Detection of apoptosis-specific autoantibodies directed against granzyme B-induced cleavage fragments of the SS-B (La) autoantigen in sera from patients with primary Sjögren's syndrome

Abstract

The objective of this study was to detect autoantibodies against granzyme B cleavage products in sera from patients with primary Sjögren's syndrome (SS). Cell lysates derived from human salivary gland (HSG) cell lines were incubated with granzyme B. The susceptibility to the generation of cleavage fragments of SS autoantigens was assayed by immunoblotting using sera from 57 primary SS patients, 17 primary SS patients with malignant lymphoma (ML), 28 systemic lupus erythematosus (SLE) patients, and 20 healthy controls. A 27 kD protein was recognized by serum autoantibodies in 8 (14.0%) of 57 primary SS patients, 5 (29.4%) of 17 SS patients with ML, 2 (7.1%) of 28 SLE patients, but not in 20 normal subjects. This protein was recognized by anti-SSB (La) monoclonal antibodies. Granzyme B-treated recombinant La protein was also shown to migrate as a discrete 27 kD protein by SDS PAGE. Blocking studies demonstrated the existence of an apoptosis-specific B cell epitope present in sera from 2 of 8 primary SS patients and in 2 of 5 primary SS patients with ML which recognized the 27 kD protein. Granzyme B-induced La fragments are generated during cytotoxicity in vitro. This is the first report describing autoantibodies in sera from primary SS patients that specifically recognize fragments of the La protein that are produced by the granzyme B protease.

Fig. 1

Screening of sera from patients with primary Sjögren's syndrome using HSG cell lysates treated with granzyme B. Lysates from HSG cells after treatment of granzyme B were prepared and subjected to 5–20% gradient SDS-PAGE and proteins were transferred to a PVDF filter. The filter was incubated in the presence of each serum sample. The serum was diluted 1 : 1000 in PBS using a multiscreen blotter after blocking with 5% nonfat dried milk in PBS-T. The filter was washed with PBS-T and incubated with goat anti-human IgG coupled to horseradish peroxidase. The enhanced chemiluminescence system was used for detection. The positions of the molecular weight markers are indicated on the right (in kD). Representative results are shown for 8 primary SS patients, 3 SLE patients, and 1 normal control. Fifty kD and 27 kD proteins are indicated by arrow and arrow head, respectively.

Fig. 2

Recombinant La protein is cleaved after treatment with granzyme B. Recombinant Ro protein, recombinant La protein, and HSG cell lysate were treated with granzyme B and were subjected to SDS-PAGE followed by Western blotting with a monoclonal antibody specific for the La protein. The positions of the molecular weight markers are indicated on the right (in kD).

Fig. 4

Granzyme B-specific La fragments are generated during cytotoxicity in vitro. 1 × 10⁵ YT cells were incubated with adherent HSG cells (1 × 10⁵ in a 6 well plate). After 6, 12, or 24 h, HSG cells were lysed with 1% NP40 buffer containing 2 mM ZnSO₄, subjected to SDS-PAGE and electropheretic transfer, and immunoblotted using a monoclonal antibody specific for the La protein. The positions of the molecular weight markers are indicated on the right (in kD).

Fig. 3

Presence of apoptosis-specific La antibodies. HSG cell lysates incubated with or without granzyme B (GB+ or Control+, respectively) were immunoblotted in the presence or absence of 2 µg/ml of recombinant La protein (Blockade + or –, respectively). Pattern A, A representative immunoblot (serum no. 3 in Fig. 1: 65-year-old female, SS + ML) using serum (1 : 10 000 dilution) recognizing both intact (50 kD) and cleaved La (27 kD) in the absence of blockade; in the presence of blockade, recognition of intact La was diminished to a greater extent than was recognition of cleaved La. Pattern B, A representative immunoblot (serum no. 2 in Fig. 1: 70-year-old female, SS + ML) using serum recognizing both intact and cleaved La in the absence of blockade; recognition of both the intact and cleaved La was equally diminished in the presence of blockade.