Cytokines and Thyroid Epithelial Integrity: Interleukin- 1a Induces Dissociation of the Junctional Complex and Paracellular Leakage in ...
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0021-972X/98/$03.00/0 Vol. 83, No. 3
Journal of Clinical Endocrinology and Metabolism Printed in U.S.A.
Copyright © 1998 by The Endocrine Society
Cytokines and Thyroid Epithelial Integrity: Interleukin-
1a Induces Dissociation of the Junctional Complex and
Paracellular Leakage in Filter-Cultured Human
Thyrocytes*
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MIKAEL NILSSON†, JOHANNA HUSMARK, ULLA BJÖRKMAN, AND
LARS E. ERICSON
Institute of Anatomy and Cell Biology, Göteborg University, S-413 90 Göteborg, Sweden
ABSTRACT paracellular flux of [3H]inulin and exogeneous 125I-Tg. This response
Locally produced proinflammatory cytokines are likely to play a to IL-1a, which was dose dependent (1–1000 U/mL) and reversible,
pathophysiological role in autoimmune thyroid disease. An important was accompanied by dramatic morphological changes of the epithelial
feature of the thyroid, not previously considered in cytokine actions, junction complex, including aberrant localization of the tight junction
is the barrier created by the follicular epithelium, which secludes two protein zonula occludens-1. At the same time, IL-1a decreased the
lumenal autoantigens [thyroglobulin (Tg) and thyroperoxidase] from apical secretion of endogeneous Tg and stimulated the basolateral
the extrafollicular space. We examined the influence of recombinant release of a novel high-molecular-mass protein. We conclude that
cytokines on the barrier function of human thyrocytes cultured as a IL-1a reduces the thyroid epithelial barrier without signs of general
tight and polarized monolayer in bicameral chambers. Whereas in- cytotoxicity. The observation suggests a mechanism by which IL-1a
terleukin (IL)-6 (100 U/mL), interferon-g (100 U/mL), tumor necrosis may promote the exposure of hidden autoantigens to the immune
factor-a (10 ng/mL), and transforming growth factor-b1 (10 ng/mL) system in thyroid autoimmunity. (J Clin Endocrinol Metab 83: 945–
had no effects, exposure to IL-1a for 24 – 48 h reduced the transepi- 952, 1998)
thelial resistance from .1000 to ,50 V 3 cm2 and increased the
T HE PATHOGENESIS of autoimmune thyroid diseases is
multifactorial, involving environmental factors and ab-
errations of both the immune system and the target tissue (1).
found to stimulate thyroid cell proliferation (7) and inhibit
several steps in the synthesis and release of thyroid hor-
mones (8 –13) (reviewed in Refs. 2 and 6). In addition, IL-1
A major sign is tissue infiltration by immune cells, which enhances the expression of major histocompatibility complex
produce autoantibodies against three major thyroid-specific class II antigen (14), intercellular adhesion molecule-1 (15),
antigens [thyroglobulin (Tg), thyroperoxidase (TPO), and the and leukocyte function antigen (16) on thyrocytes and stim-
TSH receptor] and may also generate cell-mediated cytotox- ulates the thyroidal production of other cytokines, e.g. IL-6
icity. In these autoimmune reactions, locally released proin- (17) and IL-8 (18).
flammatory cytokines are known to be critically involved (2). Thyrocytes hold a unique position among classical endo-
For instance, interleukin (IL)-1, a pleiotropic cytokine con- crine cells, in that they also exert an exocrine function and
sisting of a- and b-forms (3), accelerates the onset of lym- have a polarized, epithelial phenotype typical of exocrine
phocytic thyroiditis and insulin-dependent diabetes mellitus cells. The functional unit of the thyroid is the follicle com-
when injected to BB rat (4). However, whereas IL-1 is cyto- posed of a single-layered epithelium and a central cavity (the
toxic to the pancreatic b-cells (5), the viability of thyrocytes follicular lumen), in which Tg (the prohormone) is stored and
does not seem to be affected (2, 6), indicating that IL-1 pro- iodothyronines are synthesized (19). The junctional complex
motes thyroid autoimmunity by mechanisms other than tar- of thyroid follicular cells consists, in part, of tight junctions
get cell lysis. In different experimental systems, IL-1 has been (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
Received September 18, 1996. Revision received April 4, 1997. Re-
revision received November 14, 1997. Accepted November 24, 1997. between the extracellular compartments, the lumen and the
Address all correspondence and requests for reprints to: Mikael extrafollicular space, is critical to normal thyroid function,
Nilsson, Institute of Anatomy and Cell Biology, Göteborg Univer- because it promotes cell polarity and the establishment of
sity, Box 420, (SE) 405 30, Göteborg, Sweden. E-mail: mikael.olof. transepithelial solute gradients of, for instance, iodide and
nilsson@anat.cell.gu.se.
