French Institute of Health and Medical Research (INSERM) Unit 555, Faculté de Médecine Timone, Université de la Méditerranée, Marseille, France
Submitted 15 January 2004 ; accepted in final form 19 August 2004
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ABSTRACT |
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autoimmunity; cell-cell interactions; cell culture procedure; tight junctions
The physiological role of thyrocytes, which are polarized cells arranged in monolayers around a closed colloidal space, consists mainly of producing thyroid hormones. The iodide trapped by these cells from the peripheral circulation on the basal side of the cell is subsequently used on the apical side to iodinate the thyroglobulin (Tg) used as the substrate in the process of hormone synthesis. This process also involves the action of thyroperoxidase (TPO), a membrane-bound enzyme, and that of the H2O2 produced by a nicotinamide adenine dinucleotide phosphate oxidase. The Tg bearing the thyroid hormones is then internalized by the thyrocytes and degraded by proteases, releasing the hormones into the peripheral bloodstream (30). Cellular models have been widely used to study thyroid function, and the use of cells kept in vitro on porous filters in bicameral chambers has greatly improved the model, since these cells mimic the functional polarity of the in vivo cells and give access to the apical and basal cell membranes separately (6). Bicameral chambers have also been used to study cell-cell interactions, since they make it possible to distinguish between those mechanisms, which depend on soluble factors (11) and those involving cellular contacts (15, 27). However, cell culture systems of this kind have not yet been used in studies on thyrocyte-lymphocyte interactions. It is of paramount importance to determine whether thyrocytes, either alone or with the help of conventional APC, are able to stimulate the autologous intrathyroidal T lymphocytes (ITTL) and, if so, to determine how the stimulation process works. The present study was therefore performed with a view to determining the optimum conditions for growing human thyrocyte monolayers on the underside of large-pore filters; this makes it possible for the polarized thyrocytes to make contact through the filter with the ITTL seeded on the other side of the filter at the bottom of the upper chamber. The thyroid monolayer developed as expected on one side of the filter, and the transepithelial resistance (TER) and tight junctions observed showed that it was properly assembled. GD thyrocyte cultures prepared with thyroid stimulating hormone (TSH) expressed constitutional membrane markers in the appropriate surface orientation as well as aberrantly expressed additional markers with restricted orientations, and they stimulated the proliferation of autologous ITTL in the upper chamber without any leakage occurring through the epithelial barrier.
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MATERIALS AND METHODS |
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Isolation of intrathyroidal T lymphocytes. To obtain the autologous intrathyroidal T lymphocytes (ITTL) from each thyroid cell suspension, 1 x 107 cells from the total thyroid digest obtained after red cell lysis were grown 24 h in a 250-ml Falcon tissue culture flask (Becton Dickinson), as described above. Nonadherent cells were then harvested, extensively washed in PBS, and passed through magnetic beads coupled with a cocktail of CD11b, CD16, CD19, CD36, and CD56 antibodies for the depletion of non-T cells using the MACS LS separation column and the pan T cell isolation kit according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the ITTL preparation (>90%) was further confirmed by flow cytometry using a CD3 monoclonal antibody (AL ImmunoTools, Friesoythe, Germany). ITTL were stored frozen in FCS containing 10% DMSO until use.
Transepithelial electrical resistance.
Transepithelial electrical resistance (TER) was assessed using a Millicell electrical resistance system (Millipore, Bedford, MA) according to the manufacturer's instructions. TER was used as an index to the barrier function of the epithelial monolayer and was measured daily across the cells that bound to the filter separating the two chambers when the inserts were normally oriented in the culture plate. TER was corrected for low background level using a blank filter carrying any cells. Results were converted into ·cm2 based on the surface area of the filter.
Microscopy. Filters with confluent thyroid epithelial cells were used for light microscopy, as follows: cells bound to the filter were fixed in 2% glutaraldehyde phosphate buffer, pH 7.3, and embedded in Cryomatrix (ThermoShandon, Pittsburgh, PA). Sections 1015 µm thick were cut and stained in hematoxylin and eosin or methylene azure blue solution. Specimens were examined for monolayer formation under a Zeiss Axioplan light microscope (Lepeq, France). Before scanning electron microscopy was performed, cells bound to the filter were fixed in 2% glutaraldehyde phosphate buffer, pH 7.3, dehydrated, and coated with gold using a Jeol Fine Coat Ion Sputter JFC-1100 (Tokyo, Japan). The two sides of the filter were analyzed with a Fei Quanta 200 scanning electron microscope (Limeil-Brévannes, France).
Immunofluorescence.
