title: Tumor necrosis factor inhibitors block apoptosis of human epithelial cells of the salivary glands.
abstract: Inhibition of tumor necrosis factor-alpha (TNF-alpha) in organ-specific autoimmune disease is proving efficacious for a large number of patients. A wide array of biological agents has been designed to inhibit TNF-alpha, such as adalimumab (fully humanized) and etanercept (soluble TNF-alpha receptor fusion constructs p75 subunit). Recently, we suggested that anti-Ro and anti-La autoantibodies (Abs) isolated from patients with Sjögren's syndrome, an autoimmune rheumatic disease, are able to trigger cell death through extrinsic apoptotic mechanisms in human salivary gland epithelial cells (SGEC). We analyzed if primary human SGEC cultures, established from biopsy of labial minor salivary glands, are able to produce TNF-alpha, an inductor of the extrinsic apoptotic pathway, when treated with anti-Ro autoantibodies. A comparative study was performed to test the efficacy of adalimumab and etanercept to block TNF-alpha-mediated apoptosis. ELISA assay and RT-PCR were employed to visualize TNF-alpha production, and apoptosis was evaluated by DNA ladder and flow cytometry. We found that cell treatment with anti-Ro autoantibodies determines TNF-alpha production that reaches a maximum at 16 h and is decreased (P < 0.05) at 24 and 48 h. Adalimumab seems to be more efficacious than etanercept in blocking TNF-alpha-mediated apoptosis. The YOPRO-1 (+) and propidium iodide (-) method revealed 60% of apoptotic cells after 24 h of incubation with anti-Ro compared with 15% of apoptotic cells treated with anti-Ro plus adalimumab and 25% of apoptotic cells treated with anti-Ro plus etanercept. The antiapoptotic effect of adalimumab and etanercept was supported by inhibition of DNA laddering induced by anti-Ro Abs. These data validate the therapeutic efficacy of the anti-TNF reagents in the treatment of autoimmune disorders.
authors.affiliation: Department of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy. m.sisto@anatomia.uniba.it
authors.forename: Margherita
authors.initials: M
authors.lastname: Sisto
authors.forename: Massimo
authors.initials: M
authors.lastname: D'Amore
authors.forename: Simone
authors.initials: S
authors.lastname: Caprio
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Mitolo
authors.forename: Pasquale
authors.initials: P
authors.lastname: Scagliusi
authors.forename: Sabrina
authors.initials: S
authors.lastname: Lisi
mesh_terms: D000068879:Adalimumab; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000974:Antibodies, Antinuclear / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D053938:DNA Fragmentation / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000068800:Etanercept; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012469:Salivary Glands / Q000166:cytology*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000893:Anti-Inflammatory Agents; D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D007074:Immunoglobulin G; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; C035356:SS-A antibodies; C035357:SS-B antibodies; D014409:Tumor Necrosis Factor-alpha; D000068879:Adalimumab; D000068800:Etanercept