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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 3 945-952
Copyright © 1998 by The Endocrine Society

Original Studies

Cytokines and Thyroid Epithelial Integrity: Interleukin-1{alpha} Induces Dissociation of the Junctional Complex and Paracellular Leakage in Filter-Cultured Human Thyrocytes1

Mikael Nilsson2, Johanna Husmark, Ulla Björkman and Lars E. Ericson

Institute of Anatomy and Cell Biology, Göteborg University, S-413 90 Göteborg, Sweden

Address all correspondence and requests for reprints to: Mikael Nilsson, Institute of Anatomy and Cell Biology, Göteborg University, Box 420, (SE) 405 30, Göteborg, Sweden. E-mail: mikael.olof.nilsson{at}anat.cell.gu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Locally produced proinflammatory cytokines are likely to play a pathophysiological role in autoimmune thyroid disease. An important feature of the thyroid, not previously considered in cytokine actions, is the barrier created by the follicular epithelium, which secludes two lumenal autoantigens [thyroglobulin (Tg) and thyroperoxidase] from the extrafollicular space. We examined the influence of recombinant cytokines on the barrier function of human thyrocytes cultured as a tight and polarized monolayer in bicameral chambers. Whereas interleukin (IL)-6 (100 U/mL), interferon-{gamma} (100 U/mL), tumor necrosis factor-{alpha} (10 ng/mL), and transforming growth factor-ß1 (10 ng/mL) had no effects, exposure to IL-1{alpha} for 24–48 h reduced the transepithelial resistance from >1000 to <50 {Omega} x cm2 and increased the paracellular flux of [3H]inulin and exogeneous 125I-Tg. This response to IL-1{alpha}, which was dose dependent (1–1000 U/mL) and reversible, was accompanied by dramatic morphological changes of the epithelial junction complex, including aberrant localization of the tight junction protein zonula occludens-1. At the same time, IL-1{alpha} decreased the apical secretion of endogeneous Tg and stimulated the basolateral release of a novel high-molecular-mass protein. We conclude that IL-1{alpha} reduces the thyroid epithelial barrier without signs of general cytotoxicity. The observation suggests a mechanism by which IL-1{alpha} may promote the exposure of hidden autoantigens to the immune system in thyroid autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PATHOGENESIS of autoimmune thyroid diseases is multifactorial, involving environmental factors and aberrations of both the immune system and the target tissue (1). A major sign is tissue infiltration by immune cells, which produce autoantibodies against three major thyroid-specific antigens [thyroglobulin (Tg), thyroperoxidase (TPO), and the TSH receptor] and may also generate cell-mediated cytotoxicity. In these autoimmune reactions, locally released proinflammatory cytokines are known to be critically involved (2). For instance, interleukin (IL)-1, a pleiotropic cytokine consisting of {alpha}- and ß-forms (3), accelerates the onset of lymphocytic thyroiditis and insulin-dependent diabetes mellitus when injected to BB rat (4). However, whereas IL-1 is cytotoxic to the pancreatic ß-cells (5), the viability of thyrocytes does not seem to be affected (2, 6), indicating that IL-1 promotes thyroid autoimmunity by mechanisms other than target cell lysis. In different experimental systems, IL-1 has been found to stimulate thyroid cell proliferation (7) and inhibit several steps in the synthesis and release of thyroid hormones (8, 9, 10, 11, 12, 13) (reviewed in Refs. 2 and 6). In addition, IL-1 enhances the expression of major histocompatibility complex class II antigen (14), intercellular adhesion molecule-1 (15), and leukocyte function antigen (16) on thyrocytes and stimulates the thyroidal production of other cytokines, e.g. IL-6 (17) and IL-8 (18).