* Presented in preliminary form at the 11th International Thyroid Tg. Conversely, destruction of the paracellular barrier would
Congress, Toronto, Ontario, 10 –15 September, 1995. This work was challenge thyroid function and, in the context of autoimmu-
supported by grants from the Swedish Medical Research Council (12X- nity, might facilitate the exposure of normally secluded au-
537), the Swedish Medical Society, Assar Gabrielssons Fundation for toantigens, Tg in the follicular lumen, and TPO in the apical
Clinical Research, Jubileumsklinikens Cancer Research Fundation, and
Fundations of Magnus Bergwall and Lars Hierta.
plasma membrane, to the immune system. However, it is not
† Holds an individual postdoctoral fellowship provided by the known whether cytokines produced in autoimmune thyroid
Swedish Medical Research Council. tissue have any effect on the thyroid epithelial barrier. In
945946 NILSSON ET AL. JCE & M • 1998
Vol 83 • No 3
attempts to explore this issue, we investigated the effect of (Boehringer); 0.1 mmol/L leupeptin (Sigma); aprotinin (0.01 mmol/L)
recombinant IL-1a, IL-6, interferon-g (IFN-g), tumor necrosis (Sigma). Radiolabeled Tg was dialyzed against PBS (pH 7.0), supple-
mented with 10 mmol/L KI and 1 mmol/L methimazole. [3H]inulin (1
factor-a (TNF-a), and transforming growth factor-b1 (TGF- mCi/mL) or samples (10 mL) of the 125I-Tg dialysate (0.1 mCi/pmol) were
b1) on tight monolayers of human thyrocytes cultured on dissolved in 0.01 mol/L Tris-maleate buffer (pH 7.3), supplemented with
permeable filters in bicameral chambers. We found that IL-1a 0.13 mol/L NaCl and KCl, CaCl2, MgCl2, and glucose, according to the
was a strong negative regulator of thyroid epithelial tight- specification of Tyrode solution, and added to the apical chamber com-
ness, as evidenced by a reduced transepithelial resistance, an partment of filter-cultured thyrocytes 6 IL-1a pretreatment. As a pos-
itive control of paracellular leakiness, sets of cultures were, simulta-
increased paracellular flux of radiotracers ([3H]inulin and neously with the exposure to 125I-Tg, depleted of extracellular Ca21 by
125
I-Tg), and a rearrangement of the junctional complex in exchanging the basal medium for Ca21-free buffer containing 1 mmol/L
IL-1a-treated cells. In contrast, IFN-g and TNF-a, both ethylene glycol bis(b-aminoethyl ether)-N,N9-tetraacetic acid (EGTA;
known to interfere with the barrier function of cultured in- Sigma); this treatment is known to disrupt the epithelial junction com-
plex caused by abolition of Ca21-dependent cell-cell adhesion (see Ref.
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testinal and renal epithelial cells (20, 21), had no effect on 32). After incubation for 20 min at 37 C, the amount of [3H]inulin present
paracellular permeability and junctional morphology. This in the basal medium was analyzed in an LKB Wallac liquid scintillator
action of IL-1a has not been reported for any other (Wallac Sverige, Sollentuna, Sweden). After incubation for 20 – 60 min,
epithelium. 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
Materials and Methods for total amount of radioactivity in a Packard auto-g counter (Packard
Cytokines and antibodies Instrument Co., Dowers Grove, IL). The same media obtained from
125
I-Tg-incubated cultures were then concentrated in a microconcentra-
Human recombinant cytokines, IL-1a (53107 U/mg), IL-6 (13108 tor (Amicon Inc., Beverly, MA) and, after addition of Laemmli’s sample
U/mg), IFN-g (23107 U/mg), TNF-a (13108 U/mg), and TGF-b1, were buffer, subjected to SDS-PAGE on an 8% gel, as described below. Au-
purchased from Boehringer (Mannheim, Germany). Rabbit antihuman toradiographs of gels were prepared with Kodak BioMax MS film (East-
zonula occludens (ZO)-1 was from Zymed Laboratories (San Francisco, man Kodak, Rochester, NY).
