, such as monocytes, macrophages and Kupffer cells. Activated monocytes and macrophages release large amounts of pro-inflammatory cytokines and chemokines, including interleukin (IL)-1, tumor necrosis factor (TNF)α, and IL-8. TNFα triggers the release of a cascade of other cytokines that recruit and activate immune cells, including lymphocytes and macrophages (Bradham et al. 1998). IL-8 exhibits multiple effects on neutrophils, including the induction of lysosomal enzyme release, the increase in the expression of adhesion molecules, and rapid infiltration (Leonard et al., 1991, Baggiolini et al., 1994). In several rodent models, it was shown that the production of TNFα and neurtophil infiltration in liver play a critical role in immune-mediated liver injury by drugs such as acetaminophen, non-steroidal anti-inflammatory drugs, and antibiotics (Jaeschke, 2005, Deng et al., 2009).

Recently, it has been reported that human monocytic cell lines were useful to examine inflammatory responses mediated by drugs withdrawn from the market. In human monocytic THP-1 cells, the mRNA expression levels and/or the release of pro-inflammatory cytokines and chemokines were increased by the treatment with troglitazone or ximelagatran (Edling et al., 2008, Edling et al., 2009).

Terbinafine is an oral antifungal drug of the allylamine class and is effective for the treatment of onychomycosis and dermatophytosis (Gupta and Shear 1997). A postmarketing surveillance study showed that mild to severe gastrointestinal, skin, and taste disturbances are the most common adverse events related to oral terbinafine treatment (Hall et al. 1997). In addition, terbinafine has been also reported to cause liver injury in a small percentage of patients. Teribnafine-induced hepatic injury is classified into a mixed hepatocellular and cholestatic pattern. Liver biopsies of some patients revealed mixed cellular infiltration in portal tracts, including mononuclear cells, lymphocytes, and neutrophils (Mallat et al., 1997, Fernandes et al., 1998, Zapata Garrido et al., 2003). Moreover, some case reports showed that terbinafine-induced hepatic injury occurred in combination with hypersensitivity reactions, including fever, rash, and lymphadenopathy (Gupta and Porges 1998).

The estimated reporting incidence for the development of clinically significant signs and symptoms of hepatobiliary dysfunction for which no other cause was apparent is approximately 1:45,000–1:54,000 (Gupta et al., 1997, García Rodríguez et al., 1999). These backgrounds suggested that terbinafine-induced hepatic injury might be caused by an idiosyncratic rather than a direct hepatotoxic reaction (van't Wout et al. 1994). However, adverse reactions associated with the use of oral antifungal agents are usually mild, transient, and reversible after discontinuation. From these reasons, terbinafine was not withdrawn from the market. Routine hepatic monitoring when the duration of therapy exceeds 6 weeks could be replaced by the requirement to monitor only if symptoms or signs suggestive of hepatic injury (Gupta et al. 1997). If the underlying mechanisms of terbinafine-induced hepatic injury will be clarified, patient with high risk for hepatic injury will be predicted by using immune-related biomarkers.

Considering the reports of terbinafine-induced hepatic injury, we hypothesized that terbinafine stimulates inflammatory responses that may result in immune-mediated hepatic injury. The purpose of this study is to investigate whether terbinafine stimulates the release of pro-inflammatory cytokines and chemokines from human monocytic cells and to clarify the involvement of cell signaling in the release of pro-inflammatory cytokines and chemokines from THP-1 cells.

Section snippets

Materials

Terbinafine hydrochloride, butenafine hydrochloride, and fluconazole were purchased from Wako Pure Chemical Industries (Osaka, Japan). Primers were commercially synthesized at Hokkaido System Sciences (Sapporo, Japan). The monoclonal antibodies of anti-Thr202/Tyr204 phosphorylated extracellular signal-regulated kinase (ERK) 1/2, anti-Thr180/Tyr182 phosphorylated p38 mitogen-activated protein (MAP) kinase, and anti-Thr183/Tyr185 phosphorylated c-Jun N-terminal kinase (JNK) 1/2 were purchased

Comparative effect of antifungal drugs on human monocytic cell lines

To investigate whether antifungal drugs increased the release of IL-8 and TNFα from human monocytic cells, cells were treated with 100 μM of the antifungal drugs for 6 h and then the release of IL-8 and TNFα in the cell supernatants was measured by ELISA (Fig. 1). Butenafine was used as a drug structurally similar to terbinafine, although it used as ointment, and has never been administered orally. Fluconazole was used because it is assumed to have a lower risk of adverse events leading to

Discussion

THP-1, HL-60, and KG-1 cells are classified as human monocytic cell lines and are useful for studying the differentiation and the activation of immune cells, such as monocytes, macrophages, and immature myelocytes. In the present study, by the treatment with the antifungal drugs terbinafine and butenafine, IL-8 and TNFα release from THP-1 and HL-60 cells was significantly increased compared with the control (Fig. 1A–D). This suggested that terbinafine and butenafine have the ability to

Conclusion

In this study, we found that terbinafine stimulates human monocytic THP-1 cells resulting in IL-8 and TNFα release. It is suggested that terbinafine increases the pro-inflammatory cytokine release from monocytes and macrophages and activates the inflammatory response, which might result in immune-mediated hepatic injury. The findings presented here provide important insight concerning terbinafine-induced liver injury.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

We thank Mr. Brent Bell for reviewing the manuscript. This work was supported by Health and Labor Sciences Research Grants from the Ministry of Health, Labor, and Welfare of Japan (H20-BIO-G001).

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