,
* Department of Internal Medicine,
VA Research Service,
Molecular Microbiology & Immunology, and
Pathology, University of Missouri, School of Medicine, Columbia, Missouri
Correspondence: Dr. Helen B. Mullen, Division of Immunology & Rheumatology, University of Missouri, M450 Medical Sciences, Columbia, MO 65212. E-mail: mullenh{at}health.missouri.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
smooth-muscle actin, a
marker of myofibroblasts. Kinetic studies characterized the onset and
development of thyroid fibrosis. TGF-ß1 was increased at mRNA and
protein levels, and expression of TGF-ß1 protein paralleled G-EAT
severity. Comparison of staining patterns showed that TGF-ß1 was
expressed in areas of myofibroblast and collagen accumulation, implying
that TGF-ß1 may play a role in fibrosis in G-EAT. Further studies
demonstrated that myofibroblasts, macrophages, and thyrocytes
contributed to TGF-ß1 production. This provides an excellent model to
study the mechanisms of fibrosis associated with autoimmune
damage.
Key Words: autoimmune disease • myofibroblasts • TGF-ß1
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The thyroid infiltrating cells in G-EAT include CD4+ and CD8+ T-lymphocytes, histiocytes, macrophages, and neutrophils [22 23 24 ]. CD4+ T cells are the primary effector cells in autoimmune thyroiditis [22 , 25 ]. The inflammatory cells in the thyroid produce various cytokines [23 , 26 27 28 ], some of which may contribute to fibrosis. Transforming growth factor-ß (TGF-ß)1 is known to be involved in the development of fibrosis based on its matrix-inducing effects on stromal cells in vitro and studies demonstrating increased expression of TGF-ß1 in fibrotic tissues from a variety of organs [1 2 3 4 5 6 7 8 9 10 11 12 ]. Moreover, neutralization of TGF-ß1 has been shown to inhibit fibrosis [1 2 3 4 , 29 30 31 32 ]. Many types of cells have the potential to produce and regulate TGF-ß1 [1 2 3 , 33 , 34 ], and there are numerous inflammatory cells infiltrating G-EAT thyroids [22 23 24 ]. Thus, excessive production of TGF-ß1 thyroids could be involved in mediating fibrosis in G-EAT.
TGF-ß1 mediates a broad spectrum of biological activities important in embryogenesis, tissue repair, cell growth, and immune regulation [1 2 3 , 33 , 34 ]. The TGF-ß gene is up-regulated in response to tissue injury, and TGF-ß1 is the isoform most implicated in fibrosis [1 2 3 4 , 6 7 8 9 10 11 12 , 15 , 16 , 29 30 31 32 ]. TGF-ß1 is an important regulator of extracellular matrix (ECM), stimulating collagen deposition, blocking collagen degradation, inhibiting ECM-degrading protease, and up-regulating the synthesis of protease inhibitor [1 2 3 4 , 6 7 8 9 10 11 12 , 33 ]. TGF-ß1 is a potent chemoattractant and activator of fibroblasts also [1 2 3 , 8 , 9 , 30 , 33 34 35 36 ]. Among fibroblasts, myofibroblasts (myofbs) are generally present in fibrotic tissues and represent an activated cell phenotype with a high capacity for ECM secretion [1 2 3 , 8 , 9 , 30 , 36 37 38 39 ]. TGF-ß1 can have pro- and anti-inflammatory effects on autoimmune diseases also [33 , 34 , 40 41 42 ], and fibrosis has been shown in some patients with autoimmune disease [13 14 15 16 17 18 19 ].