CA). Mouse monoclonal antibodies against E-cadherin and catenins
were obtained from Transduction Laboratories (Lexington, KY). Horse- Metabolic labeling, SDS-PAGE, and autoradiography
radish peroxidase-conjugated rabbit antimouse IgG was from Dako A/S
(Glostrup, Denmark). Biotinylated donkey antirabbit IgG and fluores- Filter-cultured cell monolayers were washed both apically and ba-
cein-isothiocyanate-conjugated streptavidin were purchased from Am- sally with serum-free MEM devoid of methionine (MEM-met) and in-
ersham International plc (Amersham, England). Human Tg were puri- cubated with 50 mCi/mL [35S]methionine in MEM-met present in the
fied and polyclonal sera were raised by immunization of rabbits, as lower chamber, for 7 h at 37 C. The apical and basal media were then
described (22). 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
Isolation and culture of human thyrocytes analyzed for content of protein-bound radioactivity, as described below.
Equal volumes of dialysed media were also mixed with sample buffer,
Thyroid follicles were isolated by enzymatic digestion of surgically heated to 96 C for 4 min, and subjected to electrophoresis, together with
excised Graves’ (n 5 9) or normal (paradenomatous; n 5 5) thyroid [14C]methylated protein standards (Amersham), in a 4 –20% polyacryl-
tissue, following a recently described protocol (23). After being sepa- amide gradient gel (Mini-Protean II; Bio-Rad, Upplands Väsby, Swe-
rated from blood and interstitial cells by repeated centrifugation, the den). The gels were impregnated with Amplify (Amersham) and ex-
follicle segments were plated on the filter of bicameral culture inserts posed to autoradiographic film (Hyperfilm; Amersham).
(Transwell 3413; Costar Corp., Cambridge, MA) precoated with collagen
type I (Boehringer). The cells were cultured in humidified atmosphere Immunoprecipitation, immunoblotting,
(5% CO2) at 37 C in Coon’s modified Ham’s medium supplemented with
and immunofluorescence
penicillin (200 U/mL), streptomycin (200 U/mL), and fungizone (2.5
mg/mL) and enriched with 5% FCS (Gibco; Paisley, Scotland) and 5 Samples (200 mL) of dialyzed media from [35S]methionine-labeled
factors (5H medium: insulin, bovine transferrin, hydrocortisone, glycyl- cultures were mixed and incubated with purified human Tg (2.5 mg) and
l-histidyl-l-lysine acetate and somatostatin; all reagents from Sigma (St. rabbit antihuman Tg serum (15 mL) for 2 h at room temperature. Goat
Louis, MO) or 6 factors (6H medium: in addition 1029 mol/L bovine TSH antirabbit serum (10 mL) was then added, and the mixture was further
from Sigma), according to the composition of culture medium originally incubated overnight at 4 C. Immunoprecipitates were pelleted by cen-
described for FRTL cells (24). The DNA content of cultures was deter- trifugation at 3000 3 g for 15 min, washed once with PBS, and solubilized
mined fluorometrically (25). All experimental observations were made in 1 mol/L NaOH for 30 min at 60 C. Radioactivity present in precipitates
on triplicate cultures of at least three independent platings, with similar (Tg) and supernatants (non-Tg proteins) was determined by liquid
results. There were no apparent differences in the response of cells from scintillation.
