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Am J Physiol Cell Physiol 287: C1763-C1768, 2004. First published August 25, 2004; doi:10.1152/ajpcell.00024.2004
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Methods in Cell Physiology

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

Valérie Estienne, Nadège Brisbarre, Stéphanie Blanchin, Josée-Martine Durand-Gorde, Pierre Carayon, and Jean Ruf

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


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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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-µm-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}·cm2, 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 (Fc{gamma}RIIB2) 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.

autoimmunity; cell-cell interactions; cell culture procedure; tight junctions

AUTOIMMUNE THYROID DISEASES (AITD) include a whole set of conditions ranging from the hyperthyroidism associated with Graves' disease (GD) to the hypothyroidism associated with Hashimoto's thyroiditis (HT), which are the two archetypal autoimmune endocrine diseases. AITD involve genetic and environmental factors and are associated with functional impairment of both the immune system and the target organ. Identifying the initial and subsequent events involved in AITD should make it possible to develop new strategies for treating the whole range of organ-specific autoimmune disorders (32). It is now believed that in addition to the conventional immune cells, the thyrocytes themselves play an important role in the autoimmune process. During AITD, the thyrocytes are in close contact with the lymphocytes and the monocytes and macrophages that invade the gland, and in response to the release of proinflammatory cytokines such as {gamma}-interferon, they aberrantly express DR class II human leukocyte antigen (HLA-DR) (4). Autoimmune thyrocytes also express additional molecules such as the HLA-DMB, -invariant chain Ii, -DRA, and class II transactivator molecules, which are generally restricted to professional antigen presenting cells (APC) (33). We recently reported that GD thyrocytes express Fc{gamma}RIIB2 in an androgen-dependent manner; Fc{gamma}RIIB2 is an Fc receptor for the IgG present on monocytes and macrophages, which is involved in the processing of the antigen (9). It emerges from these data that thyrocytes are potentially capable of antigen presentation, but there is still some controversy as to whether they are actually involved in inducing AITD, since no B7 (CD80) costimulator molecule expression is observed either in vivo or in vitro after the cells are stimulated with multiple cytokines (29). AITD is indeed initiated by T helper lymphocyte stimulation, which occurs when the T-cell autoepitope associated with HLA class II molecules is correctly expressed together with an appropriate costimulating signal emitted by the APC. Antigen presentation by thyrocytes themselves without any B7 costimulator may result in T-cell anergy (19). Alternatively, a costimulatory signal may be provided by professional APC bystanders (20). On the other hand, B7.1 has been found to exist in HT but not in GD thyrocytes (3). The discrepancies between the data available make it rather difficult to specify the exact role played by thyrocytes in the etiology of AITD.