In this study, we demonstrated that thyroid fibrosis develops in the autoimmune disease model of G-EAT. In an attempt to elucidate the histopathologic features that underlie fibrogenesis, we studied the distribution of TGF-ß1, myofb, and collagen and the phenotypes of the cells that produced TGF-ß1 in the thyroid during development of fibrosis in G-EAT. Myofbs and collagen were present in fibrotic thyroids, and TGF-ß1 was up-regulated strongly in the granulomas and myofbs. These results suggest that TGF-ß1 may influence the fibrotic process in G-EAT thyroids, possibly by regulating collagen-producing cells.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Induction of G-EAT
Donor CBA/J or DBA/1 mice were immunized twice with 150 µg MTg
and 15 ug lipopolysaccharides (LPS; Escherichia coli
0111:B4, Sigma Chemical Co., St. Louis, MO) i.v. at 10-day intervals
[21
22
23
]. Seven days after the second injection of MTg
and LPS, spleen cells from donor mice were cultured at 107
cells/ml in RPMI 1640 containing 25 mM HEPES buffer (Cell and
Immunobiology Core Facility, University of Missouri), 5% fetal calf
serum (FCS; Sigma), 2 mM glutamine, modified Eagle’s medium (MEM)
vitamin solution, nonessential amino acids, 1 mM sodium pyruvate, and
5 x 10-5 M 2-ME. Cells were cultured
with 25 µg/ml MTg and cell culture supernatant containing anti-IL-2R
monoclonal antibody (mAb) M7/20 (5% final concentration) and 5 ng/ml
IL-12 (Intergen, Inc., Purchase, NY), as described previously
[23
]. After 72 h, cells were harvested, washed, and
injected i.v. into irradiated (600 Rad) recipient mice. DBA/1 donor
spleen cells (3–3.5x107) were transferred to DBA/1
recipient mice for induction of G-EAT, and 3.5–4 x
107 CBA/J donor spleen cells were used to induce very
severe G-EAT in CBA/J mice. Recipient thyroids were evaluated for EAT
generally 19–21 days later, the time of maximal severity of G-EAT in
this adoptive transfer model [21
22
23
], or in the
kinetics experiments, from 7–60 days following cell transfer.
Evaluation of G-EAT histopathology
Thyroids were removed from 4–5 mice/group at various times
after cell transfer, and one lobe of each thyroid was fixed with
formalin. For histologic analysis, tissues were embedded in paraffin,
sectioned (7 µm), and stained with hematoxylin and eosin. Thyroids
were scored quantitatively for G-EAT severity, using a scale of 1+–5+,
according to previously established criteria [23
].
Thyroiditis (1+) is defined as an infiltrate of at least 125 cells in
one or several foci, 2+ is 10–20 foci of cellular infiltration
involving up to 25% of the gland, 3+ indicates that 25–50% of the
gland is infiltrated, 4+ indicates that >50% of the gland is
destroyed, and 5+ indicates virtually complete destruction of the gland
with few or no remaining follicles. Thyroid lesions were evaluated
qualitatively also. In general, thyroids with 1–2+ severity scores
have infiltrates mainly consisting of lymphocytes with few neutrophils.
The more severely destroyed thyroids (4–5+) had extensive
granulomatous changes with follicular cell proliferation,
multinucleated giant cells, large numbers of histiocytes, numerous
lymphocytes, and neutrophils. They also had microabscess formation and
necrosis, as well as fibrin deposition and fibrotic features. The
granulomatous inflammation in thyroids graded 4+–5+ extended
characteristically beyond the thyroid to involve the adjacent
connective tissue and muscle. For evaluation of collagen deposition,
some thyroid sections were stained also using Masson’s trichrome.
Immunohistochemistry
Tissue sections were deparaffinized in xylene, rehydrated
through sequential ethanol, and rinsed in phosphate-buffered saline
(PBS). The immunohistochemical methods used immunoperoxidase staining,
and the intensity of immunostaining was graded semiquantitatively. All
incubation steps were performed in a humidified chamber at room
temperature, and endogenous peroxidase activity was blocked with 0.3%
hydrogen peroxide. Dilutions of antibodies were in 1% bovine serum
albumin (BSA)/PBS, pH 7.6. For TGF-ß1 staining, tissues were
pretreated with 20 µg/ml proteinase K for 20 min and blocked with 5%
normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove,
PA). Tissue sections were then incubated with chicken anti-TGF-ß1 and
diluted 1:100 (R&D Systems, Inc., Minneapolis, MN) for 1 h.
Biotin-SP-conjugated donkey antichicken antibody, 1:500 (Jackson), was
used as the secondary antibody. This was followed by incubation with
ABC (Vector Laboratories, Burlingame, CA) for 30 min. Diaminobenzidine
(DAB; Sigma) was used as chromogen. The specificity of anti-TGF-ß1
was confirmed by substitution of nonimmune chicken IgY in place of
primary antibody. The TGF-ß1 staining was assessed in frozen sections
also, which revealed a similar staining pattern as in paraffin
sections. Furthermore, we demonstrated that another antibody, 1D11.1,
derived from a mouse hybridoma against mouse active TGF-ß1
[42
], had a similar staining pattern as the chicken
anti-TGF-ß1 antibody also. The chicken anti-TGF-ß1 antibody was
raised against active TGF-ß1 originally (R&D Systems) and has been
used to detect TGF-ß1 protein in other models [11
,
12
, 43
]. TGF-ß1 can be present in the form
of an inactive complex, which consists of TGF-ß1 linked with
latency-associated protein (LAP) [33
, 34
].