paradenomatous or Graves’ tissue to the cytokines added. The patients For Western blotting, proteins from filter-cultured cells, solubilized
from which Graves’ thyroid tissue was obtained had been under treat- in Laemmli’s buffer, were separated by SDS-PAGE (4 –20%) and trans-
ment preoperatively with an antithyroid drug and T4. ferred to nitrocellulose sheets (0.45 mm) in a mini trans-blot cell (Bio-
Rad). Blots were blocked with 5% dry milk and mounted in Decaprobe
Epithelial barrier assays (Hoefer Scientific Instruments; San Fransisco, CA). Single lanes were
incubated with one of the monoclonal antibodies against E-cadherin
Paracellular tightness of cultured thyrocyte monolayers was assessed (1:5000), a-catenin (1:500), b-catenin (1:1000), and g-catenin (1:1000) for
by measurement of the transepithelial electrical resistance (RTE) across 1 h and then with horseradish peroxidase-conjugated rabbit antimouse
the filter, with a Millicell ERS ohmmeter (Millipore; Bedford, MA). IgG for 45 min at room temperature; tris-buffered saline containing 0.1%
Paracellular permeability was determined by analysis of the transepi- Tween 20, pH 7.6, was used for antibody dilution and for washings after
thelial flux of either [3H]inulin (Amersham) or 125I-Tg. For this purpose, each step of incubation. Peroxidase activity was detected by enhanced
pig Tg (5 mmol/L), purified by chromatography on a Sepharose 6B chemiluminescence (ECL; Amersham), according to the manufacturer’s
column (Pharmacia Biotech, Uppsala, Sweden) and free from low mo- instructions.
lecular mass species (as determined by SDS-PAGE), was labeled with For immunofluorescence, filter-cultured cells were fixed in ice-cold
125 2
I (Amersham) for 1 h at 37 C in the presence of lactoperoxidase (400 ethanol for 15 min, washed with PBS (pH 7.4), and preincubated at room
mU/mL; Sigma), glucose oxidase (60 mU/mL; Sigma), glucose (5 temperature with blocking buffer, consisting of 5% fat-free milk, 0.1%
mmol/L), and a mixture of protease inhibitors: 0.1 mmol/L Pefabloc gelatin, and 7.5% sucrose in PBS, for 10 min and with avidin-biotinIL-1a AND THE THYROID EPITHELIAL BARRIER 947
blocking reagents (Vector Laboratories, Burlingame, CA) for 2 3 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
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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
IL-1a 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 per- FIG. 1. Dose- and time-dependent reduction of (RTE) by recombinant
meable filter. The cultures establish a RTE, which is 200 – 400 IL-1a in filter-cultured human thyrocytes. IL-1a, at concentrations of
V 3 cm2 in the absence of TSH (5H medium) and 1000 –1500 0.1 (open circles), 1 (open squares), 10 (filled circles), and 100 (filled
V 3 cm2 in the presence of TSH (6H medium), and effectively 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
restrict the diffusion of [3H]inulin from the apical to the basal before IL-1a addition; mean 6 SD (n 5 3).
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 compart-
ment of intact follicles.
Recombinant cytokines were added to the basal medium.
As shown in Table 1, RTE and transepithelial flux of [3H]inu-
lin were not influenced by IFN-g (100 U/mL), TNF-a (10
ng/mL), IL-6 (100 U/mL), or TGF-b1 (10 ng/mL) present for
48 h. In contrast, in the same time period, IL-1a (100 U/mL)
reduced RTE to less than 100 V 3 cm2 and increased the
transfer of [3H]inulin across the cell layer (Table 1). This effect
of IL-1a was dose-dependent, in the range 1–1000 U/mL,
regarding both onset (Fig. 1) and magnitude (Fig. 2). In
addition, wash-out of IL-1a induced partial recovery of RTE
(Fig. 3). Whether the barrier dysfunction induced by IL-1a
accounts for macromolecules, as well, was estimated by an-
alyzing the transepithelial permeability of Tg. 125I-Tg was
added to the apical medium, and its appearance in the basal
TABLE 1. Effect of recombinant cytokines on the barrier function
of monolayers of human thyroid epithelial cells cultured on filter a FIG. 2. Dose-dependent increase of transepithelial flux (FTE) of
[3H]inulin by IL-1a. Cultures grown in 5H (open bars) or 6H (hatched
Transepithelial resistance Transepithelial flux c bars) medium were pretreated with 10 –1000 U/mL IL-1a for 48 h,
Treatment b (V 3 cm2) of [3H]inulin (dpm) 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
24 h 48 h
controls; mean 6 SD (n 5 3).
