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Division of Molecular and Cellular Medicine, Department of Medicine (T.J.S., H.-S.W.), Department of Biochemistry and Molecular Biology (T.J.S., H.J.C.), Albany Medical College, Samuel S. Stratton Veterans Affairs Medical Center, Albany, New York 12208; the Cancer Center and Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry (G.D.S., R.P.P.), Rochester, New York 14642; and Infectious Disease Section, Department of Medicine, State University of New York at Buffalo (C.S.B.), Department of Veterans Affairs Medical Center, Buffalo, New York 14215
Address all correspondence and requests for reprints to: Terry J. Smith, M.D., Division of Molecular and Cellular Medicine (A-175), Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208.
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Abstract |
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(500 U/ml) treatment for 48–72 h resulted
in high levels of surface HLA-DR and CD40 display. When CD40 is engaged
with CD40 ligand (CD40L), nuclear factor-
B binding activity is
up-regulated as is interleukin (IL)-6 and IL-8 expression. IL-1ß
treatment up-regulates the expression of IL-1
, IL-1ß, and
PGE2. These observations suggest that thyroid fibroblasts
possess the molecular machinery necessary for cross-talk with
immunocompetent cells such as lymphocytes and mast cells through the
CD40/CD40L complex, as well as through classic cytokine networks, and
to participate potentially in the inflammatory response of the thyroid
gland. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The thyroid gland serves a unique biological function as the principal iodine-concentrating tissue in mammals. The vast majority of investigations concerning the thyroid have focused on function and disease directly related to epithelial cells. This gland is frequently affected by inflammatory autoimmune diseases, and some of this pathology results in substantial tissue remodeling. Thus it should be of considerable interest to characterize the fibroblast population residing in thyroid tissue with regard to its role in supporting normal glandular function and potentially its contribution to disease susceptibility. Unfortunately, little is currently known about thyroid fibroblasts.
We have defined a number of attributes characteristic of human orbital fibroblasts because of their putative role in the pathogenesis of thyroid-associated ophthalmopathy (TAO). Those cells are particularly susceptible to several actions of inflammatory cytokines. For instance, the T lymphocyte product, leukoregulin, can induce the synthesis of hyaluronan (8) and the expression of plasminogen activator inhibitor type-1 (6) in orbital fibroblasts. The magnitude of these actions is substantially greater than that observed in dermal fibroblasts (6, 8). We have reported that orbital fibroblasts exhibit an exaggerated induction of PG endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase (12), and others have shown a similar inducibility of PGHS-2 in synoviocytes from patients with rheumatoid arthritis (13). We hypothesize that this exaggerated inducibility of PGHS-2 in orbital and synovial fibroblasts represents the molecular basis for the inflammatory responses observed in TAO and rheumatoid arthritis, respectively. We have also reported that orbital fibroblasts express a profile of gangliosides that differs considerably from that observed in dermal fibroblasts (14). These glycosphingolipids play important roles in cell recognition and signaling (15). Thus differences in ganglioside expression imply that fibroblasts from distant anatomic regions may engage in interplay with their molecular environment that is cell-specific.
A major conduit for the cross-talk between T and B lymphocytes was
identified with the discovery of CD40 and its natural ligand, gp39,
also known as CD40L (16). B lymphocytes are triggered by CD40
engagement, often in association with interleukin (IL)-4 (17). This
activation represents an important stimulus for B cell proliferation,
Ig production, and isotype switching (18, 19, 20). CD40 is also expressed
by non-B lymphocyte lineage cells such as monocytes, dendritic,
Langerhans, epithelial, endothelial cells, and keratinocytes (16, 21, 22, 23, 24, 25, 26). Recently, CD40 has been found on synoviocytes and certain
fibroblasts from lung and periodontal tissue (27). In these
fibroblasts, CD40 is up-regulated by interferon-. The biological
implications of CD40 expression by fibroblasts is uncertain but might
represent an important mechanism through which these cells cross-talk
with T lymphocytes and other CD40L-displaying cells.