Activation of latent TGF-ß1 is required for TGF-ß1 to exert its
diverse array of biologic activities [33
,
34
]. When anti-TGF-ß1 antibody was absorbed with
fivefold excess (by weight) of LAP (R&D Systems), the absorbed antibody
showed the same staining pattern and intensity as the nonabsorbed
anti-TGF-ß1 antibody. This suggests that the antibody recognized an
active form of TGF-ß1, as described by the manufacturers.
For staining of myofbs, pretreatment of paraffin sections using
microwave irradiation was required for antigen retrieval
[44
]. Tissue sections were immersed in boiling PBS in a
microwave oven (800 W) for two periods of 10 min, cooled down to room
temperature over 15 min, and rinsed in PBS. Mouse mAb to
smooth-muscle actin (-SMA), 1:400 (clone 1A4, Sigma), was used to
identify myofbs. Biotin-labeled goat antimouse F(ab')2,
1:4000 (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD), was
used as secondary antibody. For staining of adjacent sections, one
section was stained for TGF-ß1 as described above and the other
stained for thyrocytes. For double staining, tissues were first stained
for TGF-ß1 and visualized by DAB. The TGF-ß1-stained sections were
then stained with -SMA as described above and visualized by VIP
(Vector). Sections were counterstained with hematoxylin. Negative
controls were performed using nonimmune mouse immunoglobulin (IgG) at a
protein concentration equivalent to the respective secondary
antibodies, which showed negative staining.
Serum thyroxine (T4) assay
Serum T4 levels were determined using a T4 EIA kit (Biotecx
Labs, Houston, TX), according to the manufacturer’s instructions.
Results are expressed as µg T4/dL serum. Preliminary experiments
indicated that T4 levels in normal mouse (CBA/J or DBA/1) serum
measured with these kits ranged from 5–16 µg T4/dL serum
(mean=10.1±1.1 for 10 samples).
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
View this table: [in a new window] |
Table 1. Development of Fibrosis in G-EAT
|
 View larger version (137K): [in a new window] |
Figure 1. Histopathology emphasizing fibrotic changes in G-EAT thyroids of CBA/J
mice. Thyroiditis (3+) at day 10 showed infiltrating lymphocytes and
early proliferative changes of thyroid follicles (A), and there was
severe destruction of thyroid follicles and extensive infiltration of
inflammatory cells in 5+ G-EAT thyroids at day 21, with some areas of
fibrotic changes (B, arrow). Thyroids became atrophic and more fibrotic
at day 35 (C). Deposition of collagen was not seen in day 19 thyroids
with 3–4+ severity scores (D), was minimal at day 19 in 5+ G-EAT (E),
and was further increased at day 35 (F). Deposition of fibrin was noted
also in day 19 thyroids (E, arrow). In normal thyroids, -SMA
staining was not observed (G). Myofbs were abundant in the 5+ G-EAT
thyroids at day 21 (H) and were still present at day 35 (I). Panels
A–C, H & E staining. Panels D–F, trichrome staining (collagen is
blue). Panels G–I, -SMA staining (purple). Original magnification:
A–C and E–I, x400; D, x100.
|
To further demonstrate and characterize fibrosis in G-EAT thyroids, the
presence of collagen and myofbs was examined using trichrome and
-SMA staining, respectively. There was no collagen staining detected
within the thyroids during the early stage of G-EAT in CBA/J mice (days
7–14; Fig. 1D
), and minimal collagen was observed in day 14 thyroids
of DBA/1 mice (Table 1B)
. At the peak of G-EAT severity (days 19–21),
collagen was observed primarily around the periphery of the thyroid
(see Fig. 3A). In more severely destroyed thyroids, collagen deposition
extended into the thyroid gland also (Fig. 1E
and 1F)
. Typically, as
illustrated in Figure 1D
1E
1F
, and Table 1A
, less severely destroyed
thyroids (
4+) had minimal or no collagen staining, but collagen was
detected in 5+ G-EAT thyroids usually by 19–21 days after cell
transfer. By day 28, all 5+ G-EAT thyroids had increased collagen
accumulation, as determined by trichrome staining (unpublished
results). By days 35–60, collagen deposition was very strong in
atrophic thyroids (Fig. 1F
and Table 1
), and distributed at local sites
within the thyroid (Fig. 1F)
. The specific type of collagen in these
thyroids was not determined.