Thyrocytes hold a unique position among classical endocrine cells, in that they also exert an exocrine function and have a polarized, epithelial phenotype typical of exocrine cells. The functional unit of the thyroid is the follicle composed of a single-layered epithelium and a central cavity (the follicular lumen), in which Tg (the prohormone) is stored and iodothyronines are synthesized (19). The junctional complex of thyroid follicular cells consists, in part, of tight junctions (TJ) and adherens junctions (AJ), which encircle the cells close to their lumenal (apical) pole and limit paracellular permeability. As for other epithelial linings, a tight barrier between the extracellular compartments, the lumen and the extrafollicular space, is critical to normal thyroid function, because it promotes cell polarity and the establishment of transepithelial solute gradients of, for instance, iodide and Tg. Conversely, destruction of the paracellular barrier would challenge thyroid function and, in the context of autoimmunity, might facilitate the exposure of normally secluded autoantigens, Tg in the follicular lumen, and TPO in the apical plasma membrane, to the immune system. However, it is not known whether cytokines produced in autoimmune thyroid tissue have any effect on the thyroid epithelial barrier. In attempts to explore this issue, we investigated the effect of recombinant IL-1{alpha}, IL-6, interferon-{gamma} (IFN-{gamma}), tumor necrosis factor-{alpha} (TNF-{alpha}), and transforming growth factor-ß1 (TGF-ß1) on tight monolayers of human thyrocytes cultured on permeable filters in bicameral chambers. We found that IL-1{alpha} was a strong negative regulator of thyroid epithelial tightness, as evidenced by a reduced transepithelial resistance, an increased paracellular flux of radiotracers ([3H]inulin and 125I-Tg), and a rearrangement of the junctional complex in IL-1{alpha}-treated cells. In contrast, IFN-{gamma} and TNF-{alpha}, both known to interfere with the barrier function of cultured intestinal and renal epithelial cells (20, 21), had no effect on paracellular permeability and junctional morphology. This action of IL-1{alpha} has not been reported for any other epithelium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cytokines and antibodies

Human recombinant cytokines, IL-1{alpha} (5x107 U/mg), IL-6 (1x108 U/mg), IFN-{gamma} (2x107 U/mg), TNF-{alpha} (1x108 U/mg), and TGF-ß1, were purchased from Boehringer (Mannheim, Germany). Rabbit antihuman zonula occludens (ZO)-1 was from Zymed Laboratories (San Francisco, CA). Mouse monoclonal antibodies against E-cadherin and catenins were obtained from Transduction Laboratories (Lexington, KY). Horseradish peroxidase-conjugated rabbit antimouse IgG was from Dako A/S (Glostrup, Denmark). Biotinylated donkey antirabbit IgG and fluorescein-isothiocyanate-conjugated streptavidin were purchased from Amersham International plc (Amersham, England). Human Tg were purified and polyclonal sera were raised by immunization of rabbits, as described (22).

Isolation and culture of human thyrocytes

Thyroid follicles were isolated by enzymatic digestion of surgically excised Graves’ (n = 9) or normal (paradenomatous; n = 5) thyroid tissue, following a recently described protocol (23). After being separated from blood and interstitial cells by repeated centrifugation, the follicle segments were plated on the filter of bicameral culture inserts (Transwell 3413; Costar Corp., Cambridge, MA) precoated with collagen type I (Boehringer). The cells were cultured in humidified atmosphere (5% CO2) at 37 C in Coon’s modified Ham’s medium supplemented with penicillin (200 U/mL), streptomycin (200 U/mL), and fungizone (2.5 µg/mL) and enriched with 5% FCS (Gibco; Paisley, Scotland) and 5 factors (5H medium: insulin, bovine transferrin, hydrocortisone, glycyl-L-histidyl-L-lysine acetate and somatostatin; all reagents from Sigma (St. Louis, MO) or 6 factors (6H medium: in addition 10-9 mol/L bovine TSH from Sigma), according to the composition of culture medium originally described for FRTL cells (24). The DNA content of cultures was determined fluorometrically (25). All experimental observations were made on triplicate cultures of at least three independent platings, with similar results. There were no apparent differences in the response of cells from paradenomatous or Graves’ tissue to the cytokines added. The patients from which Graves’ thyroid tissue was obtained had been under treatment preoperatively with an antithyroid drug and T4.

