A critical role for TRAIL in resolution of granulomatous experimental autoimmune thyroiditis

Abstract

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin (MTG)-sensitized splenocytes activated in vitro with MTG and IL-12. Thyroid lesions reach maximal severity 20 days after cell transfer, and usually resolve or progress to fibrosis by day 60 depending on the extent of thyroid damage at day 20. Our previous studies indicated that neutralization of TNF-α or FasL had no effect on G-EAT induction, but neutralization of TNF-α promoted, while neutralization of FasL inhibited G-EAT resolution. TNF-related apoptosisinducing ligand (TRAIL) is a member of the TNF superfamily. This study was undertaken to define the role of endogenous TRAIL in G-EAT development and/or resolution. Neutralization of endogenous TRAIL had little effect on G-EAT induction, but significantly inhibited G-EAT resolution and increased thyroid fibrosis. This correlated with higher expression of proinflammatory cytokines and preferential expression of the pro-apoptotic molecule TRAIL, and anti-apoptotic molecules FLIP and Bcl-xL on inflammatory cells in thyroids of anti-TRAILtreated recipients. Furthermore, CD4+ T cells outnumbered CD8+ T cells in thyroids of anti-TRAIL-treated recipients. The results suggest that endogenous TRAIL is not required for G-EAT development in recipients, but is critical for G-EAT resolution. Endogenous TRAIL might promote resolution, at least in part, through modulation of the balance between pro-and antiinflammatory cytokines, and the expression pattern of pro-and anti-apoptotic molecules of thyroid epithelial cells (TEC) and inflammatory cells.

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

“…Therefore, the decreased expression of proinflammatory cytokines in CD2-FLIP transgenic mice was apparently one major factor that resulted in their overall reduced susceptibility to G-EAT. 7,21,23,24 These results would be consistent with findings by others suggesting that transgenic overexpression of FLIP in T cells results in decreased T cell activation. 37 Although transgenic overexpression of FLIP in T cells may have resulted in decreased T cell activation in our CD2-FLIP transgenic mice, the fact that depletion of donor CD8 ϩ T cells increased the ability of splenocytes from CD2-FLIP Tg ϩ mice to transfer G-EAT is consistent with the idea that apoptosis-resistant CD8 ϩ T cells are, at least in part, responsible for the relative resistance to G-EAT in recipients of cells from CD2-FLIP transgenic donors.…”

“…FLIP mRNA was highly expressed in thyroids of recipients of splenocytes from Tg ϩ donors ( Figure 6A), whereas much lower levels of FLIP were detected in thyroids of recipients of splenocytes from FLIP Tg Ϫ donors with severe G-EAT ( Figure 6A). As shown previously, 20,21,23,24 cytokine mRNA was undetectable in thyroids of normal Tg ϩ or Tg Ϫ mice ( Figure 6, B-F). Consistent with the results *Supernatants were obtained at the time cells were harvested for transfer to recipient mice.…”

“…26,27 Apoptosis of cultured splenocytes was determined by TUNEL assay using an ApopTag kit (Chemicon International, Temecula, CA), as described previously. 23,24,26 NovaRED substrate (Vector Laboratories, Burlingame, CA) was used for color development, and slides were counterstained with hematoxylin. To quantify apoptotic cells, all cells in five or six randomly selected high-power fields (magnification, ϫ400) were manually counted and positive cells were expressed as a percentage of total cells.…”

“…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).…”

“…Primer sequences were as described previously. 21,23,24 Cytokine Determination by ELISA Culture supernatants from 72-hour cultured splenocytes were collected and frozen at Ϫ20°C. IFN-␥, IL-17, TNF-␣, and IL-10 concentrations in supernatants were determined by enzyme-linked immunosorbent assay (ELISA) using mouse Ready-Set-Go ELISA kits (eBioscience), according to the manufacturer's instructions.…”