-SMA is a specific marker for myofbs, and it is expressed in smooth
muscle cells also [38
]. As shown in Figure 1G
, myofbs
were not present in normal thyroids, and only blood vessels stained
positive for -SMA (unpublished results). By day 19, many
infiltrating cells were -SMA immunopositive, demonstrating abundant
myofbs in thyroids with 5+ G-EAT severity (Fig. 1H)
. As noted for the
production of collagen, there were more myofbs in 5+ G-EAT thyroids
than in 4+ thyroids (Table 1)
. At day 35, myofbs were still present
although less frequent than in day 19 G-EAT thyroids (Table 1
and Fig. 1I
), which may be a result of decreased inflammation and atrophy of
thyroids. Many myofbs had an enlarged cytoplasm and nucleus, suggesting
an activated phenotype (unpublished results). Collectively, the
presence of collagen and myofbs in thyroids demonstrated that the onset
of fibrosis began in thyroids with 4–5+ G-EAT at day 19 in CBA/J mice.
These changes could be seen as early as day 14 in thyroids of DBA/1
mice with 5+ G-EAT but became more pronounced at day 19. Fibrosis then
progressed further through days 35–60 in CBA/J and DBA/1 mice.
TGF-ß1 expression in thyroids
TGF-ß1 plays an important role in many types of fibrosis
[1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
, 29
30
31
32
]. To determine if TGF-ß1
was produced in G-EAT thyroids during development of fibrosis,
intrathyroidal TGF-ß1 mRNA was detected at various times after cell
transfer using reverse transcription-polymerase chain reaction
(RT-PCR). TGF-ß1 mRNA expression was very low in normal thyroids but
was up-regulated during the course of G-EAT. TGF-ß1 mRNA expression
was maximal at days 13–19, but thyroids had higher levels of TGF-ß1
message than normal thyroids at day 40 (unpublished results). To
determine if TGF-ß1 protein expression in thyroids was consistent
with the mRNA level and if TGF-ß1 protein was present when thyroids
became fibrotic, expression of TGF-ß1 protein was assessed at various
times also. TGF-ß1 protein expression in normal thyroids was minimal
or absent by immunostaining (Fig. 2A
) and increased at day 15 (Fig. 2B)
with peak expression of
TGF-ß1 protein at days 19–21 (Fig. 2C
and Table 1
). Thyroids with 5+
G-EAT showed widespread TGF-ß1 immunoreactivity, with many cells
being TGF-ß1-immunopositive. Localization of TGF-ß1 was most
abundant in the thyroid granulomas (Fig. 2E
and Fig. 3C
), which were comprised of T cells, epithelioid histiocytes, and
macrophages. At day 28, 4–5+ G-EAT thyroids still exhibited strong
expression of TGF-ß1 protein, with a staining intensity comparable
with day 19 4–5+ G-EAT thyroids (unpublished results). The staining
intensity was decreased at day 35 compared with days 19–21 and further
decreased at days 50–60. In general, days 35–60 G-EAT thyroids still
had relatively strong expression of TGF-ß1 protein (Fig. 2D
and 2E
,
and Table 1
). TGF-ß1 was located generally in focal sites in these
thyroids (Fig. 2E
and unpublished results). As noted in Table 1B
,
although DBA/1 mice could develop severe G-EAT (4–5+) as early as day
10, the strongest expression of TGF-ß1 in thyroids was still at days
19–21, the peak severity of G-EAT.
 View larger version (161K): [in a new window] |
Figure 2. Representative examples of TGF-ß1 protein expression in G-EAT
thyroids of CBA/J mice. There was no staining of TGF-ß1 in normal
thyroids (A), and some staining of TGF-ß1 was noted in 3+ G-EAT
thyroids at day 15 (B). Staining for TGF-ß1 was diffuse and intense
in 5+ G-EAT thyroids at day 19 (C) and still present in atrophic
thyroids at day 45 (D) but mainly focally accumulated at that time (E).