In this study, we examine, for the first time, several aspects of the
human thyroid fibroblast phenotype in vitro. We report here
that these cells undergo the dramatic changes in morphology in response
to exogenous PGE2 that were observed previously in orbital
fibroblasts (2, 3). They express a profile of gangliosides that sets
them apart from orbital and dermal fibroblasts. Thyroid fibroblasts
express CD40 and, when exposed to CD40L, express high levels of IL-6
and IL-8. These fibroblasts also respond to exogenous IL-1ß with
regard to an up-regulation of IL-1 and IL-1ß expression and the
production of PGE2. Thus they may play an important role in
the pathogenesis of inflammatory diseases of the thyroid by virtue of
their abilities to cross-talk with immunocompetent cells. This can be
accomplished directly through the CD40/CD40L signaling conduit as well
as through multiple, classic cytokine pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was obtained from Genzyme (Cambridge, MA). Eagle’s
medium, FBS, and other culture reagents were supplied by GIBCO (Grand
Island, NY), from Hyclone (Logan, UT), or from Life Technologies
(Gaithersburg, MD).
Cell culture
Thyroid fibroblasts were obtained by mincing thyroid tissue
explants obtained at thyroidectomy for Graves’ disease or for
neoplastic disease, where normal tissue was excised. Orbital
fibroblasts were obtained during decompressive surgery for TAO. Dermal
fibroblasts were obtained by punch biopsy of normal appearing skin.
These activities have been approved by the Institutional Review Board
of the Albany Medical College. Explants were cut into small pieces and
allowed to attach to the bottom of 60-mm plastic culture plates as
described previously (28). They were covered with Eagle’s medium
supplemented with FBS (10%), glutamine, and antibiotics and maintained
in a 5% CO2, humidified incubator. When fibroblasts
emerging from the tissue became numerous, the explants were removed
carefully so as to not leave any intact thyroid tissue, which would
include thyrocytes and endothelial cells, on the plate. Fibroblast
monolayers were disrupted with trypsin/EDTA treatment. These cells
exhibit morphologies that are fibroblast-like. The cultures were
assessed for contaminating cells by immunostaining for factor VIII,
smooth muscle actin, and thyroglobulin. For experiments, cultures were
passaged approximately every 2 weeks by gentle treatment with
trypsin/EDTA. Some of the cellular material was stored at -70 C. Cells
were used between the second and 12th passage.
Phase-contrast microscopy
Fibroblasts were inoculated on sterile 22 x 22 mm glass
coverslips at a seeding density of about 104 cells per
coverslip. Cells were allowed to attach for 1 day before the initiation
of experiments. Cultures were shifted to medium supplemented with 1%
FBS without or with the test compounds. After treatment, monolayers
were fixed in 2% para-formaldehyde in PBS (pH 8.0). The coverslips
were washed twice and mounted on glass slides with glycerol/PBS (1:1).
Microscopy was performed with a Nikon Labophot (Nikon Corp., Melville,
NY) equipped with a 35-mm camera. Photographs were taken on Kodak
Tri-Max 400 film (Eastman Kodak, Rochester, NY). The final
magnification was 400x.
Flow cytometric analysis of surface HLA-DR, Thy-1, and CD40
expression by thyroid fibroblasts
Fibroblasts were prepared for flow cytometry by washing
dispersed fibroblasts in suspension in PBS with 0.1% sodium azide and
1% BSA. Cells were incubated with either antihuman HLA-DR [L243, 100
µl of hybridoma supernatant, American Type Culture Collection (ATCC),
Rockville, MD], anti-Thy-1 (F15–42-1, kindly provided by Dr. John W.
Fabre, University of London, London, UK) or anti-CD40 monoclonal
antibody (G28–5, 100 µl of hybridoma supernatant, ATCC) for 30 min
on ice. After washing, fluorescein isothiocyanate-conjugated (FITC)
goat antimouse Ig (1:100 dilution, Jackson Immunoreasearch, West Grove,
PA) was added for 30 min on ice. Samples stained only with the
secondary antibody served as negative control. Once washed, the cells
were resuspended in PBS with 0.1% sodium azide and 1% BSA and were
analyzed on a Coulter Elite flow cytometer (Coulter Corp., Hialeah,
FL). We have demonstrated that trypsin treatment, used to disrupt
fibroblast monolayers, fails to alter the detection of surface HLA-DR,
Thy-1, or CD40. Viable cells were gated on the basis of forward light
scatter, and the data were analyzed with the Cytologo software program
(Coulter). All studies were performed at least three times with
representative results being presented.
Immunofluoresence analysis of HLA-DR, CD40, and Thy-1 expression in
thyroid fibroblasts
Indirect immunofluorescence analysis of HLA-DR, CD40, and Thy-1
expression in thyroid fibroblasts was achieved by inoculating cells in
chamber slides (Nunc, Naperville, IL). They were stained under similar
conditions as those examined by flow cytometry with the exception that
they were conducted at room temperature. Slides were removed from the
chambers, coverslips were applied, and cell layers were visualized
under an Olympus BX 40 microscope (Olympus America, Lake Success, NY)
equipped with a UPlan-FL (Ph2) 40 x objective.
