Elsevier

Journal of Hepatology

Volume 47, Issue 1, July 2007, Pages 103-113
Journal of Hepatology

Gliotoxin causes apoptosis and necrosis of rat Kupffer cells in vitro and in vivo in the absence of oxidative stress: Exacerbation by caspase and serine protease inhibition

https://doi.org/10.1016/j.jhep.2007.02.024Get rights and content

Background/Aims

A potential application of gliotoxin therapy for liver fibrosis was suggested by its apoptotic effect on fibrogenic activated stellate cells. We investigated if gliotoxin exerts similar effects on hepatic macrophage Kupffer cells.

Methods

Effects of gliotoxin on Kupffer cells isolated from the normal liver and in vivo following its administration to CCl4-induced cirrhotic rats were studied.

Results

Gliotoxin caused apoptosis of cultured Kupffer cells, the effect being apparent at 0.3 μM concentration within 1 h; longer incubation caused necrosis. This effect was associated with mitochondrial cytochrome c release, caspase-3 activation and ATP depletion. Interestingly, inhibition of caspase-3 and serine proteases accelerated and augmented gliotoxin-induced cell death via necrosis. Gliotoxin stimulated nuclear translocation of NFκB, and phosphorylation of p38, ERK1/2 and JNK MAP kinases, but these signaling molecules were not involved in gliotoxin-induced death of Kupffer cells. In vivo administration of gliotoxin to cirrhotic rats caused apoptosis of Kupffer cells, stellate cells and hepatocytes. In control rats, the effect was minimal on the nonparenchymal cells and not apparent on hepatocytes.

Conclusions

In the fibrotic liver, gliotoxin nonspecifically causes death of hepatic cell types. Modification of gliotoxin molecule may be necessary for selective targeting and elimination of activated stellate cells.

Introduction

Gliotoxin, a fungal metabolite, belongs to the epipolythiodioxopiperazine class of compounds. Gliotoxin (0.3–7.5 μM) was reported to cause apoptosis of activated rat and human hepatic stellate cells (HSCs) by inducing mitochondrial permeability, cytochrome c release and caspase-3 activation [1], [2]; higher concentrations (>7.5 μM) caused necrosis of activated human HSCs in association with increased oxidative stress [2]. These were important observations as activated HSCs play a central role in liver fibrosis and sinusoidal component of portal hypertension [3]. HSCs produce several growth factors, cytokines and chemokines [4], [5] responsible for initiation and progression of liver pathology. Therefore, strategies to remove selectively activated HSCs from the fibrotic liver are intensely investigated.

Gliotoxin possesses immunomodulating activity: it stimulates lymphocyte proliferation, and induces T cell cytotoxicity and lymphokine release by T cells [6], [7], [8], [9]. Gliotoxin (10–100 nM) was found to inhibit phagocytic activity of peritoneal and circulating macrophages [6], [7], [9] at concentrations lower than those required to cause apoptosis of HSCs. High concentration of gliotoxin (>1 μM) induces reactive oxygen species (ROS)-mediated apoptosis of peritoneal macrophages, independent of the inhibition of phagocytosis [8]. The role of hepatic macrophages Kupffer cells extends from defense against invading microbes and clearance of toxic substances to liver growth and immune regulation [10], [11], [12], [13]. These functions of Kupffer cells are even more critical in disease states as their removal can significantly augment pathological development [14]. Since gliotoxin-induced elimination of activated HSCs could be therapeutic strategy for liver fibrosis, it is important to rule out its adverse effects on other hepatic cell types. Therefore, we investigated the effects of gliotoxin on rat Kupffer cells in vitro, and in control and CCl4-induced cirrhotic rats in vivo.

Section snippets

Materials and methods

Gliotoxin, TPCK, MG132 (Sigma, St. Louis, MO); PD98059, SB203580, SP600125 and PDTC (Calbiochem, La Jolla, CA); Z-DEVD-fmk (R&D, Minneapolis, MN); Mn-TBAP (Calbiochem, La Jolla, CA); anti-rabbit NFκB-p65, ERK1/2 and caspase-3, mouse anti-rat desmin antibodies and CY-3-conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-rabbit monoclonal P-ERK1/2, P-p38, p38, P-JNK and JNK antibodies (Cell Signaling, Danvers, MA); anti-rat ED2 antibody (Serotech,

Gliotoxin decreases Kupffer cell viability

Viability of control cells did not change over 24 h (Fig. 1A). Gliotoxin (0.03 μM) caused significant reduction in viability only at 24 h. At 0.3 and 3.0 μM, gliotoxin caused time-dependent loss of viability that was significant at 3 and 1 h, respectively (Fig. 1A); incubation beyond 3 h at both concentrations caused detachment of the majority of cells. Gliotoxin caused equivalent loss of viability of Kupffer cells even in the presence of serum (Fig. 1B). Pancreatic elastase (apoptotic agent for

Discussion

Here we report that gliotoxin causes death of cultured Kupffer cells via apoptosis and secondary necrosis. Previously, activated rat and human HSCs were found to undergo apoptosis and exclude trypan blue at 0.3–7.5 μM gliotoxin [1], [2] but necrosis was observed at concentration above 7.5 μM [2]. Hepatocytes are resistant to lower gliotoxin concentration but undergo necrosis at high concentration (50 μM). Apoptosis of Kupffer cells by lower concentration of gliotoxin (0.03–0.3 μM) indicates their

Acknowledgements

This work was supported by two grants from National Institutes of Health: RO1-DK54401 to CRG and CA76541 to DBS. We thank Dr. C. Thirunavukkarasu for assistance with apoptotic assays and Mr. Mark Ross for immunostaining of liver sections.

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    The authors who have taken part in this study declared that they have no relationship with the manufacturers of the drugs involved either in the past or present and did not receive funding from the manufacturers to carry out their research. The authors received funding from the National Institutes of Health, USA, which enabled them to carry out their study.

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