Filters carrying confluent thyroid epithelial cells were washed with PBS, pH 7.4, containing 0.9 mM CaCl2 and 0.45 mM MgCl2 (PBS+) and fixed for 30 min with 2% paraformaldehyde. After a quenching step with 50 mM cold NH4Cl in PBS for 10 min, unspecific binding sites were blocked for 30 min with 10% FCS in PBS+. Primary antibody was then added to either the upper or lower chamber for basolateral or apical labeling, respectively. Unlabeled murine monoclonal antibody (MAb) to human ZO-1 tight junction protein (Zymed Laboratories, San Francisco, CA), human TPO (MAb 47) (28), and human TSH receptor (MAb A9) (23) were used at appropriate dilutions in PBS+, 10% FCS. Rhodamine-labeled anti-mouse secondary antibodies (Immunotech, Marseille, France) were used for fluorescence labeling. FITC-labeled anti-human HLA-DR and FcRIIB2 antibodies (BD Biosciences Pharmingen, San Diego, CA) were used directly without any secondary antibodies. Stained cells were mounted in Mowiol (Calbiochem) and viewed in an Olympus fluorescence microscope using a x50 oil-immersion lens. Pictures were acquired with the Kodak DC290 Zoom Digital Camera equipped with the Kodak MDS290 software program (Eastman Kodak, New Haven, CT). The Photoshop software program (v6; Adobe System) was used for image editing.
Thyrocyte-lymphocyte coculture. Inserts carrying confluent thyroid epithelial cells on the lower or upper surface of the filter were used. Freshly thawed ITTL (1 x 105 cells/500 µl) were seeded into the upper or lower chamber and incubated for 24 h at 37°C in 6H medium to allow them to sediment and establish (or not) close contacts with the thyroid monolayer. Control tests were run without the thyrocyte monolayer. Viable ITTL from the lower or upper chambers were then counted using the Trypan blue dye exclusion method with a Malassez cytometer.
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RESULTS AND DISCUSSION |
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The pattern of thyrocyte staining obtained on day 8 with a specific MAb directed against ZO-1, a tight junction protein thought to be involved in the establishment and maintenance of epithelial barriers (10), was found to be distributed throughout the cell-cell contacts, delineating the entire cell borders (Fig. 4C). This finding further showed that the human thyroid epithelium was successfully organized and suggested that the filter-grown cells formed a tight barrier between the culture chambers, as previously reported to occur with human thyrocytes plated onto permeable collagen-coated filters (25).
The thyrocyte monolayer showed functional polarity. Numerous studies have been performed on pig (7) and human (25) thyrocyte monolayers, based on the fact that these cells secrete Tg vectorially into the apical culture medium and thus mimic the polarity of the in vivo cells. Cell polarity was assessed here by identifying cell-surface markers with specific MAb. Functional TPO is present in the apical membrane facing the colloidal space, and the TSH receptor is located in the basolateral membrane, which is in contact with the bloodstream (30). As expected, the cell-surface immunolocalization of TPO (Fig. 5A) and TSH receptor (Fig. 5B) in filter-cultured human thyrocytes was found to be strictly apical and basolateral, respectively.
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We recently established that GD thyrocytes express FcRIIB2 (9), an IgG Fc receptor that mediates the internalization and lysosomal degradation of IgG-antigen complexes in monocytes and macrophages (17). Fc
RIIB2 was found to increase the efficiency of the antigen bound to IgG before being presented by HLA class II by facilitating its uptake and lysosomal delivery, and hence its proteolytic processing (2, 18). Since the question arose as to whether Fc
RIIB2 might be involved in the thyrocyte antigen presentation process, we analyzed the expression of this molecule in our polarized model, as previously done with the HLA-DR molecule. The expression of Fc
RIIB2 was found to be restricted to the apical membrane of the thyrocytes (Fig. 5D), where iodinated Tg is normally internalized into the cell and used for hormone production. The latter finding was in complete agreement with the recently reported modulatory role of IgG in the Fc receptor-mediated Tg presentation process (8).
The thyrocyte monolayer established a close contact with ITTL. This model was used to test the possibility that ITTL directly contact the thyrocytes through the large pores of the filter. This physical contact may result in the establishment of an immunological synapse (5) able to stimulate ITTL proliferation and differentiation. To distinguish between the direct effects generated by cell-cell contacts and indirect effects induced by cytokines, we also used our model in a more conventional way, i.e., with the thyrocyte monolayer on the upper side of the filter at the bottom of the upper chamber and the ITTL seeded into the lower chamber. In both situations, the basal pole of the thyrocytes faced the ITTL. Control experiments were performed with ITTL seeded into cell culture wells without the inserts containing thyrocyte monolayer. Figure 6 clearly shows that ITTL proliferated for 24 h after contacting the thyrocyte monolayer. By contrast, under paracrine conditions preventing cell-cell contacts, the ITTL did not proliferate, and some of them died as under the control conditions (Fig. 6).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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