Epithelial barrier assays

Paracellular tightness of cultured thyrocyte monolayers was assessed by measurement of the transepithelial electrical resistance (RTE) across the filter, with a Millicell ERS ohmmeter (Millipore; Bedford, MA). Paracellular permeability was determined by analysis of the transepithelial flux of either [3H]inulin (Amersham) or 125I-Tg. For this purpose, pig Tg (5 µmol/L), purified by chromatography on a Sepharose 6B column (Pharmacia Biotech, Uppsala, Sweden) and free from low molecular mass species (as determined by SDS-PAGE), was labeled with 125I- (Amersham) for 1 h at 37 C in the presence of lactoperoxidase (400 mU/mL; Sigma), glucose oxidase (60 mU/mL; Sigma), glucose (5 mmol/L), and a mixture of protease inhibitors: 0.1 mmol/L Pefabloc (Boehringer); 0.1 µmol/L leupeptin (Sigma); aprotinin (0.01 µmol/L) (Sigma). Radiolabeled Tg was dialyzed against PBS (pH 7.0), supplemented with 10 mmol/L KI and 1 mmol/L methimazole. [3H]inulin (1 µCi/mL) or samples (10 µL) of the 125I-Tg dialysate (0.1 µCi/pmol) were dissolved in 0.01 mol/L Tris-maleate buffer (pH 7.3), supplemented with 0.13 mol/L NaCl and KCl, CaCl2, MgCl2, and glucose, according to the specification of Tyrode solution, and added to the apical chamber compartment of filter-cultured thyrocytes ± IL-1{alpha} pretreatment. As a positive control of paracellular leakiness, sets of cultures were, simultaneously with the exposure to 125I-Tg, depleted of extracellular Ca2+ by exchanging the basal medium for Ca2+-free buffer containing 1 mmol/L ethylene glycol bis(ß-aminoethyl ether)-N,N'-tetraacetic acid (EGTA; Sigma); this treatment is known to disrupt the epithelial junction complex caused by abolition of Ca2+-dependent cell-cell adhesion (see Ref.32). After incubation for 20 min at 37 C, the amount of [3H]inulin present in the basal medium was analyzed in an LKB Wallac liquid scintillator (Wallac Sverige, Sollentuna, Sweden). After incubation for 20–60 min, the basal media of 125I-Tg-exposed cultures were collected and diluted to 1 mL with PBS (pH 7.0) containing 2 mmol/L methimazole, 0.1 mmol/L KI, and protease inhibitors (listed above) and then determined for total amount of radioactivity in a Packard auto-{gamma} counter (Packard Instrument Co., Dowers Grove, IL). The same media obtained from 125I-Tg-incubated cultures were then concentrated in a microconcentrator (Amicon Inc., Beverly, MA) and, after addition of Laemmli’s sample buffer, subjected to SDS-PAGE on an 8% gel, as described below. Autoradiographs of gels were prepared with Kodak BioMax MS film (Eastman Kodak, Rochester, NY).

Metabolic labeling, SDS-PAGE, and autoradiography

Filter-cultured cell monolayers were washed both apically and basally with serum-free MEM devoid of methionine (MEM-met) and incubated with 50 µCi/mL [35S]methionine in MEM-met present in the lower chamber, for 7 h at 37 C. The apical and basal media were then collected separately in the presence of protease inhibitor (0.5 mmol/L Pefabloc), dialyzed against large volumes of PBS (pH 7.0) at 4 C, and analyzed for content of protein-bound radioactivity, as described below. Equal volumes of dialysed media were also mixed with sample buffer, heated to 96 C for 4 min, and subjected to electrophoresis, together with [14C]methylated protein standards (Amersham), in a 4–20% polyacrylamide gradient gel (Mini-Protean II; Bio-Rad, Upplands Väsby, Sweden). The gels were impregnated with Amplify (Amersham) and exposed to autoradiographic film (Hyperfilm; Amersham).

Immunoprecipitation, immunoblotting, and immunofluorescence

Samples (200 µL) of dialyzed media from [35S]methionine-labeled cultures were mixed and incubated with purified human Tg (2.5 µg) and rabbit antihuman Tg serum (15 µL) for 2 h at room temperature. Goat antirabbit serum (10 µL) was then added, and the mixture was further incubated overnight at 4 C. Immunoprecipitates were pelleted by centrifugation at 3000 x g for 15 min, washed once with PBS, and solubilized in 1 mol/L NaOH for 30 min at 60 C. Radioactivity present in precipitates (Tg) and supernatants (non-Tg proteins) was determined by liquid scintillation.

For Western blotting, proteins from filter-cultured cells, solubilized in Laemmli’s buffer, were separated by SDS-PAGE (4–20%) and transferred to nitrocellulose sheets (0.45 µm) in a mini trans-blot cell (Bio-Rad). Blots were blocked with 5% dry milk and mounted in Decaprobe (Hoefer Scientific Instruments; San Fransisco, CA). Single lanes were incubated with one of the monoclonal antibodies against E-cadherin (1:5000), {alpha}-catenin (1:500), ß-catenin (1:1000), and {gamma}-catenin (1:1000) for 1 h and then with horseradish peroxidase-conjugated rabbit antimouse IgG for 45 min at room temperature; tris-buffered saline containing 0.1% Tween 20, pH 7.6, was used for antibody dilution and for washings after each step of incubation. Peroxidase activity was detected by enhanced chemiluminescence (ECL; Amersham), according to the manufacturer’s instructions.

For immunofluorescence, filter-cultured cells were fixed in ice-cold ethanol for 15 min, washed with PBS (pH 7.4), and preincubated at room temperature with blocking buffer, consisting of 5% fat-free milk, 0.1% gelatin, and 7.5% sucrose in PBS, for 10 min and with avidin-biotin blocking reagents (Vector Laboratories, Burlingame, CA) for 2 x 10 min. The cells were then incubated in sequence with anti-ZO-1 (1:400) for 1 h, biotinylated donkey antirabbit IgG (1:400) for 30 min, and fluorescein-isothiocyanate-conjugated streptavidin (1:300) for 30 min. Filters with immunolabeled cells were cut out of the filter inserts, mounted on glass with Vectashield (Vector), and examined in a Nikon Microphot FXA epifluorescence microscope.