Negative control of the same thyroid shown in C (F). Magnification: A,
B, and E, x400; C, D, and F, x100.
|
 View larger version (145K): [in a new window] |
Figure 3. Staining pattern of TGF-ß1, collagen, and myofbs. The primary areas
of collagen deposition were around the periphery of the thyroid (A) and
surrounding the granulomas (B); these areas corresponded to areas with
myofbs (E and F) and TGF-ß1 expression (C). In very severely damaged
thyroid, TGF-ß1 was expressed throughout the thyroid (D); myofbs were
present also throughout the gland (G) where collagen was deposited (A
and B). A and B, Trichrome staining (collagen is blue). C and D,
TGF-ß1 staining (brown). E–G, -SMA staining for myofbs (purple).
Note that C and F represent adjacent sections from the same thyroid. H,
Double-staining of -SMA (purple) and TGF-ß1 (brown) showed myofbs
expressing TGF-ß1 (C, arrows). Original magnification: A, D, E, and
G, x100; B, C, and F, x400; H, x1000. Representative photos are
shown from DBA/1 mice at day 21; similar results were obtained for
CBA/J mice.
|
Identification of TGF-ß1-producing cells in G-EAT thyroids
Identification of the cellular source of TGF-ß1 is needed to
determine what may control the transition of inflammatory G-EAT to
fibrosis. Immunostaining revealed that many cells contributed to the
production of TGF-ß1, but some cells showed a higher intensity of
TGF-ß1 staining (Fig. 3C
and unpublished results), suggesting that
excess production of TGF-ß1 by some cells could contribute to the
development of thyroid fibrosis. Staining of adjacent sections or dual
staining was used to identify the phenotype of some of the
TGF-ß1-positive cells. Myofbs (Fig. 3H)
and enlarged thyroid
follicular cells (unpublished results) had strong TGF-ß1 staining
intensity at the peak of inflammation. Macrophages were predominant in
granulomas, and some macrophages in the granulomas had strong TGF-ß1
staining (unpublished results). Although neutrophil infiltration was
extensive, neutrophils were usually TGF-ß1-negative (Fig. 3C
and
unpublished results). At days 35–60 when thyroids became atrophic,
TGF-ß1 was accumulated locally, with strong expression by some cells
morphologically resembling macrophages, and thyroid follicles were no
longer a major source of TGF-ß1 (unpublished results).
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Fibrosis is characterized by ECM deposition, of which collagen is the major constituent. Myofbs contribute greatly to the production of ECM and have been associated with progressive fibrosis in various organs [1 , 2 , 8 , 9 , 30 , 37 , 38 ]. In G-EAT, accumulation of myofbs and collagen deposition was first evident at days 19–21 (the peak of disease) and increased as thyroids became atrophic at days 35–60. Fibrin deposition was noted also in most severely damaged thyroids, particularly at 19–21 days. Myofbs and collagen first appeared at the periphery of G-EAT thyroids, which has been observed also in other fibrosis models [3 , 7 , 38 ]. Myofbs were concentrated in areas of collagen deposition, were most prominent in 5+ thyroids at days 19–21, and persisted at days 35–60 when there was increased collagen deposition. Thus, fibrosis began to develop when disease severity was maximal (days 19–21), and myofbs and collagen appeared concomitantly with severe destruction of thyroid follicles.
Production of TGF-ß1 increases during inflammation, and TGF-ß1 has potent immunomodulatory functions [33 , 34 ] as well as fibrosis-inducing effects [1 2 3 4 , 6 7 8 9 10 11 12 , 15 , 16 , 29 30 31 32 ]. This suggests that overproduction of TGF-ß1 could involve fibrosis in G-EAT where there is marked infiltration of the thyroid by inflammatory cells and production of Th1 and Th2 cytokines [23 , 26 27 28 ]. TGF-ß1 protein expression in the thyroid was highest at the peak of disease, and 5+ G-EAT thyroids expressed more TGF-ß1 protein than 4+ G-EAT thyroids (Table 1) . Even at day 45, there was sustained expression of TGF-ß1 in fibrotic thyroids. In normal healing or resolution of inflammation, TGF-ß1 expression is increased only transiently, and in progressive fibrosis, TGF-ß1 is increased and sustained [1 2 3 ]. Because high levels of TGF-ß1 were present in thyroids during fibrosis, and its expression paralleled G-EAT severity, increased production of TGF-ß1 could play a role in the development of thyroid fibrosis. Further studies using anti-TGF-ß1 antibody to reduce TGF-ß1 in G-EAT thyroids are in progress to determine if TGF-ß1 overproduction is involved in thyroid fibrosis in this model.