2 1,275 6 126 1,250 6 86 835 6 64
IL-1 a 706 6 174 72 6 10 2,007 6 227
IL-6 1,642 6 206 1,542 6 57 846 6 104
TNF-a 1,525 6 100 1,658 6 164 765 6 51 medium after short term (20 – 60 min) incubation was deter-
IFN-g 1,258 6 152 1,350 6 90 965 6 178 mined. As shown in Fig. 4, 125I-Tg was undetectable in the
TGF-b 1 1,308 6 83 1,406 6 102 752 6 47 basal medium of untreated cultures, whereas large amounts
a
Growth to confluence in 5H medium, then switched to 6H (TSH- of radiolabeled Tg appeared basally in cultures pretreated
containing) medium, which promotes epithelial tightness; mean 6 SD with 100 U/mL IL-1a for 48 h. At the highest concentration
(n 5 4). tested (1000 U/mL), IL-1a induced a .40-fold increase in
b
Cytokines added to 6H medium (100 U/ml: IL-1 a, IL-6 and IFN-g;
10 ng/ml: TNF-a and TGF-b 1). transepithelial flux of 125I-Tg; the radioactivity recovered in
c
Apical-to-basal flux of radiotracer (1 mCi/ml), measured for a the basal medium was 290 6 12 vs. 13408 6 728 cpm/well
20-min period after 48 h of cytokine exposure. (mean 6 sd; n 5 4) in control and IL-1a-treated cultures.948 NILSSON ET AL. JCE & M • 1998
Vol 83 • No 3
cells also displayed distinct assemblies of ZO-1 in the cyto-
plasm, which were less frequent in control cultures (not
shown). The altered distribution of ZO-1 induced by IL-1a
was not reproduced by the other cytokines examined.
Ultrastructural derangement of the thyroid junction
complex by IL-1a
Sections cut perpendicular to the cell layer and filter (ver-
tical sections) or crossing the apical pole of the cells (hori-
zontal sections) were examined by electron microscopy. The
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junctional complex, composed of TJ and AJ, was found to be
located at the apical end of the intercellular space (Fig. 6A),
which conforms with its native location in the thyroid fol-
licular epithelium (19). In untreated cells, both junctions had
a smooth and rectilinear appearance, as demonstrated most
favorably in the horizontal sections (Fig 6B). Typically, the
cytoplasmic facet of the AJ exhibited a coat of dense material
(Fig. 6B), which is known as the junctional plaque, consisting
FIG. 3. Recovery of barrier dysfunction induced by IL-1a. Filter-cul-
of proteins involved in the attachment of the actin-based
tured thyrocytes, plated and grown in 5H for 5 days and then in 6H cytoskeleton to the plasma membrane (27).
for 2 days, were exposed to 1, 10, or 100 U/mL IL-1a for 24 h, after The ultrastructure of the junctional complex was not af-
which incubation continued in 6H medium. The dotted curve indicates fected by low concentrations (0.01–1 U/mL) of IL-1a (Fig. 6C)
the development of (RTE) in cultures not exposed to IL-1a; mean 6 SD
(n 5 3).
or by treatment with IL-6, TNF-a, or IFN-g (not shown). In
contrast, in cells exposed to 10 U/mL of IL-1a 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. 7A). In addition,
plaque-like material was found to be spread out in the cy-
toplasm beneath the apical plasma membrane (Fig. 7A). This
response to IL-1a was even more obvious in horizontal sec-
tions (Fig. 7B), which also revealed extensive bundles of
microfilaments present in the apical cytoplasm, in associa-
tion with the patches of dense material, or being anchored to
FIG. 4. Transepithelial flux of 125I-Tg. Confluent cultures, grown in the AJ. However, despite these dramatic changes, the TJ
6H medium, were exposed to 100 U/mL IL-1a for 48 h and then
examined for epithelial leakiness to 125I-Tg in the apical-to-basal seemed to be largely intact after treatment with 10 U/mL
direction (for details, see Materials and Methods). As a positive con- IL-1a. Cells given 100 U/mL IL-1a for 48 h displayed more
trol, paracellular leakage was induced by removal of extracellular generalized changes in the morphology of the junctional
Ca21 in the basal medium (exchange for Ca21-free medium containing complex (Fig. 7C), occasionally comprising a complete lack
1 mmol/L EGTA) during the flux experiment; this treatment is known
to disrupt the epithelial junction complex caused by disturbance of of both AJ and TJ, which created free communication be-
E-cadherin-based cell-cell adhesion (32). Protein-bound radioactivity, tween the apical compartment and the intercellular space
appearing in the basal medium of untreated (lane 1), Ca21-chelated (Fig. 7D). There were no ultrastructural signs of cytotoxicity,
(lane 2), and IL-1a-treated (lane 3) cultures, was determined by SDS- such as generalized vacuolization and membrane blebbing,
PAGE, followed by autoradiography. The arrow points at a .300-kDa
protein corresponding to pig Tg; radiolabeled proteins of lower mo- in cells exposed to IL-1a.