Electron microscopy

Cultures were fixed for 1 h in 2.5% glutaraldehyde in 0.05 mol/L sodium cacodylate, pH 7.4, followed by postfixation for 1 h in 1% OsO4, dehydration in ethanol series, and embedding in epoxy resin. Ultrathin sections, cut either perpendicular to the cell layer and filter (vertical sections) or crossing the apical pole of the cells (horizontal sections), were contrasted with uranyl acetate and lead citrate and examined in a Philips 400 T electron microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-1{alpha} induces paracellular leakage in cultured human thyroid epithelium

As recently described (23), human thyrocytes form a tight and polarized epithelium when grown to confluence on permeable filter. The cultures establish a RTE, which is 200–400 {Omega} x cm2 in the absence of TSH (5H medium) and 1000–1500 {Omega} x cm2 in the presence of TSH (6H medium), and effectively restrict the diffusion of [3H]inulin from the apical to the basal chamber compartment. As an example of cell polarization, Tg is secreted vectorially into the apical culture medium (23), which, in the model, corresponds to the lumenal compartment of intact follicles.

Recombinant cytokines were added to the basal medium. As shown in Table 1Go, RTE and transepithelial flux of [3H]inulin were not influenced by IFN-{gamma} (100 U/mL), TNF-{alpha} (10 ng/mL), IL-6 (100 U/mL), or TGF-ß1 (10 ng/mL) present for 48 h. In contrast, in the same time period, IL-1{alpha} (100 U/mL) reduced RTE to less than 100 {Omega} x cm2 and increased the transfer of [3H]inulin across the cell layer (Table 1Go). This effect of IL-1{alpha} was dose-dependent, in the range 1–1000 U/mL, regarding both onset (Fig. 1Go) and magnitude (Fig. 2Go). In addition, wash-out of IL-1{alpha} induced partial recovery of RTE (Fig. 3Go). Whether the barrier dysfunction induced by IL-1{alpha} accounts for macromolecules, as well, was estimated by analyzing the transepithelial permeability of Tg. 125I-Tg was added to the apical medium, and its appearance in the basal medium after short term (20–60 min) incubation was determined. As shown in Fig. 4Go, 125I-Tg was undetectable in the basal medium of untreated cultures, whereas large amounts of radiolabeled Tg appeared basally in cultures pretreated with 100 U/mL IL-1{alpha} for 48 h. At the highest concentration tested (1000 U/mL), IL-1{alpha} induced a >40-fold increase in transepithelial flux of 125I-Tg; the radioactivity recovered in the basal medium was 290 ± 12 vs. 13408 ± 728 cpm/well (mean ± SD; n = 4) in control and IL-1{alpha}-treated cultures. Taken together, the data show that the epithelial integrity of human thyrocytes in culture is reversibly impaired by IL-1{alpha}.


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  		Table 1. Effect of recombinant cytokines on the barrier function of monolayers of human thyroid epithelial cells cultured on filter1

 


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  		Figure 1. Dose- and time-dependent reduction of (RTE) by recombinant IL-1{alpha} in filter-cultured human thyrocytes. IL-1{alpha}, at concentrations of 0.1 (open circles), 1 (open squares), 10 (filled circles), and 100 (filled squares) U/mL, were added to 7-day-old cultures at time zero in the abscissa. Culture proceeded in TSH-containing (6H) medium 2 days before IL-1{alpha} addition; mean ± SD (n = 3).

 


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  		Figure 2. Dose-dependent increase of transepithelial flux (FTE) of [3H]inulin by IL-1{alpha}. Cultures grown in 5H (open bars) or 6H (hatched bars) medium were pretreated with 10–1000 U/mL IL-1{alpha} for 48 h, after which the apical-to-basal transfer of [3H]inulin, exposed to the cells for 20 min, was analyzed. Data are presented as percent of controls; mean ± SD (n = 3).

 


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  		Figure 3. Recovery of barrier dysfunction induced by IL-1{alpha}. Filter-cultured thyrocytes, plated and grown in 5H for 5 days and then in 6H for 2 days, were exposed to 1, 10, or 100 U/mL IL-1{alpha} for 24 h, after which incubation continued in 6H medium. The dotted curve indicates the development of (RTE) in cultures not exposed to IL-1{alpha}; mean ± SD (n = 3).