Ganglioside analysis
Gangliosides were purified from fibroblasts as reported
previously (14, 29). Briefly, monolayers were disrupted mechanically
and cells rinsed in 0.31 M pentaerythritol and centrifuged.
Pelleted cells were continuously inverted for 18 h in
chloroform-methanol (1:1, all vol/vol) in glass tubes. Total lipid
extracts were rotary evaporated to dryness and applied to
diethylaminoethyl-Sephadex A-25 columns (bed vol, 3 ml) in
chloroform-methanol-water (30:60:8). Acidic lipids were eluted with
chloroform-methanol: 0.8 N sodium acetate (30:60:8),
evaporated to dryness, and solubilized in 0.1 N NaOH.
Samples were then neutralized to pH 4–5 with 0.1 N HCl and
desalted by reverse phase column chromatography (Sep-Pak, Waters
Associates, Waltham, MA). Lipids were recovered and applied to
Iatrobeads 6RS-8060 (Iatron Laboratories, Tokyo). Less polar lipids
were eluted with chloroform-methanol (85:15). Gangliosides were eluted
with chloroform-methanol (1:2) and dried, and purity was confirmed on
thin layer chromatograms sprayed with resorcinol.
Two-dimensional TLC was performed on each sample, containing 3–5 µg ganglioside-bound sialic acid. Standard markers of murine brain ganglioside were included in each dimension for reference. The solvent system for the first dimension TLC was chloroform-methanol-0.25% KCl (50:45:10), and that of the second was chloroform-methanol-2.5 N NH3 in aqueous 0.025% KCl (50:40:10). After the chromatogram was dried, gangliosides were visualized by spraying with resorcinol reagent and heating uniformly to 92 C. Relative quantification of individual ganglioside peaks was performed (30) with a two-dimensional analytical scanning system (Molecular Dynamics, Sunnyvale, CA). To compensate for differences in the quantity of individual samples, individual peaks are expressed as relative percentages of the total signal. Each chromatogram was loaded with 3–5 µg ganglioside-bound sialic acid. Because of the small quantities of materials, significance of differences in the abundance of the minor peaks could not be assessed. Thus, differences in peaks containing less than 6% of the total (i.e. <0.4 µg sialic acid) were not assigned levels of significance. Mean relative percentages of homologous peaks contained in Table 1 are expressed as the mean ± SEM. Statistical differences between groups was determined using Student’s one-tailed t test and confirmed by post hoc ANOVA using Scheffe’s F test.
RT-PCR
Fibroblasts were either treated with nothing or with
interferon- (500 U/ml) for 72 h, after which the monolayers
were scraped with a rubber policeman, and total cellular RNA was
extracted with Tri-Reagent (1 ml per 106 cells, Molecular
Research Center, Cincinnati, OH) following the manufacturer’s
protocol. RNA was solubilized in nuclease-free water, concentrations
were determined spectrophotometrically, and 5 µg of each sample were
subjected to reverse transcription with Moloney murine leukemia virus
reverse transcriptase (200 U/reaction, Life Technologies) and oligo-dT
primer (Pharmacia, Piscataway, NJ) as described previously (31). RT was
withheld from replicate samples that served as negative controls
(RT-). PCR reactions for CD40 and ß-actin included 5 µl of the
above generated complementary DNA (cDNA), PCR buffer (5 µl of 10X
stock, Boehringer Mannheim, Indianapolis, IN), deoxynucleotides (1
µM each) primers (1 µM each),
Taq polymerase (2.5 U, Boehringer Mannheim) in a total
volume of 50 µl. The CD40 primer sequences were
3'-CGTACAGTGCCAGCCTTCTTC and 5'-ATGGTTCGTCTGCCTCTG CAG yielding a 330
bp product and the ß-actin primers were 3'-CTCCTTAATGTCACG CACGATTTC
and 5'-GTGGGGCGCCCAGGCACCA, which generated a 539-bp product. Samples
underwent 30 cycles of amplification in a M.J. Research PTC-200 DNA
Engine (Watertown, MA), and each cycle included denaturation at 94
C for 30 sec, annealing at 63 C for 30 sec, and primer extension at 72
C for 60 sec. The products were electrophoresed on 2% agarose gels and
visualized by ethidium bromide staining. A 100-bp ladder (Life
Technologies) was used for size determination.