Myofbs and collagen were detected in G-EAT thyroids at days 19–21 when
expression of TGF-ß1 protein was maximal. TGF-ß1 induces
proliferation and activation of fibroblasts [1
2
3
,
8
, 9
, 35
, 36
] and
induces and maintains -SMA content in fibroblasts
[35
]. In G-EAT, elevated TGF-ß1 protein in
proliferating thyroid follicular cells and granulomas may have created
a favorable environment for recruitment and activation of fibroblasts,
leading to production of myofbs. Furthermore, the secreted TGF-ß1 can
bind matrix and act as a persistent stimulus [1
,
2
, 33
], resulting in the rapid and abundant
accumulation of myofbs (Figs. 1H
and 3G)
. Myofbs can produce TGF-ß1
also (Fig. 3H)
[7
], thereby magnifying the fibrotic
effect. TGF-ß1 can accelerate ECM production in thyrocytes also
[45
, 46
]. Moreover, TGF-ß1 has been shown
to promote disruption of follicles and induce apoptosis in quiescent
thyroid cells [47
, 48
]. Therefore, the
association among TGF-ß1, myofb, and ECM could be involved in the
induction of thyroid fibrosis. TGF-ß1 is known to have potent
immunosuppressive effects [3
, 33
,
34
]. TGF-ß1 might be elevated initially in an attempt
to suppress the autoimmune response in G-EAT thyroids, but intense
inflammation induced by CD4+ T cells and macrophages may result in
abnormal expression of TGF-ß1 in the thyroid.
Neutralization of TGF-ß1 can prevent scarring and fibrosis in animal models of kidney, lung, and liver fibrosis [1 , 29 30 31 32 ] and inhibited inflammation in an animal model of arthritis [42 ]. Angiotensin-converting enzyme (ACE) inhibitors or angiotensin-receptor antagonists, which can also reduce production of TGF-ß1 [2 , 7 ], prevented granuloma formation in schistosomiasis [49 ] and reduced fibrosis in myocardial infarction, scleroderma, and kidney disease [1 , 2 , 13 ]. Conversely, increasing production of TGF-ß1 by gene transfer led to development of fibrosis [8 ]. These studies show directly that TGF-ß1 can promote fibrosis. Currently, blocking TGF-ß1 by anti-TGF-ß1 antibody and ACE inhibitors is being used to address directly the role of TGF-ß1 in thyroid fibrosis and to determine if modulation of TGF-ß1 may be an effective approach to prevent and treat fibrosis.
Identification of the cells responsible predominantly for TGF-ß1 production in the thyroid might help define the cellular signal for triggering fibrosis. Fibrosis is preceded generally by inflammation and is associated with the repair of tissues following inflammation [1 2 3 , 5 , 7 ]. In this study, severely damaged thyroids had extensive inflammation, and many cells in these thyroids could produce TGF-ß1 and/or express high-affinity TGF-ß1 receptors. Some enlarged thyroid follicular cells expressed TGF-ß1 at the peak of disease, in agreement with experiments of others, demonstrating that TGF-ß1 can be synthesized by thyroid epithelial cells [50 , 51 ]. Myofbs produced TGF-ß1 at day 21 also (Fig. 3H) . TGF-ß1 expression was strongest within granulomas, which contained many macrophages also, and dual staining showed that many of the macrophages expressed TGF-ß1 (unpublished results). Macrophages in day 45 thyroids still stained strongly for TGF-ß1, accounting for the local strong expression of TGF-ß1 in the fibrotic thyroids, suggesting that sustained production of TGF-ß1 by macrophages may be involved in development of fibrosis [6 , 7 , 10 , 15 ]. Collectively, our data showed that myofbs, macrophages, and proliferating thyrocytes may contribute to TGF-ß1 production at the peak of disease, and macrophages and myofbs may be critical to maintain high levels of TGF-ß1 at the later stage. Because the stimulus that triggers expression of TGF-ß1 is mediated by an autoimmune response, the effector CD4+ T cells may be essential for the initial induction of TGF-ß1. Our preliminary results suggest that CD4+ T cells produce TGF-ß1 at the peak of G-EAT, and studies are underway to determine if CD4+ T cells express and regulate TGF-ß1 expression differentially during development of fibrosis.