 


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  		Figure 4. Transepithelial flux of 125I-Tg. Confluent cultures, grown in 6H medium, were exposed to 100 U/mL IL-1{alpha} for 48 h and then examined for epithelial leakiness to 125I-Tg in the apical-to-basal direction (for details, see Materials and Methods). As a positive control, paracellular leakage was induced by removal of extracellular Ca2+ in the basal medium (exchange for Ca2+-free medium containing 1 mmol/L EGTA) during the flux experiment; this treatment is known to disrupt the epithelial junction complex caused by disturbance of E-cadherin-based cell-cell adhesion (32). Protein-bound radioactivity, appearing in the basal medium of untreated (lane 1), Ca2+-chelated (lane 2), and IL-1{alpha}-treated (lane 3) cultures, was determined by SDS-PAGE, followed by autoradiography. The arrow points at a >300-kDa protein corresponding to pig Tg; radiolabeled proteins of lower molecular mass were not detected.

 
Redistribution of TJ protein ZO-1 by IL-1{alpha} in thyrocytes

ZO-1 is a TJ protein proposed to be involved in the establishment and maintenance of epithelial barriers (26). In untreated cultures, ZO-1 immunoreactivity was distributed all along the cell-cell contacts (Fig. 5AGo), indicating the circumferential position of the TJ and a complete sealing of the intercellular space. In contrast, after treatment with IL-1{alpha} (100 U/mL) for 48 h, ZO-1 present at the cell borders showed a markedly zigzaggy course and was often discontinuous (Fig. 5BGo), as if the TJ had been partly broken. IL-1{alpha}-treated cells also displayed distinct assemblies of ZO-1 in the cytoplasm, which were less frequent in control cultures (not shown). The altered distribution of ZO-1 induced by IL-1{alpha} was not reproduced by the other cytokines examined.



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  		Figure 5. Immunolocalization of ZO-1 in filter-cultured human thyrocytes. A, Cells grown in 5H to establish confluence and then in 6H for 4 days. A uniform ZO-1 immunoreactivity delineates the entire cell borders. B, Cells, cultured as in (A), with IL-1{alpha} (100 U/mL) present during the last 48 h. ZO-1 follows a highly irregular and partly interrupted course (arrows). Bar = 20 µm.

 
Ultrastructural derangement of the thyroid junction complex by IL-1{alpha}

Sections cut perpendicular to the cell layer and filter (vertical sections) or crossing the apical pole of the cells (horizontal sections) were examined by electron microscopy. The junctional complex, composed of TJ and AJ, was found to be located at the apical end of the intercellular space (Fig. 6AGo), which conforms with its native location in the thyroid follicular epithelium (19). In untreated cells, both junctions had a smooth and rectilinear appearance, as demonstrated most favorably in the horizontal sections (Fig 6BGo). Typically, the cytoplasmic facet of the AJ exhibited a coat of dense material (Fig. 6BGo), which is known as the junctional plaque, consisting of proteins involved in the attachment of the actin-based cytoskeleton to the plasma membrane (27).



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  		Figure 6. Electron micrographs of human thyrocytes isolated from Graves’ tissue and grown as a complete monolayer on filter in 6H medium (AM, apical medium; F, filter). A, Vertical section of untreated cells forming a tight and polarized epithelium. The apical cell surface is furnished with microvilli (mv) and the junctional complex, indicated by the dense plaque (arrows), is located in the most apical portion of the intercellular cleft (bar = 3 µm). B, Horizontal section across the apical pole of untreated cells. Both TJ and AJ, the latter identified by the presence of a junctional plaque (jp), have a rectilinear course. Desmosomes (D) are located close to the AJ (bar = 1 µm). C, Horizontal section at the junctional level of cells exposed to 1 U/mL of IL-1{alpha} for 48 h. There are no apparent ultrastructural alterations of TJ and AJ. The numerous phagolysosomes present in the upper cell profile are not specific for cultures treated with IL-1{alpha}. Bar = 1 µm.