Electrophoretic mobility shift assay (EMSA) for nuclear factor
(NF)-B activity
Fibroblasts were allowed to proliferate to confluence in 100-mm
culture plates covered in medium supplemented with 10% FBS. They were
treated with interferon- (500 U/ml) or nothing for 72 h. The
cultures were then washed extensively and incubated overnight with
medium with 1% FBS. Cells were then stimulated for 2 h at 37 C
with medium alone, control insect cell membranes containing glutathione
S-transferase (GST) or insect cell membranes containing
human CD40L, generously provided by Dr. Marilyn Kehry (Boehringer
Ingelheim, Ridgefield, CT) prepared as described previously (32).
Nuclear extracts were prepared as described (33), and proteins were
normalized using a BCA protein assay (Pierce, Rockford, IL). To assess
NF-B binding activity, 2.5 µg of the extract were incubated with
32P-labeled double- stranded oligonucleotide probe
representing the consensus sequence for the NF-B-binding site
following the supplier’s instructions (Promega, Madison, WI). Samples
were electrophoresed on 4% polyacrylamide gels, and the DNA/protein
complexes were visualized by autoradiography. Supershift assays were
performed by preincubating the nuclear extracts with anti-p65
antibodies or control rabbit IgG (Santa Cruz Biotechnology, Santa
Cruz, CA).
Cytokine expression in response to CD40 engagement with CD40L
Thyroid fibroblasts were seeded into 96-well tissue
culture plates (5000 cells per well) and once established were
pretreated with nothing or interferon- (500 U/ml) for 72 h in
medium supplemented with 10% FBS. Monolayers were washed extensively
with fresh medium and then stimulated as triplicate wells for 2 h
with medium alone, control insect cell membranes containing GST, or
human CD40L. Cell-free culture media were harvested, and the levels of
IL-6 and IL-8 secreted by the monolayers were determined by specific
enzyme-linked immunosorbent assay (ELISA). These were diluted with
sample buffer so that the cytokine concentrations would fall in the
linear range of the assays. Neither cytokine was detectable in fresh
medium without or with the various additives.
With regard to the IL-6 assay, Immulon 3 flat bottom 96-well plates (Dynatech, Chantilly, VA) were coated overnight at 4 C with 2 µg/ml antihuman IL-6 (capture antibody, Pharmingen, San Diego, CA) diluted in 0.1 M sodium bicarbonate (pH 8.2). The plates were then washed three times with PBS/Tween (0.5 ml Tween-20 per liter PBS containing 0.1% sodium azide). Plates were blocked with ELISA buffer (PBS containing 5% BSA (wt/vol), 5% calf serum (vol/vol) and 0.1% sodium azide) for 2 h at room temperature. Plates were washed three times with PBS/Tween. Samples and IL-6 standards (Pharmingen) were incubated in the plates for 2 h at 37 C followed by three washes with PBS/Tween. Detection antibody (2 µg/ml biotinylated antihuman IL-6, Pharmingen) was diluted in ELISA buffer and incubated in the plates for 1 h at room temperature. Plates were washed three times and incubated 1 h at 37 C with streptavidin-alkaline phosphatase (1:500 dilution in ELISA buffer, Southern Biotechnologies, Birmingham, AL). After three washes, plates were incubated with p-nitrophenyl phosphate (1 mg/ml) in DEA buffer (10 µM MgCl2, 30 µM NaN3, and 1% (vol/vol) diethanolamine, pH 9.0). The reaction was stopped with 3 N NaOH, and the optical density of each well was determined at 405 nm with a Dynatech plate reader.
The assay for IL-8 resembled that for IL-6 with the following exceptions. The capture antibody (anti-IL-6, Endogen, Cambridge, MA) was at a concentration of 1 µg/ml. The IL-8 standard was from Biosource, and the biotinylated detection antibody (antihuman IL-8, Endogen) was at a concentration of 0.5 µg/ml. Detection antibody was added 15 min after the addition of sample to the plates and incubated for 1 h at room temperature. All studies were performed at least three times, and results from representative experiments are shown.