lecular mass were not detected.
Taken together, the data show that the epithelial integrity of
human thyrocytes in culture is reversibly impaired by IL-1a.
Redistribution of TJ protein ZO-1 by IL-1a in thyrocytes
ZO-1 is a TJ protein proposed to be involved in the es-
tablishment and maintenance of epithelial barriers (26). In
untreated cultures, ZO-1 immunoreactivity was distributed
all along the cell-cell contacts (Fig. 5A), indicating the cir-
cumferential position of the TJ and a complete sealing of the FIG. 5. Immunolocalization of ZO-1 in filter-cultured human thyro-
intercellular space. In contrast, after treatment with IL-1a cytes. A, Cells grown in 5H to establish confluence and then in 6H for
4 days. A uniform ZO-1 immunoreactivity delineates the entire cell
(100 U/mL) for 48 h, ZO-1 present at the cell borders showed borders. B, Cells, cultured as in (A), with IL-1a (100 U/mL) present
a markedly zigzaggy course and was often discontinuous during the last 48 h. ZO-1 follows a highly irregular and partly in-
(Fig. 5B), as if the TJ had been partly broken. IL-1a-treated terrupted course (arrows). Bar 5 20 mm.IL-1a AND THE THYROID EPITHELIAL BARRIER 949
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FIG. 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 un-
treated 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 5 3 mm). 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 5 1 mm). C,
Horizontal section at the junctional level of cells exposed to 1 U/mL
of IL-1a 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-1a. Bar 5 1 mm.
FIG. 7. Electron micrographs of filter-cultured human thyrocytes ex-
posed to IL-1a for 48 h. A and B, Cells treated with 10 U/mL IL-1a;
Unchanged levels of E-cadherin and catenins in IL-1a-
A, vertical section. Neighboring cells show cytoplasmic projections (a,
treated cells b, and c), which extend on top of each other in the junctional area close
In view of the pronounced ultrastructural changes taking to the apical surface. The junctional complex has an irregular course
and displays large amounts of plaque material. In addition, the cy-
place mainly in the AJ, we examined whether IL-1a had any toplasm underneath the apical plasma membrane contains dense
effect on the expression of AJ-associated molecules, i.e. the material associated with microfilaments (arrows). Microvilli are not
cadherin-catenin complex, which previously have been present (bar 5 1 mm). B, Horizontal section. The membranes forming
shown to be down-regulated by phorbol ester (28), and TJ and AJ have a twisted course but are not separated from each
other. The apical cytoplasm contains large amounts of microfila-
TGF-b (29) in other cell types. However, we found that the ments, which appear either as a web (w) or as bundles (bu). Patches
protein levels of E-cadherin and the catenins in Western blots of dense material (arrows) are scattered among these filaments,
were not altered by IL-1a treatment for 48 h (Fig. 8). Immu- which (according to the orientation of the section) seem to run in
nolocalized E-cadherin and catenins were mainly distributed parallel to the apical plasma membrane (bar 5 2 mm). C and D, Cells
along the cell-cell contacts (not shown). treated with 100 U/mL IL-1a; 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
Independent action of IL-1a on apically and basally space (bar 5 0.5 mm). D, vertical section. Both TJ and AJ are lacking
secreted proteins at their expected position apical to the desmosome (D). Bar 5 0.5 mm.