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.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Primary human thyrocyte cell cultures. Thyroid tissues were obtained from various patients who underwent thyroidectomy for GD, who gave their informed consent. The study protocol was approved by the local ethics committee. Each patient's tissue was used to prepare a single primary cell culture as previously described (26). Briefly, the tissue was minced into small fragments. The tissue fragments were washed in Coon's modified Ham's F-12 medium (Sigma, St. Louis, MO) containing 2.6 g/l sodium carbonate (Merck, Darmstadt, Germany), penicillin/streptomycin (100 µg/ml) (Life Technologies, Grand Island, NY), fungizone (2.5 µg/ml) (Life Technologies) and kanamycin (100 µg/ml) (Life Technologies). To separate the epithelial cells from the connective tissue, the fragments were digested with collagenase I (1 mg/ml) (Life Technologies) and dispase II (2.4 U/ml) (Roche Diagnostics, Meylan, France) in a calcium- and magnesium-free phosphate-buffered saline (PBS), pH 7.4, at 37°C for 15 min. The enzymatic digestion procedure was repeated for 60 min with fresh enzyme mixture. The digested tissue was filtered though sterile gauze. Culture medium was added and the cells were washed by performing several centrifugations at 100 g for 5 min. Contaminating red cells were lysed for 5 min at 37°C with 0.16 M NH4Cl, 0.17 M Tris buffer, pH 7.2, and cells from thyroid were washed again and grown at 37°C (in a humidified air atmosphere containing 5% CO2) in the initial medium supplemented with 5% fetal calf serum (FCS) (Valbiotech, Paris, France), in the presence of the following six factors: bovine TSH (1 U/l) (Sigma); human insulin (10 mg/l) (Boehringer); somatostatin (10 µg/l) (Novabiochem, Läufelfingen, Switzerland); human transferrin (6 mg/l) (Roche Diagnostics); hydrocortisone (10–8 M) (Calbiochem, La Jolla, CA); and glycyl-histidyl-lysine acetate (10 µg/l) (Calbiochem); this medium corresponds to the 6H medium described by Ambesi-Impiobato et al. (1). The culture medium also contained L-glutamine, 2 mM (Life Technologies) and nonessential amino acids (ICN Pharmaceuticals, Costa Mesa, CA). Sterile Falcon cell culture inserts (Becton Dickinson, Franklin Lakes, NJ), with a 0.3-cm2 permeable polyethylene terephthalate filter having a pore size of 8 µm were first immersed in the inverted position in wells of deep well plates of Biocoat inserts (Becton Dickinson) containing culture medium up to the level of the lower face of the filter. Cells from thyroid patients were then seeded at a density of 0.75 x 106 cells/60 µl medium on the upper face of the filter and grown for 24 h. Nonadherent cells were then discarded and inserts were returned to their normal orientation before being placed in the wells of the Multiwell companion culture plates (Becton Dickinson). Fresh medium was added to the lower (1 ml) and upper (500 µl) chambers to obtain identical fluid levels in both chambers, thus preventing the formation of a hydrostatic pressure gradient. The adherent cells were subsequently grown for 9 days, resulting in a thyrocyte monolayer with the basolateral surface exposed on the underside of the insert filter. Culture medium was exchanged every 3 days. In each primary cell culture, only filters which developed confluent thyroid epithelial cells with a TER greater than 1,000 {Omega}·cm2 were subsequently used.

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 {Omega}·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 10–15 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 Fc{gamma}RIIB2 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.


   RESULTS AND DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
Thyrocytes forming a monolayer on one side of the large- pore filter. In the present cell-cell interaction model based on a bicameral chamber, the thyrocyte monolayer was grown on the underside of the filter of the porous chamber, using an inverted insert as depicted in Fig. 1. We found the polyethylene terephtalate filter from Becton Dickinson Labware particularly suitable for use with thyrocyte growth procedures; this filter abolishes the need for the collagen coating required to improve pig (13) and human (25) thyrocyte cultures on other membrane supports. Interestingly, the large pore size (8 µm) of the filter made it possible to establish cellular contacts across the filter between polarized thyrocytes and lymphocytes. We found the constant use of the 6H medium in the presence of 5% FCS to provide the most suitable culture conditions for efficiently growing GD thyrocytes in a monolayer on this kind of material. Thyrocytes were plated at a density (0.75 x 106 cells/60 µl medium) that minimized cell division and prevented the formation of multilayers.



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  		Fig. 1. Diagram of the coculture model. Human thyrocytes are grown on the underside of 8-µm-pore polyethylene terephtalate culture insert filters. When lymphocytes are added to the upper chamber, they make close contacts across the filter with the basal membrane of the thyrocytes.

 
To study the morphology of the thyrocytes grown in vitro on this material, cells bound to the filter were subjected to light and electron microscopy. Under light microscopy, the thyroid monolayer was found to have developed on the lower side of the filter without any signs of cell multilayering (Fig. 2A). The cytoplasm of the plated cells had a bright appearance with well-delineated nucleus (Fig. 2B). The multiple pores of the filter (1 x 105 pores/cm2) visible through the transparent blue-colored thyrocytes on one side of the filter obviously allowed the passage of ITTL cocultured at the opposite side of the filter. Because the size of a thyrocyte was really larger than that of a pore, each thyrocyte was able to come into contact with several lymphocytes (Fig. 2B).



