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Original Studies |
Induces Dissociation of the Junctional Complex and Paracellular Leakage in Filter-Cultured Human Thyrocytes1
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(100 U/mL), tumor
necrosis factor-
(10 ng/mL), and transforming growth factor-ß1 (10
ng/mL) had no effects, exposure to IL-1 for 24–48 h reduced the
transepithelial resistance from >1000 to <50 
x cm2
and increased the paracellular flux of [3H]inulin and
exogeneous 125I-Tg. This response to IL-1, 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 decreased the apical secretion
of endogeneous Tg and stimulated the basolateral release of a novel
high-molecular-mass protein. We conclude that IL-1 reduces the
thyroid epithelial barrier without signs of general cytotoxicity. The
observation suggests a mechanism by which IL-1 may promote the
exposure of hidden autoantigens to the immune system in thyroid
autoimmunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- 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, IL-6, interferon-
(IFN-), tumor necrosis factor- (TNF-), 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 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-treated cells. In
contrast, IFN- and TNF-, 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 has not been reported for any other
epithelium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human recombinant cytokines, IL-1 (5x107 U/mg),
IL-6 (1x108 U/mg), IFN- (2x107 U/mg),
TNF- (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 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- 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), -catenin (1:500), ß-catenin (1:1000), and
-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.
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Results |
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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 x
cm2 in the absence of TSH (5H medium) and 1000–1500 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 1
, RTE and transepithelial
flux of [3H]inulin were not influenced by IFN- (100
U/mL), TNF- (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
(100 U/mL) reduced RTE to less than 100 x
cm2 and increased the transfer of [3H]inulin
across the cell layer (Table 1). This effect of IL-1 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-1
induced partial recovery of RTE (Fig. 3). Whether the barrier dysfunction
induced by IL-1 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. 4, 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 for 48 h. At the highest
concentration tested (1000 U/mL), IL-1 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-treated cultures. Taken together, the data show
that the epithelial integrity of human thyrocytes in culture is
reversibly impaired by IL-1.
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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. 5A), indicating the circumferential position of the TJ and a
complete sealing of the intercellular space. In contrast, after
treatment with IL-1 (100 U/mL) for 48 h, ZO-1 present at the
cell borders showed a markedly zigzaggy course and was often
discontinuous (Fig. 5B), as if the TJ had been partly broken.
IL-1-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 was not reproduced
by the other cytokines examined.
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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. 6A), 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 6B). Typically, the cytoplasmic facet of the AJ exhibited a coat
of dense material (Fig. 6B), 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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(Fig. 6C
) or by treatment with
IL-6, TNF-, or IFN- (not shown). In contrast, in cells exposed to
10 U/mL of IL-1 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 cytoplasm beneath the apical plasma
membrane (Fig. 7A). This response to IL-1 was even more obvious in
horizontal sections (Fig. 7B), 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. Cells given 100 U/mL IL-1 for
48 h displayed more generalized changes in the morphology of the
junctional complex (Fig. 7C), occasionally comprising a complete lack
of both AJ and TJ, which created free communication between the apical
compartment and the intercellular space (Fig. 7D). There were no
ultrastructural signs of cytotoxicity, such as generalized
vacuolization and membrane blebbing, in cells exposed to IL-1.
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-treated
cells
In view of the pronounced ultrastructural changes taking place
mainly in the AJ, we examined whether IL-1 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 treatment for
48 h (Fig. 8). Immunolocalized
E-cadherin and catenins were mainly distributed along the cell-cell
contacts (not shown).
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on apically and basally secreted
proteins
Previous studies indicate that the expression of thyroid-specific
proteins is down-regulated by IL-1 (9, 10). In agreement with this,
we found that IL-1 reduced the amount of Tg released into the apical
medium of [35S]methionine-labeled cells (Fig. 9). At the same time, IL-1 increased
severalfold the secretion of radiolabeled non-Tg proteins into the
basal medium (Fig. 9). 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 was a
high-molecular-mass protein of hitherto unknown identity (Fig. 10). 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 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].
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. In contrast, IFN- and TNF- 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
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-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,
the ultrastructural changes were confined mainly to the AJ and its
submembranous plaque. This suggests that the effect of IL-1 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 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. We found here that the protein expression of E-cadherin was
not altered by IL-1 treatment for 48 h. Also, the cellular
amounts of -, ß-, and -catenins, which regulate the binding of
E-cadherin to AJ-associated actin (27), were unchanged. Therefore, if
the IL-1-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 inhibits thyroid hormonogenesis.
Locally produced IL-1 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 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 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--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 in
thyroid autoimmunity, may be examined in further detail.
Accompanying the barrier dysfunction, the IL-1-treated cells showed
an altered pattern of synthesis and release of secretory proteins. In
agreement with previous reports (8, 9), IL-1 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. 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 in
filter-cultured human thyrocytes, remain to be elucidated.
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Acknowledgments |
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Footnotes |
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2 Holds an individual postdoctoral fellowship provided by the
Swedish Medical Research Council.
Received September 18, 1996.
Revised April 4, 1997.
Revised November 14, 1997.
Accepted November 24, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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