Previous studies indicate that the expression of thyroid-
specific proteins is down-regulated by IL-1a (9 –10). In agree- ments, IL-1a did not influence the DNA content [1.76 6 35
ment with this, we found that IL-1a reduced the amount of vs. 1.74 6 38 mg/filter (mean 6 sd; n 5 5) in the presence or
Tg released into the apical medium of [35S]methionine- absence of 100 U/mL IL-1a].
labeled cells (Fig. 9). At the same time, IL-1a increased sev-
Discussion
eralfold the secretion of radiolabeled non-Tg proteins into
the basal medium (Fig. 9). Autoradiographs of secreted pro- In the intact thyroid follicle, epithelial cells form a tight
teins, run on SDS-PAGE, confirmed the reduction of Tg and barrier between the extrafollicular space and the follicular
revealed that the major component released basally in re- lumen in which hormonogenesis and prohormone storage
sponse to IL-1a was a high-molecular-mass protein of hith- take place (19). The purpose of the present work was to
erto unknown identity (Fig. 10). Despite the fact that the total elucidate whether the thyroid epithelial barrier is influenced
synthesis of secretory proteins was increased, ranging be- by proinflammatory cytokines. Using filter-cultured human
tween 1.4 – 4.5 times the control level in different experi- thyrocytes, we found that, from the five important cytokines950 NILSSON ET AL. JCE & M • 1998
Vol 83 • No 3
FIG. 8. Western blotting of E-cadherin (lanes 1 and 2), a-catenin
(lanes 3 and 4), b-catenin (lanes 5 and 6), and g-catenin (lanes 7 and FIG. 10. Autoradiograph of [35S]methionine-labeled secretory pro-
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8) prepared from untreated cells (lanes 1, 3, 5, and 7) or cells exposed teins, separated by SDS-PAGE in a 4 –20% gradient gel. The gel shows
to 100 U/mL IL-1a for 48 h (lanes 2, 4, 6, and 8). The IL-1a-treated proteins released from cells cultured in 5H (lanes 1– 6) or 6H (lanes
cultures showed a reduced epithelial barrier function before being 8 –13) medium and which, before metabolic labeling, were untreated
solubilized. Proteins were separated by SDS-PAGE on a 4 –20% gra- (lanes 1, 4, 8, and 11) or exposed to 100 U/mL IL-1a (lanes 2, 5, 9, and
dient gel. Right margin indicates Mr 3 103. 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 me-
dium, as compared with that present in 5H medium (lanes 1– 6). Tg
(arrowhead); high-molecular-mass protein of unknown identity (ar-
row). Right margin indicates Mr 3 103 (lane 7, [14C]-labeled standard
proteins).
changes were confined mainly to the AJ and its submem-
branous plaque. This suggests that the effect of IL-1a 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 Ca21 (32). As a result of reduced
Ca21-dependent cell-cell adhesion, the AJ is gradually dis-
integrated, and the plasma membrane is displaced by re-
FIG. 9. Apical and basal secretion of metabolically labeled proteins. tracting actin filaments that normally are firmly bound to the
Cultures grown in 6H were incubated with or without 100 U/mL IL-1a AJ. The tensile forces thus generated may in turn negatively
for 48 h and then labeled with [35S]methionine (50 mCi/mL) in MEM- influence the integrity of TJ. In the present study, IL-1a
met) for an additional 7 h. Dialyzed media (API-apical; BAS-basal)
were immunoprecipitated with anti-Tg, and the radioactivity content caused similar ultrastructural changes of the AJ and the
of pellet (Tg) and supernatant (non-Tg) was counted. Data are from microfilaments in the apical cytoplasm.
