Murine FLIP Transgene Expressed on Thyroid Epithelial Cells Promotes Resolution of Granulomatous Experimental Autoimmune Thyroiditis in DBA/1 Mice

Abstract

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin-sen

Effect of Transgenic Overexpression of FLIP on Lymphocytes on Development and Resolution of Experimental Autoimmune Thyroiditis

“…FVB mice, like DBA/1 mice, express the H-2 q major histocompatibility complex, but are less susceptible to EAT. 21 The transgenic female FVB founder was therefore crossed with an EAT-susceptible DBA/1 male, and Tg ϩ F1 offspring were selected by PCR amplification of tail DNA. Tg ϩ F1 mice were backcrossed six times to DBA/1 mice, and offspring were selected at each generation for expression of the transgene and the DBA/1 coat color.…”

“…Western blotting was performed as described previously, 21 using 30 g of protein loaded on a 10% SDS-PAGE gel.…”

“…21,23 Polyclonal anti-FLIP, anti-FLAG (Abcam), and active caspase-3 (BD Pharmingen, San Diego, CA) antibodies, together with rat anti-CD4, anti-CD8 or anti-B220 monoclonal antibody, were used on frozen sections of spleens. FLAG, FLIP, and active caspase-3 were visualized with Alexa Fluor 568 (red; Molecular Probes-Invitrogen, Eugene, OR).…”

“…21,23,24 To determine the relative initial amounts of target cDNA, each cDNA sample was serially diluted 1/5 and 1/25. 21,23,24 PCR products were electrophoresed in 2% agarose gel, visualized by UV light after staining with ethidium bromide, and normalized between samples relative to levels of hypoxanthine phosphoribosyltransferase (HPRT) or ␤-actin using IS-1000 digital imaging system software (Alpha Innotech-Cell Biosciences, Santa Clara, CA).…”

“…17 Expression of transgenic FLIP on thyroid epithelial cells promotes earlier resolution of G-EAT by protecting thyroid epithelial cells from Fas-mediated apoptosis. 20,21 Because CD4 ϩ T cells are the primary effector cells for G-EAT, we hypothesized that if transgenic FLIP were expressed on lymphocytes, CD4 ϩ effectors would be protected from Fas-mediated apoptosis, and resolution would be inhibited, resulting in chronic inflammation. Transgenic (Tg) mice overexpressing FLIP under the CD2 promoter were generated to test this hypothesis.…”