Liver, Pancreas and Bilary Tract
Hepatitis B virus particles preferably induce Kupffer cells to produce TGF-β1 over pro-inflammatory cytokines

https://doi.org/10.1016/j.dld.2011.11.005Get rights and content

Abstract

Background

Kupffer cells and related cytokines are thought to play a critical role in liver fibrosis; however, the role played by Kupffer cells in hepatitis B virus-related fibrogenesis is unknown.

Methods

Primary rat Kupffer cells were cultured with different titres of hepatitis B virus particles and the concentrations of transforming growth factor (TGF)-β1, interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-α in the culture supernatant were measured every 24 h for 7 days. The mRNA and protein levels of these cytokines in Kupffer cells were also analysed using quantitative real-time polymerase chain reaction and western blotting, respectively.

Results

Kupffer cells maintained normal morphology and function throughout the 7-day exposure to hepatitis B virus. The concentration of TGF-β1 secreted by hepatitis B virus-stimulated Kupffer cells (6 log IU/ml hepatitis B virus) increased 5.38- and 7.75-fold by Days 3 and 7, respectively (p < 0.01). Western blotting showed that TGF-β1 expression in Kupffer cells exposed to high titres of hepatitis B virus increased 1.80- and 2.42-fold by Days 3 and 7, respectively (p < 0.01). In contrast, Kupffer cell expression and secretion of pro-inflammatory cytokines (IL-6, IL-1 and TNF-α) was unchanged throughout the experiment.

Conclusion

Hepatitis B virus preferentially stimulates Kupffer cells to produce the pro-fibrogenic/anti-inflammatory cytokine TGF-β1 rather than the pro-inflammatory cytokines IL-6, IL-1 and TNF-α. This may partly explain why overt liver fibrosis still presents in cases of chronic hepatitis B virus infection with minimal (or no) necro-inflammation.

Introduction

Chronic infection associated with hepatitis B virus (HBV) is a major cause of liver fibrosis and cirrhosis in many Asian countries, including China [1], [2]. Although the general theory regarding the pathogenesis of fibrosis suggests that chronic necro-inflammation activates hepatic stellate cells (HSC) to differentiate into myofibroblast-like cells, which produce excess extracellular matrix (ECM), the mechanisms underlying HBV-induced liver fibrosis have not yet been fully elucidated [3], [4], [5]. Indeed, some cases of chronic HBV infection with minimal or no evidence of necro-inflammation show overt fibrosis [2], [3]. Recent studies in models of toxic or cholestatic liver fibrosis show that Kupffer cells, the largest group of resident liver macrophages, promote fibrogenesis by producing pro-fibrogenic mediators such as transforming growth factor (TGF)-β1 [6], [7], [8]. Toll-like receptors expressed by murine Kupffer cells also interact with HBV [9], [10]. A recent study concluded that HBV interferes with murine liver macrophage function, suggesting that Kupffer cells recognize and engulf HBV particles [11]. However, it is still not known whether HBV directly stimulates Kupffer cells to produce pro-fibrotic mediators without causing significant levels of necro-inflammation. To address this issue, we stimulated rat Kupffer cells with different titres of purified HBV virions and observed the resulting pro-fibrogenic/anti-inflammatory and pro-inflammatory cytokine profiles. The results showed that high HBV titres stimulated Kupffer cells to produce the pro-fibrogenic/anti-inflammatory mediator, TGF-β1, rather than pro-inflammatory cytokines such as interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-α. Taken together, these results may provide a novel explanation for the relationship between HBV and liver fibrosis.

Section snippets

Isolation and purification of HBV particles

HBV particles were isolated and purified from the serum of chronic hepatitis B patients with serum HBV DNA levels around 7 log IU/ml as measured by quantitative real-time polymerase chain reaction (RT-PCR). As previously described [12], 2 ml of serum was carefully layered onto a 10%/20%/30%/40%/50% (w/w) sucrose gradient in TNE buffer (20 mM Tris–HCl, pH 7.5, 0.15 M NaCl, 1 mM EDTA) in a 12 ml ultracentrifuge tube, and ultracentrifuged at 200,000 × g for 16 h at 4 °C in a Beckman Sw41Ti rotor (Beckman

Effects of HBV on Kupffer cell proliferation and morphology

Both high-dose (6 log IU/ml) and low-dose (4 log IU/ml) HBV-treated Kupffer cells showed a similar levels of proliferation to those of untreated Kupffer cells over the 7 days (Fig. 3A). ED1 expression levels were also similar over the same time period (Fig. 3B). Cellular protein and mRNA were isolated on Days 3 and 7, at which point both treated and untreated Kupffer cells (≥95% pure) showed similar morphology and ED1 levels (Fig. 3C). These results show that HBV was neither cytotoxic nor

Discussion

The results of the present study show that purified HBV induces the production of TGFβ1 rather than IL-1, IL-6 and TNFα by rat Kupffer cells. This finding may offer a novel explanation for the pathogenesis of HBV-related fibrosis.

It is widely thought that necro-inflammation of hepatocytes related to HBV activates HSC and promotes liver fibrosis [4], [16]. Indeed, liver function and HBV DNA levels correlate with the progression of liver fibrosis in many patients. However, many HBV carriers

Conflict of interest statement

The authors have no conflicts of interest to declare.

Acknowledgements

This work was partly supported by the National High Technology Research and Development Program of China (2006AA02A410), Major State Basic Research Development Program of China (2007CB512802) and Municipal Key Laboratory of Beijing for Regulation of Liver Protection and Regeneration.

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