single cultures from one of three experiments, showing similar re- Ca21-dependent cell-cell adhesion in epithelia is mainly
sults. Open bars, control; filled bars, IL-1a. mediated by E-cadherin, which plays a central role in the
formation and maintenance of a cohesive epithelial sheet
examined, only IL-1a reduced the epithelial tightness and (33). Conversely, down-regulation or inhibited function of
caused paracellular leakage. The effect was obvious after 24 h E-cadherin is associated with loss of epithelial junctions, as
and further augmented after 48 h of exposure, without mor- found in tumor progression towards a more malignant phe-
phological signs of cytotoxicity. In addition, the synthesis of notype of carcinoma cells (34). There are no previous reports
a novel high-molecular-mass protein, released mainly in the addressing the question of whether cadherins are influenced
basal direction, was stimulated, further indicating that cell by IL-1a. We found here that the protein expression of E-
viability was not challenged by IL-1a. In contrast, IFN-g and cadherin was not altered by IL-1a treatment for 48 h. Also,
TNF-a were ineffective at concentrations which open TJs in the cellular amounts of a-, b-, and g-catenins, which regulate
intestinal (20) and renal (21) epithelial cells. Likewise, IL-6, the binding of E-cadherin to AJ-associated actin (27), were
which has been found to reduce the intercellular contact of unchanged. Therefore, if the IL-1a-induced disruption of
mammary carcinoma cells (30), had no such effect, indicating thyroid junctions is related to altered function of E-cadherin
that cytokine-induced loss of epithelial integrity is target or catenins, posttranslational modification, as recently
cell-dependent. The dysregulating effect of IL-1a on the bar- shown to occur in response to src oncoprotein (35) and pep-
rier function of the thyroid epithelium is a novel finding not tide growth factors (36), must be considered. In agreement
previously reported for other epithelial cells unless being with the present findings, Tamm et al. (31) found that IL-6
related to cell damage (31). caused dissociation of mammary carcinoma cells without
The IL-1a-induced loss of barrier function was accompa- altering the expression of E-cadherin.
nied by altered distribution of the TJ protein ZO-1 and dis- Because iodide organification normally takes place exclu-
organization of the junctional complex, as revealed by elec- sively inside the follicular lumen at the apical surface of the
tron microscopy. However, unless the cells were treated with thyroid epithelial cells (19), the integrity of the follicular wall
a high concentration (100 U/mL) of IL-1a, the ultrastructural is of considerable importance for thyroid function. Iodide isIL-1a AND THE THYROID EPITHELIAL BARRIER 951
actively transported across the epithelium to the lumen by protein of similar size, produced by thyrocytes and secreted
basolateral uptake and apical efflux mechanisms (37), and Tg in the same polarized manner, is thrombospondin (42), an
is secreted predominantly in the apical direction (38). A oligomeric, multifunctional extracellular matrix component.
prerequisite for maintaining the lumenal content of Tg and However, thrombospondin is resolved to a monomeric form
iodide is that paracellular leakage down-hill from their con- of approximately 190 kDa under reducing SDS-PAGE, which
centration gradients is restricted by the presence of TJ. In were the conditions used for protein separation in the present
addition, TJ is known to act as a fence, which prevents the study. Moreover, IL-1 is known to down-regulate the pro-
mixing of integral membrane proteins specific for either of duction of thrombospondin, e.g. in endothelial cells (43).
the apical and basolateral plasma membranes (39). It is likely Thus, the molecular identity and possible function(s) of
that this fence function of TJ supports the polarized dis- the high-molecular-mass secretory protein, stimulated by
tribution of both the iodide transporters and the thyroid- IL-1a in filter-cultured human thyrocytes, remain to be
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specific enzymes, H2O2-generating NADPH oxidase and elucidated.
TPO, which catalyze the iodination of Tg at the apical cell
surface. Consequently, agents that disrupt thyroid follicular
Acknowledgments
integrity would be a serious threat to the production of
thyroid hormones. Previous studies (8 –9) show that the syn- We would like to thank Drs. Å. Krogh Rasmussen and J. P. Banga for
thesis of Tg is reduced by IL-1. The present findings suggest valuable criticism of the manuscript; Dr. L. E. Tisell and colleagues at the
Department of Surgery, Sahlgrenska University Hospital, Göteborg, for
that a loss of the thyroid epithelial barrier may be another providing human thyroid tissue; and G. Bokhede, T. Carlsson, and Y.
mechanism by which IL-1a inhibits thyroid hormonogenesis. Josefsson for superb technical assistance.
Locally produced IL-1a is likely to be involved in the
development of autoimmune thyroid disease (1, 2), although
its precise pathophysiologic role has not been clarified. An References
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