Elsevier

Journal of Hepatology

Volume 53, Issue 2, August 2010, Pages 318-325
Journal of Hepatology

Research Article
Modulation of the microenvironment by senescent biliary epithelial cells may be involved in the pathogenesis of primary biliary cirrhosis

https://doi.org/10.1016/j.jhep.2010.03.008Get rights and content

Background & aims

Biliary epithelial cells (BECs) in damaged small bile ducts in primary biliary cirrhosis (PBC) show senescent features. Given that senescent cells modulate the microenvironment by expressing senescence-associated secretory phenotypes (SASP), including inflammatory cytokines and chemokines, we investigated the possible involvement of SASP in the pathogenesis of PBC.

Methods

We examined the chemokine profiles and the induced migration of RAW264.7 cells in senescent BECs induced by oxidative stress, DNA damage, and serum deprivation. We also immunohistochemically examined the expression of CCL2 and CX3CX1 in livers taken from patients with PBC (n = 37) and control livers (n = 75).

Results

Senescent BECs induced by oxidative stress, DNA damage, or serum deprivation expressed a significantly higher level of chemokines to various degrees, when compared with control BECs. Senescent BECs significantly facilitated the migration of RAW264.7 cells (p <0.01), and neutralizing antibodies against CCL2 and CX3CX1 partially blocked the migration induced by senescent BECs (p <0.01). The expression of CCL2 and CX3CL1 was significantly higher in BECs in inflamed and damaged small bile ducts in PBC, when compared with non-inflamed bile ducts and control livers (p <0.01). The expression of CCL2 and CX3CL1 was co-localized with the expression of senescent markers.

Conclusions

Senescent BECs displayed an upregulated expression of various chemokines and chemotactic activities. The expression of CCL2 and CX3CL1 was increased in senescent BECs in PBC. These findings suggest that the senescent BECs may modulate the microenvironment around bile ducts by expressing SASP and contribute to the pathogenesis of bile duct lesions in PBC.

Introduction

Cellular senescence is defined as a condition in which a cell no longer has the ability to proliferate. Senescent cells are irreversibly arrested at the G1 phase of the cell cycle and do not respond to various external stimuli, but remain metabolically active. Senescent cells display several characteristics, including histological changes in vitro and in vivo [1], [2], shortened telomeres, increased expression of p16INK4 and p21WAF1/CIP, and increased activity of senescence-associated β-galactosidase (SA-β-gal) [3]. Cellular senescence can be triggered by multiple mechanisms, including telomere shortening, the epigenetic de-repression of the INK4a/ARF locus, and DNA damage [4]. Recent studies suggest that senescent cells play an important role in modulating the microenvironment by secreting biologically active molecules, such as cytokines [interleukin (IL)-6, IL-1 and so on], chemokines [CXCL8/IL-8, CCL2/monocyte chemotactic protein-1 (MCP)-1] and so on), growth factors and profibrogenic factors [5], [6], [7], [8], [9]. In fact, studies in human beings with biliary disorders and in animal models of biliary fibrosis have shown that the ductal epithelium can express a number of profibrogenic and chemotactic proteins (for example, IL-1, IL-6, CXCL8/IL-8 and CCL2/MCP-1), the latter capable of attracting and activating cells of both inflammatory and stellate cell lineage [10], [11], [12], [13].

Primary biliary cirrhosis (PBC) is an organ specific autoimmune disease and presents with chronic, progressive cholestasis and liver failure [14], [15], [16]. PBC is characterized histologically as a cholangitis of small bile ducts (chronic non-suppurative destructive cholangitis; CNSDC) eventually followed by the extensive loss of small bile ducts [15], [16], [17] Although there have been many studies on the immunopathological features [18], [19], [20], there have been few studies on the pathogenesis of bile duct loss in PBC. We have reported the cellular senescence of biliary epithelial cells (BECs) with shortened telomeres, the expression of SA-β-gal, and the augmented expression of p16INK4a and p21WAF1/Cip in damaged small bile ducts in PBC, and suggested that cellular senescence may be involved in the pathogenesis of progressive bile duct loss in PBC [21], [22]. The possible association of oxidative stress and decreased expression of the polycomb group protein Bmi-1 was suggested to be involved in the pathogenesis of cellular senescence in PBC [22], [23], [24]. Similar biliary epithelial senescence was reported in chronic liver allograft rejection, in which intrahepatic bile ducts are diminished [25]. Recent progress in the field of hepatology also revealed that cellular senescence is involved in the pathophysiology of various chronic liver diseases [22], [23], [24], [25], [26], [27], [28] and hepatocarcinogenesis [29], [30].

Previous reports, including those from our group, have reported the upregulation of several cytokines and chemokines in the damaged bile ducts in PBC [11], [12], [31], and recent studies have shown that most of these factors are known to belong to SASP [5], [6], [7], [8], [9]. We hypothesized that senescent biliary epithelial cells may secrete various proinflammatory cytokines and chemokines, and may contribute to the modulation of the microenvironment around bile ducts and the pathophysiology of bile duct lesions in PBC. We examined the chemokine profiles in cultured senescent BECs and their chemotactic capacity. As previous studies suggested, the significance of intraepithelial infiltration of monocytes and T cells in the bile duct lesion in PBC [12], [13], [16], [22], we focused on chemokines which are chemotactic for these cells, especially on CCL2 and CX3CL1 [12], [13]. We also examined the correlation between the expression of senescent features and chemokine profiles in human PBC livers by immunohistochemistry. This is the first study suggesting the involvement of senescent BECs in the modulation of the inflammatory microenvironment around affected small bile ducts in PBC.

Section snippets

Cell culture and treatments of mouse intrahepatic BECs

Mouse intrahepatic BECs were isolated from 8-week-old female BALB/c mice and were purified and cultured as described previously [32]. The confluency of the cells was less than 80% during experiments. In several experiments, BECs were treated with 100 μM H2O2 for 2 h, washed thoroughly to remove H2O2, and cultured in fresh medium. DNA damage was induced by treatment with Etoposide (Sigma Chemical, Co., 100 μM for 4 days). Recombinant CCL2 (20 or 50 ng/ml for 4 days, R&D, Minneapolis, MN) and CX3CL1

Induction of cellular senescence in BECs treated with H2O2, Etoposide and serum deprivation

Cellular senescence was induced in BECs by treatment with H2O2 (100 μM for 2 h), Etoposide (100 μM for 4 days), or serum deprivation for 4 days. The percentage of cells positive for SA-β-gal was significantly higher in cultured BECs treated with H2O2 (SA-β-gal labeling index, 26.4 ± 3.9), Etoposide (36.3 ± 6.3) and serum deprivation (46.4 ± 14.2), when compared with the control without treatment (8.0 ± 4.0) (p <0.01) (Fig. 1A). These data confirmed the effective induction of cellular senescence by H2O2,

Discussion

The data obtained in this study are summarized as follows: (1) Senescent BECs induced by oxidative stress, DNA damage, and serum deprivation showed the upregulated expression of various chemokines, such as CCL2 and CX3CL1. (2) Treatment with CCL2 and CX3CL1 induced cellular senescence in BECs. (3) Senescent BECs induced the chemotactic migration of RAW264.7 cells, and the neutralizing antibodies against CCL2 and CX3CL1 effectively blocked the migration. (4) The expression of CCL2 and CX3CL1 was

Conflicts of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Acknowledgement

This study was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports and Science and Technology of Japan (18590325).

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