An in vitro model based on cell monolayers grown on the underside of large- pore filters in bicameral chambers for studying thyrocyte-lymphocyte interactions

Abstract

In the processes underlying thyroid autoimmunity, thyrocytes probably act as antigen-presenting cells exposing T-cell epitopes to intrathyroid lymphocytes. To study the interactions between lymphocytes and thyrocytes, which are arranged in a tight, polarized monolayer, we developed a new in vitro model based on human thyrocytes grown on the underside of a filter placed in a bicameral chamber. Thyrocytes from Graves' disease glands were plated onto the upper face of a 8-mum-pore polyethylene terephthalate culture insert filter placed in the inverted position and grown for 24 h before the insert was returned to the normal position for a week in the cell culture plate wells. Thyrocytes grown in the presence of thyroid stimulating hormone, forming a homogeneous monolayer on the underside of the filter, reached confluence after 8 days in vitro. The cells developed a transepithelial electrical resistance >1,000 Omega.cm(2), and the ZO-1 tight junction protein showed a junctional pattern of distribution. Thyrocytes showed a polarized pattern of thyroperoxidase and thyroid stimulating hormone receptor expression in the apical and basolateral positions, respectively. They were also found to aberrantly express DR class II human leukocyte antigen and an Fc immunoglobulin receptor (FcgammaRIIB2) in the basolateral and apical positions, respectively. Autologous intrathyroidal T lymphocytes cocultured for 24 h across the filter with the thyrocyte monolayer proliferated and remained in the upper chamber without any leakage occurring through the epithelial barrier, which makes this model particularly suitable for studying the cell-cell interactions involved in antigen processing.

Interaction of Lymphocytes and Thyrocytes in Graves’ Disease and Nonautoimmune Thyroid Diseases in Immunohistochemical and Ultrastructural Investigations

“…Graves’ disease patients seem to have mixed Th1/Th2 profiles [23]. In the in vitro investigations, Estienne et al [24] indicated the possibility of forming a so-called immunological synapse of a tight junction between lymphocytes and thyrocytes with participation of adhesive proteins. This physical contact may result in the establishment of an immunological synapse [25] able to stimulate intrathyroid T lymphocyte proliferation and differentiation [24].…”

“…In the in vitro investigations, Estienne et al [24] indicated the possibility of forming a so-called immunological synapse of a tight junction between lymphocytes and thyrocytes with participation of adhesive proteins. This physical contact may result in the establishment of an immunological synapse [25] able to stimulate intrathyroid T lymphocyte proliferation and differentiation [24]. In our immunohistochemical investigations, it was the CD8+ lymphocytes that most frequently entered the basal membrane of thyroid follicles.…”

Graves’ Disease - The Interaction of Lymphocytes and Thyroid Cells

“…Presentation of antigens by thyrocytes without the costimulatory molecule B7 does not lead to activation of T-cells [Marelli-Berg et al, 1997]. The expression APC characteristic antigens are dependent on TSH [Todd et al, 1987, Estienne et al, 2004. Thyrocytes may produce HLA I under the influence of cytokines of lymphocytes present in the thyroid.…”

Endogenous and exogenous citrate transport and release in prostatic preparations: semi-polarized two-dimensional cultures of human PNT2-C2 cells and isolated tubules and segments of rat prostate

“…PNT2-C2 cells were derived from luminal epithelial cells of normal human prostate [18][19][20]. In the micro-pore filter/Ussing chamber approach generally, cells were ''induced'' to polarize in the epithelial monolayer in a number of different, sometimes complex ways [21,22]. In the present study, no specific form of induction was used apart from pre-treatment of the membrane with serum which, to some extent, could have influenced the polarization of the epithelial layer.…”