Quantification of IL-1 and IL-1ß expression and
PGE2 production by thyroid fibroblasts
Thyroid fibroblasts for the IL-1 and IL-1ß assays were
cultured in 24-well plates to confluence in medium supplemented with
10% FBS. Cultures were then shifted to medium with 1% FBS for the
next 16 h. IL-1 or IL-1ß were added to some of the wells at
the times indicated in the figures. After treatment, monolayers were
washed extensively with PBS and cells were taken up in lysis buffer
containing 20 mM Tris-HCl (pH 7.5), 15 mM
3-[(3-chloamidopropyl)dimethylammonio]-1-propanesulfonic acid, 1
mM EDTA, 10 µM phenylmethylsulfonyl fluoride,
and 10 U/ml soybean trypsin inhibitor. Cellular protein (10 µg) was
subjected to assay using either IL-1 or IL-1ß ELISA kits
(Immunotech, Westbrook, ME) according to the instructions of the
manufacturer. We have determined that the exogenous IL-1ß used in the
treatment of cultures does not contribute to the cytokine measured in
the cell lysates. With regard to quantification of PGE2
production, confluent fibroblasts in 24-well plates were shifted to
medium with 1% FBS for 16 h, and then some received IL-1ß (10
ng/ml) for 16 h. Cultures were then shifted to PBS (150 µl/well)
for the final 30 min of incubation in the absence or presence of the
cytokine. PBS was collected and subjected to RIA as described
previously (12) for PGE2 content.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, panel A). When exposed to
PGE2 (100 nM, panel B) or 8-br-cAMP (1
mM, panel C) they become stellate. This response is similar
to that reported previously in orbital fibroblasts (Fig. 1, panels E
and F) (2, 3). In contrast, abdominal wall fibroblasts fail to exhibit
an altered cell morphology in response to either compound (panels H and
I). Thus the vulnerability of the cytoarchitecture previously
recognized in orbital fibroblasts is shared by thyroid cells.
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.
Each chromatogram contains 10 to 11 discrete, resorcinol-positive
peaks, occupying the same TLC positions, relative to the standards, as
gangliosides from orbital and dermal fibroblasts (14). A typical
chromatogram is presented in Fig. 3. Some
variations occur between individuals with regard to specific
ganglioside peaks resolvable on thin layer chromatograms. In some
samples, it is not possible to accurately distinguish borders between
peaks 1 and 2 and between peaks 3 and 4, although each pair appears as
two separate, overlapping peaks. These peaks are designated as
peak 1/2 and peak 3/4. There are two predominant
ganglioside peaks in every sample from normal and Graves’ thyroids,
consistent with what had been reported previously in orbital and dermal
fibroblasts (14). The most abundant in all strains examined is peak
1/2. The material migrating in this complex accounts for
62.8 ± 1.4% of the total. This is not significantly different
from the fractional contribution of the peak found in dermal
fibroblasts (63.9 ± 2.9%) (14). However, as with dermal
fibroblasts, it is significantly different from the relative percentage
of peak 1/2 of orbital fibroblasts (35.3 ± 2.4%,
P < 0.01) (14). Peak 1/2 migrated slightly
ahead of the GM1 standards in both dimensions. The other
major ganglioside complex in thyroid fibroblasts, peak 7, migrates
slightly ahead of GD1a standards in both dimensions and
made up 16.8 ± 0.6% of the total. Comparison to the relative
percent of peak 7 of dermal (11.1 ± 1.3%) and orbital (36.8
± 2.4%) reveals significant differences with both (P
< 0.05 and P < 0.01, respectively). The relative
abundance of several other peaks varies between the two profiles. The
composite ratio of peak 1/2 to peak 7 in all thyroid fibroblast
strains is 3.8 ± 0.2 (mean ± SEM). As can be
seen in Fig. 4, this ratio differs from
the ratios found between the two peaks in both the orbital (0.98
± 0.1, P < 0.01) and dermal (6.1 ± 0.7,
P < 0.7, P < 0.01) fibroblasts
(14).
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Human thyroid fibroblasts express Thy-1 uniformly
Dermal fibroblasts uniformly express Thy-1, a glycoprotein on the
surface of the cell (9). In contrast, orbital fibroblasts derived from
the connective/adipose tissue are comprised of cells that express Thy-1
and those that do not (9). The fraction of cells expressing Thy-1 in
orbital cultures ranges from 54–71% in the strains examined.
Moreover, the Thy-1 expression status of human fibroblasts is
faithfully retained over prolonged time in culture. Thyroid fibroblasts
resemble dermal fibroblasts with regard to a uniform surface display of
Thy-1. Flow cytometric analysis of intact thyroid fibroblasts confirms
this near-uniformity. There is a homogeneous fluorescent signal on the
surfaces of the cells under both unstimulated and
interferon--treated culture conditions (Fig. 5). It would appear from both
immunofluorescent studies and flow cytometry that Thy-1 expression is
invariant with respect to interferon- treatment.
