Elsevier

Journal of Autoimmunity

Volume 27, Issue 4, December 2006, Pages 232-241
Journal of Autoimmunity

T cell targeting and phagocytosis of apoptotic biliary epithelial cells in primary biliary cirrhosis

https://doi.org/10.1016/j.jaut.2006.11.004Get rights and content

Abstract

Primary biliary cirrhosis (PBC) is characterized by loss of tolerance against ubiquitously expressed mitochondrial autoantigens followed by biliary and salivary gland epithelial cell (BEC and SGEC) destruction by autoreactive T cells. It is unclear why BECs and SGECs are targeted. Previous work demonstrated that the reduced form of the major PBC autoantigen predominated in apoptotic BECs and SGECs as opposed to an oxidized form in other apoptotic cells. This led to the hypothesis that presentation of novel self-peptides from phagocytosed apoptotic BECs might contribute to BEC targeting by autoreactive T cells. The effect of autoantigen redox status on self-peptide formation was examined along with the phagocytic ability of BECs. Oxidation of PBC autoantigens first was shown to be due to protein S-glutathionylation of lipoyllysine residues. Absence of protein S-glutathionylation generated novel self-peptides and affected T cell recognition of a lipoyllysine containing peptide. Liver biopsy staining revealed BEC phagocytosis of apoptotic BECs (3.74 ± 2.90% of BEC) was present in PBC (7 of 7 cases) but not in normal livers (0 of 3). BECs have the ability to present novel mitochondrial self-peptides derived from phagocytosed apoptotic BECs. Apoptotic cell phagocytosis by non-professional phagocytes may influence the tissue specificity of autoimmune diseases.

Introduction

The hallmarks of primary biliary cirrhosis (PBC) are progressive bile duct and salivary gland epithelial cell damage, elevated alkaline phosphatase levels and loss of tolerance against ubiquitously expressed mitochondrial autoantigens [1]. This loss of self-tolerance to mitochondrial autoantigens precedes biliary and salivary gland epithelial cell damage (BEC and SGEC), often by many years [2], [3], [4]. Autoantibodies against the major PBC autoantigen, the E2 subunit of the mitochondrial pyruvate dehydrogenase complex (PDC-E2), are present in 95% of PBC cases and are highly specific for PBC. The autoantibodies recognize the inner lipoyl domain of PDC-E2 as well as other mitochondrial proteins that contain lipoyllysine residues. The PDC-E2 self-peptide recognized by autoreactive T cells in PBC also includes the lipoyllysine residue [5]. The destruction of bile duct and salivary gland epithelial cells characteristic of PBC appears to be mediated by autoreactive T cells [6], [7], [8], [9]. Why these cell types are specifically targeted is uncertain.

Similar to other epithelial cells, BECs and SGECs potentially act as antigen presenting cells. Extra-mitochondrial staining by some anti-PDC-E2 antibodies of PBC patient BECs and SGECs suggest a molecular mimic of PDC-E2 may be present in these cell types [10], [11]. This extra-mitochondrial “PDC-E2” may be a source of unique PDC-E2 self-peptides presented by PBC patient BECs and SGECs. T cell mediated destruction of these cell types may also in part be due to increased basolateral expression of MHC class I and II molecules [12], [13], which enhance peptide presentation. BECs in PBC do not have the capacity to activate primary (or naive) autoreactive T cells, but are merely the targets of destruction [14], [15]. Identification of potential sources of extra-mitochondrial “PDC-E2” may aid both in understanding the pathogenesis of PBC and in its treatment.

Apoptotic cells phagocytosed by BECs and SGECs are an obvious potential exogenous source of extra-mitochondrial PDC-E2. Other epithelial cell types are known to phagocytose neighboring apoptotic cells [16], [17], [18]. During apoptosis, many autoantigens associated with systemic autoimmune diseases cluster at the cell surface and are known to undergo either proteolytic or non-proteolytic modification, which may lead to generation of unique self-peptides [19], [20], [21], [22]. These findings have led to a number of preliminary studies examining the effect of apoptosis on PBC autoantigens. For example, MacDonald et al. have reported PDC-E2 is present on the cell surface of cultured apoptotic BECs [23]. Apoptosis specific proteases cleave purified PBC autoantigens [24], however, only oxidative modification of PDC-E2 has been detected in apoptotic cells to date [25]. Interestingly, oxidative modification appears to be cell type specific in that PDC-E2 is spared in apoptotic BECs and SGECs. It is unknown whether lack of oxidative modification may alter subsequent PDC-E2 self-peptide formation. Additionally, bile-induced apoptosis is unique with regard to its activation of the cathepsin B protease [26], which may also generate novel self-peptides. In the current study, BEC and SGEC apoptosis and phagocytosis are examined in order to define their role in the tissue specificity of autoreactive T cell targeting in PBC.

Section snippets

Sera and antibodies

Sera were obtained from patients diagnosed with PBC. The diagnosis of PBC was confirmed by biochemical, serologic, and histological criteria in all cases. The specificity of the sera autoantibodies was confirmed by western blotting and ELISA as previously described [27]. Informed consent in writing was obtained from each participant. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Institutional Review Board.

Purified PDC-E2 is cleaved by cathepsin B in vitro, but not during cathepsin B-dependent apoptosis

Unlike many other autoantigens, cleavage of PBC autoantigens has not been detected during apoptosis [25]. However, Roberts et al. have shown that apoptosis induced by toxic bile salts (e.g. GCDC) occurs via a novel pathway involving release of the lysosomal protease cathepsin B [26]. In biliary diseases such as PBC, this novel pathway is particularly relevant.

Purified PDC-E2 (500 ng) was first incubated with purified cathepsin B (300 nM) in vitro to determine if it is a potential substrate (Fig. 1

Discussion

These results demonstrate for the first time that apoptotic cells are phagocytosed by BECs and consequently are an exogenous source of autoantigens in BECs. Protease digestion of the reduced form of PDC-E2, which predominates in apoptotic BECs and SGECs, unlike other cell types, yields distinctive self-peptides. These findings support the paradigm that tissue specific damage in some autoimmune diseases, such as PBC, is due to cell type specific differences in apoptosis and phagocytosis of

Acknowledgements

The first two authors made equivalent contributions to the manuscript and are listed in alphabetical order. We are grateful for the assistance of Dr Mary Ann Gawinowicz for MALDI-MS analysis and the Columbia University Protein Core Facility, New York, NY. Helpful advice and assistance from Drs Nancy Bach, Toren Finkel and Carmen Stanca was much appreciated. This study was supported by an NIH grants DK59653 (JAO) and DK 39588 (MEG) and the Artzt Family PBC Foundation (JAO). Microscopy was

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