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  		Fig. 2. Light micrograph of thyrocyte monolayer on a porous filter. Thyrocytes were plated onto the filter and grown until they reached confluence. The filter was then processed for light microscopy. A: cross section of hematoxylin-eosin-colored thyrocytes grown on a porous filter. B: overview of the methylene/azure blue-colored thyrocyte monolayer plated on the filter. Note the large pores of the filter (arrows). Scale bar: 20 µm.

 
To check the integrity of the cell monolayer, filters were left until they were overgrown confluently by the thyrocytes (as indicated by the TER value) and observed by performing scanning electron microscopy. Cells were found to be homogeneously attached to the underside of the filter, masking the large pores (Fig. 3A), whereas no cells were present on the other side of the filter, where the pores were still clearly visible (Fig. 3B).



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  		Fig. 3. Scanning electron micrograph of thyrocytes bound to the filter. A: confluent thyrocyte culture on the underside of the filter. B: the other side is devoid of thyrocytes, and the pores of the filter (arrows) are therefore visible. Scale bar: 20 µm.

 
Thyrocytes formed a tight epithelium when grown to confluence on permeable filters. The barrier function of the thyrocyte monolayer was checked by measuring TER across the filter. After the cells were plated, the TER value gradually increased, reaching a maximum on day 8 ranging from 1,000 to 1,600 {Omega}·cm2 based on cells from five different cell cultures, and then decreased to 200 {Omega}·cm2 on day 10 (Fig. 4A). Like lymphocytes, GD thyrocytes themselves were found to express immunological molecules such as the proinflammatory cytokine IL-1{alpha} (12). The fact that 24-h exposure of IL-1{alpha} reduces the thyroid epithelial tightness in filter-cultured human thyrocytes (24) was thought to possibly explain the rapid decrease in the TER values observed here when the tightly assembled confluent thyroid monolayer was exposed to secreted IL-1{alpha}.



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  		Fig. 4. Barrier function measurement of the human thyrocyte monolayer. Thyrocytes were plated onto porous filters and grown in 6H medium. Representative data obtained with primary cell cultures from 5 different Graves’ disease (GD) tissues are shown. A: time course and transepithelial resistance (TER) values of thyrocytes grown on porous filters. The maximum TER, reflecting the epithelial tightness, was consistently obtained after 8 days of culture. B: intrathyroidal T lymphocyte (ITTL) leakage assay. 1 x 105 viable ITTL were recounted after undergoing 24 h of culture in the upper chamber with or without the presence of the thyrocyte monolayer grown for 8 days. C: fluorescence micrograph of thyrocyte monolayer on filters stained with ZO-1 MAb. Filter-cultured thyrocytes grown 8 days in 6H medium with a TER of 1,200 {Omega}·cm2 are shown. Immunoreactivity of ZO-1 is present at the cell periphery: the cell borders have a smooth, hexagonal appearance, which indicates the presence of tight junctions around each cell.

 
The barrier function of the thyrocyte monolayer was therefore checked to determine whether it allowed the passage of intact ITTL seeded into the upper chamber on day 8 through the thyrocyte barrier. ITTL were counted in the lower chamber after 24 h of exposure to the filter with or without the thyrocyte monolayer. ITTL were detected only in the lower chamber on day 9, when the thyrocyte monolayer was missing (Fig. 4B). This study showed the great permeability of the filter that allowed the passage of lymphocytes through the large pores in the absence of the thyrocyte monolayer closing the gaps.

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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  		Fig. 5. Indirect immunofluorescence staining of human thyrocyte monolayer with various antibodies used as specific cell-surface markers. Staining was performed on either the apical or the basolateral surface of thyrocytes forming a tight monolayer on porous filters. Representative polarized pattern of expression of thyroperoxidase (TPO) on the apical membrane (A), thyroid stimulating hormone receptor (TSH-R) on the basolateral membrane (B), human leukocyte antigen (HLA-DR) on the basolateral membrane (C), and Fc{gamma}RIIB2 on the apical membrane (D).