 
The ultrastructure of the junctional complex was not affected by low concentrations (0.01–1 U/mL) of IL-1{alpha} (Fig. 6CGo) or by treatment with IL-6, TNF-{alpha}, or IFN-{gamma} (not shown). In contrast, in cells exposed to 10 U/mL of IL-1{alpha} for 48 h, the membrane portions of the AJ were generally twisted and distorted, and the thickness of the submembranous AJ plaque was substantially increased (Fig. 7AGo). In addition, plaque-like material was found to be spread out in the cytoplasm beneath the apical plasma membrane (Fig. 7AGo). This response to IL-1{alpha} was even more obvious in horizontal sections (Fig. 7BGo), which also revealed extensive bundles of microfilaments present in the apical cytoplasm, in association with the patches of dense material, or being anchored to the AJ. However, despite these dramatic changes, the TJ seemed to be largely intact after treatment with 10 U/mL IL-1{alpha}. Cells given 100 U/mL IL-1{alpha} for 48 h displayed more generalized changes in the morphology of the junctional complex (Fig. 7CGo), occasionally comprising a complete lack of both AJ and TJ, which created free communication between the apical compartment and the intercellular space (Fig. 7DGo). There were no ultrastructural signs of cytotoxicity, such as generalized vacuolization and membrane blebbing, in cells exposed to IL-1{alpha}.



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  		Figure 7. Electron micrographs of filter-cultured human thyrocytes exposed to IL-1{alpha} for 48 h. A and B, Cells treated with 10 U/mL IL-1{alpha}; A, vertical section. Neighboring cells show cytoplasmic projections (a, b, and c), which extend on top of each other in the junctional area close to the apical surface. The junctional complex has an irregular course and displays large amounts of plaque material. In addition, the cytoplasm underneath the apical plasma membrane contains dense material associated with microfilaments (arrows). Microvilli are not present (bar = 1 µm). B, Horizontal section. The membranes forming TJ and AJ have a twisted course but are not separated from each other. The apical cytoplasm contains large amounts of microfilaments, which appear either as a web (w) or as bundles (bu). Patches of dense material (arrows) are scattered among these filaments, which (according to the orientation of the section) seem to run in parallel to the apical plasma membrane (bar = 2 µm). C and D, Cells treated with 100 U/mL IL-1{alpha}; C, horizontal section. The cells are connected by an abnormal AJ, which is associated with large amounts of microfilaments (mf). TJ is not possible to identify. IC, Intercellular space (bar = 0.5 µm). D, vertical section. Both TJ and AJ are lacking at their expected position apical to the desmosome (D). Bar = 0.5 µm.

 
Unchanged levels of E-cadherin and catenins in IL-1{alpha}-treated cells

In view of the pronounced ultrastructural changes taking place mainly in the AJ, we examined whether IL-1{alpha} had any effect on the expression of AJ-associated molecules, i.e. the cadherin-catenin complex, which previously have been shown to be down-regulated by phorbol ester (28), and TGF-ß (29) in other cell types. However, we found that the protein levels of E-cadherin and the catenins in Western blots were not altered by IL-1{alpha} treatment for 48 h (Fig. 8Go). Immunolocalized E-cadherin and catenins were mainly distributed along the cell-cell contacts (not shown).



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  		Figure 8. Western blotting of E-cadherin (lanes 1 and 2), {alpha}-catenin (lanes 3 and 4), ß-catenin (lanes 5 and 6), and {gamma}-catenin (lanes 7 and 8) prepared from untreated cells (lanes 1, 3, 5, and 7) or cells exposed to 100 U/mL IL-1{alpha} for 48 h (lanes 2, 4, 6, and 8). The IL-1{alpha}-treated cultures showed a reduced epithelial barrier function before being solubilized. Proteins were separated by SDS-PAGE on a 4–20% gradient gel. Right margin indicates Mr x 103.

 
Independent action of IL-1{alpha} on apically and basally secreted proteins

Previous studies indicate that the expression of thyroid-specific proteins is down-regulated by IL-1{alpha} (9, 10). In agreement with this, we found that IL-1{alpha} reduced the amount of Tg released into the apical medium of [35S]methionine-labeled cells (Fig. 9Go). At the same time, IL-1{alpha} increased severalfold the secretion of radiolabeled non-Tg proteins into the basal medium (Fig. 9Go). Autoradiographs of secreted proteins, run on SDS-PAGE, confirmed the reduction of Tg and revealed that the major component released basally in response to IL-1{alpha} was a high-molecular-mass protein of hitherto unknown identity (Fig. 10Go). Despite the fact that the total synthesis of secretory proteins was increased, ranging between 1.4–4.5 times the control level in different experiments, IL-1{alpha} did not influence the DNA content [1.76 ± 35 vs. 1.74 ± 38 µg/filter (mean ± SD; n = 5) in the presence or absence of 100 U/mL IL-1{alpha}].



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  		Figure 9. Apical and basal secretion of metabolically labeled proteins. Cultures grown in 6H were incubated with or without 100 U/mL IL-1{alpha} for 48 h and then labeled with [35S]methionine (50 µCi/mL) in MEM-met) for an additional 7 h. Dialyzed media (API-apical; BAS-basal) were immunoprecipitated with anti-Tg, and the radioactivity content of pellet (Tg) and supernatant (non-Tg) was counted. Data are from single cultures from one of three experiments, showing similar results. Open bars, control; filled bars, IL-1{alpha}.