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express surface
HLA-DR
) and with immunostaining followed by flow
cytometry (Fig. 6). Addition of
interferon- (500 U/ml) for 72 h, however, results in a strong
induction of the protein on the surface of the fibroblasts. It would
appear that this response occurs uniformly among the fibroblast
population. This result is consistent with our findings in orbital and
dermal fibroblasts where interferon- induced HLA-DR (34). The
expression of HLA-DR in thyroid fibroblasts treated with interferon-
was dramatic in cultures derived from both Graves’ and normal
thyroids, suggesting that the disease was not a major determinant of
response.
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. The log fluorescence intensity of staining is plotted
vs. cell number for each sample analyzed. Fibroblasts from
Graves’ and normal tissue express CD40. We confirmed this expression
of the protein by assessing CD40 mRNA levels by RT-PCR in untreated
thyroid fibroblasts as well as those treated with interferon- (Fig. 7). Equivalent amounts of RNA were
reverse transcribed, and the cDNA generated was then amplified for 30
cycles with primers specific for CD40 and ß-actin. Thyroid
fibroblasts derived from normal and Graves’ tissue express CD40 mRNA
under basal conditions. Exposure to interferon- appears to
up-regulate the mRNA levels in both types of fibroblasts; however, the
RT-PCR was not quantitative. This issue was assessed further by
examining the levels of surface CD40 protein display. They are found to
be increased substantially by interferon- (Figs. 5 and 6). When
stimulated with the cytokine for 72 h, there is a dramatic 10-fold
up-regulation of CD40 protein in thyroid fibroblasts as assessed by
flow cytometry.
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B in thyroid fibroblastsB (35). We therefore performed a series of EMSAs on the nuclear
extracts of thyroid fibroblasts that had been pretreated with
interferon- to up-regulate surface CD40 display. Cells were
stimulated for 2 h with control insect membranes or those
containing CD40L. Nuclear proteins were then incubated with a
32P-labeled double-stranded oligonucleotide probe
representing the consensus sequence for the NF-B-binding site. As
the electrophoretic pattern shown in Fig. 8 demonstrates, there is a substantial
up-regulation of NF-B-binding activity. The activity is similar to
that elicited by tumor necrosis factor-, an established activator of
NF-B (data not shown). Identity of the protein/DNA complex was
established by adding 200-fold excess of unlabeled NF-B
oligonucleotide, which quenched the signal, as expected. A supershift
EMSA was performed by preincubating the reaction mixture with anti-p65
(RelA) polyclonal antiserum. Addition of the antibody resulted in the
supershift of the NF-B/DNA complex, confirming the presence of the
p65-transactivating subunit (data not shown). Addition of preimmune
serum failed to supershift the complex. These results indicate that
thyroid fibroblasts preincubated with interferon- display functional
CD40 on their surface and that engagement with CD40L results in the
activation and nuclear translocation of NF-B.
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B sites. Moreover, the
expression of both IL-6 and IL-8 genes can be transactivated by
p65-containing dimers (36, 37, 38). IL-6 is an important trigger for the
activation of B lymphocytes (39) and is a fibroblast autocrine growth
factor (40). IL-8 is a powerful chemoattractant molecule for
neutrophils and T lymphocytes (41). We therefore examined the influence
of CD40 activation on IL-6 and IL-8 expression in thyroid fibroblasts.
Confluent monolayers were incubated in the absence or presence of
interferon- (500 U/ml) for 72 h to up-regulate surface CD40
display. Cultures were then treated with fresh medium with insect
membranes containing GST (control) or CD40L for 72 h, and then the
media were collected for cytokine quantification using ELISA assays.
CD40 engagement with CD40L in these cells provokes a substantial
increase in IL-6 release into the culture medium. The magnitude of this
response is 3.1-fold and 2.5-fold in normal and Graves’ fibroblasts,
respectively, as Fig. 9 (left
panel) suggests. Priming the cultures with interferon- (500
U/ml) pretreatment enhances the impact of CD40 engagement, as expected.