 
In various inflammatory conditions, HLA class II molecules have been observed on the surface of epithelial cells such as thyroid (14) and intestinal epithelial cells (21). Based on these findings, it has been suggested that thyroid (4) and intestinal (16) cells may act like APC. It has been further demonstrated that HLA class II expression is restricted to the basolateral surface of intestinal epithelial cells (15). Because we were using thyrocyte monolayers of autoimmune origin, we performed immunofluorescence studies to determine the polarity of the endogenously expressed HLA-DR molecules on the thyrocyte surface. Interestingly, the pattern of staining obtained with a specific antibody showed that there was a basolateral expression of HLA-DR in the cells (Fig. 5C). It was previously observed that the pattern of HLA-DR expression of GD thyrocytes is preserved in medium containing TSH (31), whereas the restricted pattern of basolateral HLA-DR distribution observed here in human thyrocytes conflicts with previous data showing that these cell surface molecules have a nonpolarized pattern of distribution (22).

We recently established that GD thyrocytes express Fc{gamma}RIIB2 (9), an IgG Fc receptor that mediates the internalization and lysosomal degradation of IgG-antigen complexes in monocytes and macrophages (17). Fc{gamma}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{gamma}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{gamma}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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  		Fig. 6. ITTL stimulation assay. ITTL were isolated from nonadherent cells and grown in 6H medium. Representative data obtained with primary cell cultures from 5 different GD tissues are shown. 1 x 105 viable ITTL were recounted after undergoing 24 h of coculture in the upper or lower chamber in the presence of the autologous thyrocyte monolayer cultured on the lower or upper surface of the filter for 8 days. Under these conditions, either ITTL contacted the thyrocytes through the large pores of the filter (+contact) or all contact was prevented from occurring between the ITTL seeded into the bottom of the lower chamber and the thyrocytes kept at a distance over the filter in the upper chamber (–contact), respectively. Control experiments were run by growing ITTL in the wells without any insert: they reached 0.91 x 105 ± 0.23 x 105 ITTL after 24 h (means ± SD; n = 5).

 
A new model for thyroid autoimmunity. In conclusion, the cell culture method used in the present study provides a useful model for the cellular interactions occurring between polarized thyroid epithelial cells and resident lymphocytes. To our knowledge, this is the first time the strictly polarized pattern of expression of HLA-DR and Fc{gamma}RIIB2 has been brought to light in human thyrocytes growing in vitro in monolayers. Interestingly, our pilot coculture experiments showed that cell-cell contacts actually occurred in our model, which made it possible for ITTL to be stimulated upon contacting thyrocytes. The method described here should provide a useful tool for further studies on the role of epithelial cells in antigen processing mechanisms and those designed to take a closer look at the antigenic material internalized on the apical side and the T-cell epitopes expressed on the basal side of polarized cells.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
REFERENCES
 
V. Estienne, N. Brisbarre, and S. Blanchin are recipients of grants from the Société Française d'Endocrinologie and the Fondation pour la Recherche Médicale, the Association pour le Développement des Recherches Biologiques et Médicales, and the Association pour le Développement des Recherches Biologiques et Médicales and the Fondation pour la Recherche Médicale, respectively.


   ACKNOWLEDGMENTS
 
We extend special thanks to Mr. Coquelet (Becton Dickinson France S.A.) for helpful technical advice about cell culture materials. We also thank H. Borghi and C. Allasia, who performed microscope examinations. We gratefully acknowledge the help of Prof. J. F. Henry and Dr. C. De Micco, who provided the thyroid specimens, Dr. J. P. Banga, who generously provided aliquots of the TSH-receptor MAb A9, and Dr. O. Chabaud for fruitful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Ruf, French Institute of Health and Medical Research (INSERM) Unit 555, Faculté de Médecine Timone, Université de la Méditerranée, 27, Boulevard Jean Moulin, F-13385 Marseille Cedex 5, France (E-mail: Jean.Ruf{at}medecine.univ-mrs.fr)

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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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
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