 


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  		Figure 10. Autoradiograph of [35S]methionine-labeled secretory proteins, separated by SDS-PAGE in a 4–20% gradient gel. The gel shows proteins released from cells cultured in 5H (lanes 1–6) or 6H (lanes 8–13) medium and which, before metabolic labeling, were untreated (lanes 1, 4, 8, and 11) or exposed to 100 U/mL IL-1{alpha} (lanes 2, 5, 9, and 12) or IL-6 (lanes 3, 6, 10, and 13) for 48 h. Proteins released into either of the apical (lanes 1–3 and 8–10) and basal (lanes 4–6 and 11–13) media were analyzed. The autoradiographic detection in lanes 8–13 is overexposed, making the Tg band difficult to quantify, because of a much larger amount of radiolabeled proteins recovered in 6H medium, as compared with that present in 5H medium (lanes 1–6). Tg (arrowhead); high-molecular-mass protein of unknown identity (arrow). Right margin indicates Mr x 103 (lane 7, [14C]-labeled standard proteins).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the intact thyroid follicle, epithelial cells form a tight barrier between the extrafollicular space and the follicular lumen in which hormonogenesis and prohormone storage take place (19). The purpose of the present work was to elucidate whether the thyroid epithelial barrier is influenced by proinflammatory cytokines. Using filter-cultured human thyrocytes, we found that, from the five important cytokines examined, only IL-1{alpha} reduced the epithelial tightness and caused paracellular leakage. The effect was obvious after 24 h and further augmented after 48 h of exposure, without morphological signs of cytotoxicity. In addition, the synthesis of a novel high-molecular-mass protein, released mainly in the basal direction, was stimulated, further indicating that cell viability was not challenged by IL-1{alpha}. In contrast, IFN-{gamma} and TNF-{alpha} were ineffective at concentrations which open TJs in intestinal (20) and renal (21) epithelial cells. Likewise, IL-6, which has been found to reduce the intercellular contact of mammary carcinoma cells (30), had no such effect, indicating that cytokine-induced loss of epithelial integrity is target cell-dependent. The dysregulating effect of IL-1{alpha} on the barrier function of the thyroid epithelium is a novel finding not previously reported for other epithelial cells unless being related to cell damage (31).

The IL-1{alpha}-induced loss of barrier function was accompanied by altered distribution of the TJ protein ZO-1 and disorganization of the junctional complex, as revealed by electron microscopy. However, unless the cells were treated with a high concentration (100 U/mL) of IL-1{alpha}, the ultrastructural changes were confined mainly to the AJ and its submembranous plaque. This suggests that the effect of IL-1{alpha} on TJ might be indirect caused by the gross changes appearing in the juxtapositioned AJ. A functional connection between AJ and TJ is previously known from studies on cultured cells depleted of extracellular Ca2+ (32). As a result of reduced Ca2+-dependent cell-cell adhesion, the AJ is gradually disintegrated, and the plasma membrane is displaced by retracting actin filaments that normally are firmly bound to the AJ. The tensile forces thus generated may in turn negatively influence the integrity of TJ. In the present study, IL-1{alpha} caused similar ultrastructural changes of the AJ and the microfilaments in the apical cytoplasm.

Ca2+-dependent cell-cell adhesion in epithelia is mainly mediated by E-cadherin, which plays a central role in the formation and maintenance of a cohesive epithelial sheet (33). Conversely, down-regulation or inhibited function of E-cadherin is associated with loss of epithelial junctions, as found in tumor progression towards a more malignant phenotype of carcinoma cells (34). There are no previous reports addressing the question of whether cadherins are influenced by IL-1{alpha}. We found here that the protein expression of E-cadherin was not altered by IL-1{alpha} treatment for 48 h. Also, the cellular amounts of {alpha}-, ß-, and {gamma}-catenins, which regulate the binding of E-cadherin to AJ-associated actin (27), were unchanged. Therefore, if the IL-1{alpha}-induced disruption of thyroid junctions is related to altered function of E-cadherin or catenins, posttranslational modification, as recently shown to occur in response to src oncoprotein (35) and peptide growth factors (36), must be considered. In agreement with the present findings, Tamm et al. (31) found that IL-6 caused dissociation of mammary carcinoma cells without altering the expression of E-cadherin.

