calpain and Sjogren-s-Syndrome

Autoantibodies specific for alpha-fodrin fragments are found in the tissues of persons afflicted with Sjögren's syndrome (SS). However, the mechanism for alpha-fodrin degradation remains elusive. The following experiments utilized Par C5 cells to examine the role of P2X7 receptor (P2X7R) in apoptosis, particularly in the cleavage and release of alpha-fodrin, an apparent SS autoantigen. Five mM ATP stimulation induced apoptotic cell death with a sustained Ca2+ influx, which was mimicked in HEK cells transfected with P2X7R. ATP also induced cleavage of alpha-fodrin mediated by caspase-3 and calpain, releasing alpha-fodrin fragments through membrane blebs. However, both apoptotic cell death and alpha-fodrin cleavage were inhibited in the presence of 300 microM oxidized-ATP (ox-ATP), an irreversible blocker of P2X7R, or in Ca(2+)-free solution. We concluded that P2X7R plays an important role in apoptosis and alpha-fodrin degradation in salivary epithelial cells, providing an important clue elucidating the presence of alpha-fodrin fragments in SS tissues.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Autoantigens; Calcium; Calpain; Carrier Proteins; Caspase 3; Cell Death; Cell Line; Cell Surface Extensions; Epithelial Cells; Humans; Membrane Proteins; Microfilament Proteins; Neuropeptides; Parotid Gland; Purinergic P2 Receptor Antagonists; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X4; Receptors, Purinergic P2X7; Signal Transduction; Sjogren's Syndrome; Spectrin

The alpha-fodrin N-terminal portion (AFN) autoantigen mediates in vivo immunoregulation of autoimmune responses in primary Sjögren's syndrome (SS). We further examined this process and found that cleavage products of AFN were frequently detected in the salivary gland duct cells of SS patients. In in vitro studies using human salivary gland HSY cells, anti-Fas-induced apoptosis resulted in specific cleavage of alpha-fodrin into the 120-kd fragment, in association of alpha-fodrin with mu-calpain, and activation of caspase 3. Significant proliferative responses against AlphaFN autoantigen were observed in the peripheral blood mononuclear cells (PBMCs) from SS patients with higher pathological score (grade 4) and with short duration from onset (within 5 years). In vivo roles of AFN peptides were investigated using PBMCs from patients with SS, systemic lupus erythematosus, and rheumatoid arthritis. Significant proliferative T-cell responses of PBMCs to AFN peptide were detected in SS but not in systemic lupus erythematosus or rheumatoid arthritis. AFN peptide induced Th1-immune responses and accelerated down-regulation of Fas-mediated T-cell apoptosis in SS. Our data further elucidate the in vivo role of AFN autoantigen on the development of SS and suggest that the AFN autoantigen is a novel participant in peripheral tolerance.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Apoptosis; Arthritis, Rheumatoid; Autoantigens; Blotting, Western; Calpain; Carrier Proteins; Case-Control Studies; Caspase 3; Caspases; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cells, Cultured; Coculture Techniques; Enzyme Activation; fas Receptor; Female; Furans; Glutathione Transferase; Humans; Immunohistochemistry; Japan; Leukocytes, Mononuclear; Lupus Erythematosus, Systemic; Microfilament Proteins; Molecular Sequence Data; Molecular Weight; Parotid Gland; Recombinant Fusion Proteins; Sjogren's Syndrome; Thymidine