After interferon- pretreatment, the increases are 15-fold and 4-fold
in the two types of fibroblasts. The effects of interferon- on basal
IL-6 expression are negligible. IL-8 is a chemoattractant and thus
potentially of importance in the trafficking of neutrophils and T
lymphocytes to the thyroid in inflammatory states. It is expressed by
both bone marrow-derived and nonimmunocompetent cells at sites of
tissue injury and inflammation (41, 42, 43). As was the case with regard to
IL-6, thyroid fibroblasts express low levels of IL-8 constitutively,
and mRNA encoding the cytokine is detectable by RT-PCR (data not
shown). Il-8 synthesis increases dramatically in thyroid fibroblasts
after CD40 activation (Fig. 9, right panel). CD40L elicits a
5.7-fold and 30.4-fold increase in IL-8 production in normal and
Graves’ thyroid fibroblasts not pretreated with interferon-. As can
be seen, pretreatment of the cultures with the cytokine enhances
substantially the impact of CD40 engagement. The magnitude of the
induction is 15.5-fold and 30-fold above baseline in the respective
pretreated culture strains. Thus CD40 engagement in thyroid fibroblasts
from Graves’ and normal tissue results in substantial increases in
IL-6 and IL-8 expression.
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and IL-1ß in response to
exogenous IL-1 (44). We examined thyroid fibroblasts for basal and
cytokine-stimulated IL-1 and IL-1ß production. As Fig. 10 suggests, IL-1 is expressed in
untreated Graves’ fibroblasts but is at the limits of detection in the
cultures from normal thyroid tissue. In contrast, IL-1ß is
undetectable in both types of untreated fibroblasts. When the cultures
were exposed to exogenous IL-1, the levels of both IL-1 and IL-1ß
are up-regulated substantially. The levels of IL-1 achieved after
16 h of IL-1 and IL-1ß exposure are 17.3 ± 0.9 and
7.2 ± 1.6 pg/µg protein in fibroblasts from normal tissue
(100-fold and 42-fold increases, respectively) and 23.1 ± 4 and
14.6 ± 2.4 pg/µg protein (3.8-fold and 2.4-fold, respectively)
in cultures of Graves’ fibroblasts. With regard to IL-1ß expression,
IL-1 and IL-1ß induce the cytokine’s levels to 12.5 ± 1.7
and 35.9 ± 2 pg/µg protein, respectively, in normal fibroblasts
and 55 ± 4.3 and 74.6 ± 3.7 pg/µg protein, respectively,
in Graves’ cultures. Thus fibroblasts from Graves’ and normal thyroid
tissue exhibit substantial expression of IL-1 and IL-1ß when
treated with exogenous IL-1.
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demonstrate, these cells produce detectable levels of the
prostanoid under basal culture conditions. PGE2 levels were
192 ± 10 pg in normal fibroblasts and 480 ± 74 pg in
fibroblasts from Graves’ tissue. When the cultures are treated for
16 h with IL-1ß (10 ng/ml), PGE2 levels were
increased modestly to 672 ± 70 pg and 960 ± 100 pg in the
two cell types, respectively (3.5-fold and 2-fold increases). These
values are similar to those observed in dermal fibroblasts where
control levels of PGE2 are 120 ± 10 pg and those
after treatment with IL-1ß for 16 h increase to 1200 ± 15
pg. In contrast, PGE2 levels in orbital cultures are
44 ± 8.7 pg before and 3000 ± 74 pg after IL-1ß
treatment. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We hypothesize that differences in fibroblast phenotype reflect peculiarities in function and susceptibility to disease manifested by the tissues from which the cells derive. Moreover, we believe that fibroblast subtypes, as have been demonstrated in the lung (10), spleen (11), and orbit (9), may serve different roles in tissue repair and normal remodeling. This notion of fibroblast specialization differs from the prevailing concept of more or less equivalence among these cells. Substantial evidence, some dating 30 yr, suggests that the site of derivation is a crucial determinant of fibroblast behavior in vitro. By virtue of their extensive biosynthetic repertoires, fibroblasts almost certainly impact on the normal function of neighboring cells. This occurs through characteristic extracellular matrix elaboration and by releasing small regulatory molecules, such as growth factors and cytokines, into the extracellular space. Thus differences in fibroblast responses to molecular cues could account for variations in wound healing and inflammatory responses observed in dissimilar tissues.