Because iodide organification normally takes place exclusively inside the follicular lumen at the apical surface of the thyroid epithelial cells (19), the integrity of the follicular wall is of considerable importance for thyroid function. Iodide is actively transported across the epithelium to the lumen by basolateral uptake and apical efflux mechanisms (37), and Tg is secreted predominantly in the apical direction (38). A prerequisite for maintaining the lumenal content of Tg and iodide is that paracellular leakage down-hill from their concentration gradients is restricted by the presence of TJ. In addition, TJ is known to act as a fence, which prevents the mixing of integral membrane proteins specific for either of the apical and basolateral plasma membranes (39). It is likely that this fence function of TJ supports the polarized distribution of both the iodide transporters and the thyroid-specific enzymes, H2O2-generating NADPH oxidase and TPO, which catalyze the iodination of Tg at the apical cell surface. Consequently, agents that disrupt thyroid follicular integrity would be a serious threat to the production of thyroid hormones. Previous studies (8, 9) show that the synthesis of Tg is reduced by IL-1. The present findings suggest that a loss of the thyroid epithelial barrier may be another mechanism by which IL-1{alpha} inhibits thyroid hormonogenesis.

Locally produced IL-1{alpha} is likely to be involved in the development of autoimmune thyroid disease (1, 2), although its precise pathophysiologic role has not been clarified. An unsolved question in thyroid autoimmunity is that of how the autoantigens, especially Tg and TPO secluded inside the follicles, are made accessible and presented to the immune cells that produce the autoantibodies. One possible mechanism is antigen release caused by target cell lysis, but this does not explain the presence of autoantibodies in patients in which there are no apparent signs of a cytotoxic reaction in the thyroid. The data reported in this paper suggest several other possibilities by which IL-1{alpha} might promote antigen exposure. First, a reduced gate function of the TJ may be followed by paracellular leakage of colloid and release of Tg to the extrafollicular space at a concentration sufficient to elicit or maintain an immune response. That the barrier dysfunction induced by IL-1{alpha} indeed allows macromolecules to leak through the epithelium in apical-to basal direction was shown for exogeneous 125I-Tg added to the filter-cultured cells. Second, if the fence function of TJ was lost, TPO present in the apical plasma membrane might be translocated by lateral diffusion to the basolateral pole of the thyrocytes and recognized by interstitial macrophages and dendritic cells, which often are located in direct contact with the epithelium in autoimmune thyroids (40). A third possibility is that dissociation of the entire junctional complex might allow antigen-presenting cells to transmigrate across the follicular epithelium to the lumen, similar to that recently demonstrated for neutrophils interacting with IFN-{gamma}-stimulated intestinal epithelial cells (41). The in vitro model for culture of human thyrocytes, as a complete epithelium in bicameral chambers, provides a means by which these hypotheses, pointing on a new role for IL-1{alpha} in thyroid autoimmunity, may be examined in further detail.

Accompanying the barrier dysfunction, the IL-1{alpha}-treated cells showed an altered pattern of synthesis and release of secretory proteins. In agreement with previous reports (8, 9), IL-1{alpha} was found to inhibit the production of Tg. In addition, the synthesis of another protein with a predicted molecular mass of approximately 500 kDa, which was mainly secreted in the basal direction, was strongly stimulated by IL-1{alpha}. A protein of similar size, produced by thyrocytes and secreted in the same polarized manner, is thrombospondin (42), an oligomeric, multifunctional extracellular matrix component. However, thrombospondin is resolved to a monomeric form of approximately 190 kDa under reducing SDS-PAGE, which were the conditions used for protein separation in the present study. Moreover, IL-1 is known to down-regulate the production of thrombospondin, e.g. in endothelial cells (43). Thus, the molecular identity and possible function(s) of the high-molecular-mass secretory protein, stimulated by IL-1{alpha} in filter-cultured human thyrocytes, remain to be elucidated.


   Acknowledgments
 
We would like to thank Drs. Å. Krogh Rasmussen and J. P. Banga for valuable criticism of the manuscript; Dr. L. E. Tisell and colleagues at the Department of Surgery, Sahlgrenska University Hospital, Göteborg, for providing human thyroid tissue; and G. Bokhede, T. Carlsson, and Y. Josefsson for superb technical assistance.


   Footnotes
 
1 Presented in preliminary form at the 11th International Thyroid Congress, Toronto, Ontario, 10–15 September, 1995. This work was supported by grants from the Swedish Medical Research Council (12X-537), the Swedish Medical Society, Assar Gabrielssons Fundation for Clinical Research, Jubileumsklinikens Cancer Research Fundation, and Fundations of Magnus Bergwall and Lars Hierta. Back

2 Holds an individual postdoctoral fellowship provided by the Swedish Medical Research Council. Back

Received September 18, 1996.

Revised April 4, 1997.

Revised November 14, 1997.

Accepted November 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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