Bcl-2–dependent oxidation of pyruvate dehydrogenase-E2, a primary biliary cirrhosis autoantigen, during apoptosis
Joseph A. Odin, … , Nicholas F. LaRusso, Antony Rosen
Joseph A. Odin, … , Nicholas F. LaRusso, Antony Rosen
Published July 15, 2001
Citation Information: J Clin Invest. 2001;108(2):223-232. https://doi.org/10.1172/JCI10716.
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Article

Bcl-2–dependent oxidation of pyruvate dehydrogenase-E2, a primary biliary cirrhosis autoantigen, during apoptosis

Abstract

The close association between autoantibodies against pyruvate dehydrogenase-E2 (PDC-E2), a ubiquitous mitochondrial protein, and primary biliary cirrhosis (PBC) is unexplained. Many autoantigens are selectively modified during apoptosis, which has focused attention on apoptotic cells as a potential source of “neo-antigens” responsible for activating autoreactive lymphocytes. Since increased apoptosis of bile duct epithelial cells (cholangiocytes) is evident in patients with PBC, we evaluated the effect of apoptosis on PDC-E2. Autoantibody recognition of PDC-E2 by immunofluorescence persisted in apoptotic cholangiocytes and appeared unchanged by immunoblot analysis. PDC-E2 was neither cleaved by caspases nor concentrated into surface blebs in apoptotic cells. In other cell types, autoantibody recognition of PDC-E2, as assessed by immunofluorescence, was abrogated after apoptosis, although expression levels of PDC-E2 appeared unchanged when examined by immunoblot analysis. Both overexpression of Bcl-2 and depletion of glutathione before inducing apoptosis prevented this loss of autoantibody recognition, suggesting that glutathiolation, rather than degradation or loss, of PDC-E2 was responsible for the loss of immunofluorescence signal. We postulate that apoptotic cholangiocytes, unlike other apoptotic cell types, are a potential source of immunogenic PDC-E2 in patients with PBC.

Authors

Joseph A. Odin, Robert C. Huebert, Livia Casciola-Rosen, Nicholas F. LaRusso, Antony Rosen

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Figure 3

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PDC-E2 staining by PBC patient sera in apoptotic cells does not localize...
PDC-E2 staining by PBC patient sera in apoptotic cells does not localize to cell membrane blebs or apoptotic bodies. Both control cells and cells treated with UV-B to induce apoptosis were examined by confocal, immunofluorescence microscopy. Cells were stained with DAPI (blue) (a–h) to distinguish apoptotic cells (cells labeled with stars) with characteristic condensed, fragmented chromatin from nonapoptotic cells (unlabeled cells). Control NRC were costained with PBC patient serum monospecific for PDC-E2 (green) (a) and a mAb specific for COX-1 (red) (e). Immunoreactivity against PDC-E2 and COX-1 colocalized in mitochondria. In apoptotic NRCs, immunoreactivity against PDC-E2 (green) (b) remained perinuclear and colocalized with COX-1 (red) (f). Preincubation of PBC patient sera with purified PDC blocked staining of PDC-E2 (green) in both nonapoptotic and apoptotic NRCs (g). Cells were stained with PI (red) to distinguish cell membrane blebs or apoptotic bodies in apoptotic cells (c and d). Staining of PDC-E2 (green) in apoptotic NRCs did not localize to cell membrane blebs or apoptotic bodies (c). In contrast, immunoreactivity against PDC-E2 (green) was not detected in apoptotic HeLa cells (d), although immunoreactivity against COX-1 (red) was detected (h). Each experiment was repeated twice with each of the monospecific PBC patient sera with identical results. Bar, 20 μm. Representative images are shown.