We have demonstrated previously that a major departure in fibroblast phenotype, related to the anatomic region of derivation, relates to the profile of gangliosides they express (14). Thyroid fibroblasts exhibit gangliosides in proportions that differ from those in other fibroblast types. They do not appear to display novel, abundant glycolipids not found in the other fibroblasts. Among the abundant gangliosides, it is peak 7 material that differs most among fibroblast strains. Gangliosides are sialic acid-containing glycosphingolipids with diverse biological functions (47). Their putative roles in human autoimmunity (48) make the observations concerning diversity among fibroblasts, with respect to ganglioside expression, particularly interesting. Because gangliosides play important roles in cell signaling (15, 47), differences in expression may represent important and cell-specific recognition determinants. The presence of antibodies against the Forssman glycolipid (Gb5) in patients with Graves’ disease and Hashimoto’s thyroiditis (49) and the apparent alterations in ganglioside content in membranes collected from whole thyroid glands (50) suggest potential relevance of these findings in fibroblasts to thyroid autoimmunity. It is intriguing that the variability in relative ganglioside abundance is restricted to the monosialogangliosides. The material in peaks 5 and 6 may represent ganglioside species similar to those in peaks 3 and 4, differing perhaps in the lengths of fatty acyl chains of the ceramide moiety. Interestingly, neuroblastoma gangliosides with shorter fatty acyl chains are considerably more active immunologically than are those with longer chains (51).
We have demonstrated that thyroid fibroblasts treated with
interferon- express detectable cell-surface HLA-DR (Fig. 5). Thus
these fibroblasts are capable of antigen presentation in the setting of
inflammation. Thyroid fibroblasts also display uniformly high levels of
CD40 on their surface when treated with interferon-. Moreover, they
respond to CD40L with regard to a marked up-regulation of IL-6 and IL-8
expression. We hypothesize that the expression of CD40 in fibroblasts
underlies, in some way, the propensity for the thyroid to become
inflamed. This unexpected result suggests a previously unrecognized
pathway whereby resident fibroblasts can receive molecular information
from cells expressing CD40L, such as T lymphocytes and mast cells, both
of which have been implicated in the pathogenesis of autoimmune thyroid
diseases. Mast cells are considered normal residential cells in thyroid
tissue (52). Thus it is possible that cross-talk between mast cells and
fibroblasts through CD40 occurs under physiological conditions as
well.
Our finding that IL-6 is strongly expressed in CD40-engaged
fibroblasts may be of considerable relevance to the pathogenesis of
autoimmunity of the thyroid such as Graves’ disease and Hashimoto’s
thyroiditis. IL-6 is a potent activator of B lymphocytes and has been
shown to enhance B cell proliferation and Ig production (39). Moreover,
it has been suggested that IL-6 acts as an autocrine growth factor in
fibroblasts (40). Serum IL-6 and soluble IL-6 receptor have been
reported to be elevated in Graves’ disease (53, 54), and elevated
levels have been associated with toxic nodular goiter. The mRNAs
encoding IL-6, as well as IL-2, IL-4, IL-5, IL-10, and interferon-,
have been demonstrated by RT-PCR in glands affected by both
Hashimoto’s thyroiditis and Graves’ disease (55). With regard to
Graves’ disease, the IL-6 synthesis occurring in fibroblasts could
contribute substantially to the proliferation and activation of
intrathyroidal B lymphocytes. This would potentially enhance
thyroid-stimulating Ig production. IL-8 is also strongly up-regulated
through CD40 engagement. IL-8 is an important chemattractant for
neutrophils and T lymphocytes (41, 42, 56). The ability for CD40
activation to induce IL-6 and IL-8 expression defines a previously
unidentified circuit for the coordination by the fibroblast of both B
and T lymphocyte activities within the gland. IL-1 and IL-1ß are
also highly inducible in thyroid fibroblasts. IL-1 is thought to be an
important coactivational molecule with IL-2 in Th1 lymphocytes (57),
and thus the IL-1ß released by the thyroid fibroblasts is of
potential importance as a determinant of intrathyroidal lymphocyte
function and cytokine production.
The current observations cast the thyroid fibroblast in a previously unrecognized and potentially central role in the pathogenesis of thyroid autoimmunity. Coupled with our recent (unpublished) finding that thyroid and orbital fibroblasts also express IL-16, a CD4+-specific chemoattractant (58), these fibroblasts may well engage in diverse sets of molecular communication with immunocompetent cells and thus orchestrate and amplify the local immune response. We suggest that thyroidal fibroblasts might function as an integral component of the immune system. Insight into the scope of involvement of these cells in states of health and disease in this tissue, as well as other endocrine organs, awaits further studies.
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Acknowledgments |
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Footnotes |
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2 Current address: Duke University Medical Center, Box 3258, Durham,
North Carolina 27710.
3 Current address: Department of Anatomy, School of Life Science,
National Yang-Ming University, Taipei, Taiwan.
Received June 4, 1997.
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in cultured fibroblasts
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