The murine model of G-EAT is, to our knowledge, the only well-characterized animal model of an autoimmune disease in which a granulomatous form of histopathology can be experimentally induced. Although this model is not physiologic, it may be relevant for increasing our understanding of the pathologic features of several human granulomatous autoimmune diseases. Fibrosis is a major pathologic feature of systemic sclerosis and can be present in granulomatous tissue [7 , 9 , 13 14 15 16 , 32 ]. Our results showed that severe damage induced by an experimental autoimmune disease can result in fibrosis. Production of TGF-ß1 in inflamed thyroids correlated closely with progression of fibrosis, characterized by deposition of collagen and emergence of myofbs. These studies suggest that targeting TGF-ß1 and steps involved in TGF-ß1 regulation could lead to antifibrogenic therapeutic strategies, as shown recently in an animal model of scleroderma [32 ]. The G-EAT model can be used to study the efficacy of such therapies, and such studies are currently in progress.
Received April 6, 2000; revised July 12, 2000; accepted July 14, 2000.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-smooth muscle actin in fibroblasts Am. J. Pathol. 154,871-882This article has been cited by other articles:
 |
|
 |
|
  S. Yu, G. C. Sharp, and H. Braley-Mullen TGF-{beta} Promotes Thyroid Epithelial Cell Hyperplasia and Fibrosis in IFN-{gamma}-Deficient NOD.H-2h4 Mice J. Immunol., August 1, 2008; 181(3): 2238 - 2245. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Fang, V. G. DeMarco, G. C. Sharp, and H. Braley-Mullen Expression of Transgenic FLIP on Thyroid Epithelial Cells Inhibits Induction and Promotes Resolution of Granulomatous Experimental Autoimmune Thyroiditis in CBA/J Mice Endocrinology, December 1, 2007; 148(12): 5734 - 5745. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Fang, Y. Wei, V. DeMarco, K. Chen, G. C. Sharp, and H. Braley-Mullen Murine FLIP Transgene Expressed on Thyroid Epithelial Cells Promotes Resolution of Granulomatous Experimental Autoimmune Thyroiditis in DBA/1 Mice Am. J. Pathol., March 1, 2007; 170(3): 875 - 887. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Chen, Y. Wei, A. Alter, G. C. Sharp, and H. Braley-Mullen Chemokine expression during development of fibrosis versus resolution in a murine model of granulomatous experimental autoimmune thyroiditis J. Leukoc. Biol., September 1, 2005; 78(3): 716 - 724. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen Balance of proliferation and cell death between thyrocytes and myofibroblasts regulates thyroid fibrosis in granulomatous experimental autoimmune thyroiditis (G-EAT) J. Leukoc. Biol., February 1, 2005; 77(2): 166 - 172. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen Mechanisms of Spontaneous Resolution versus Fibrosis in Granulomatous Experimental Autoimmune Thyroiditis J. Immunol., December 1, 2003; 171(11): 6236 - 6243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen Inhibition of TGF{beta}1 by Anti-TGF{beta}1 Antibody or Lisinopril Reduces Thyroid Fibrosis in Granulomatous Experimental Autoimmune Thyroiditis J. Immunol., December 1, 2002; 169(11): 6530 - 6538. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Clancy, A. D. Askanase, R. P. Kapur, E. Chiopelas, N. Azar, M. E. Miranda-Carus, and J. P. Buyon Transdifferentiation of Cardiac Fibroblasts, a Fetal Factor in Anti-SSA/Ro-SSB/La Antibody-Mediated Congenital Heart Block J. Immunol., August 15, 2002; 169(4): 2156 - 2163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Braley-Mullen, K. Chen, Y. Wei, and S. Yu Role of TGF{beta} in Development of Spontaneous Autoimmune Thyroiditis in NOD.H-2h4 Mice J. Immunol., December 15, 2001; 167(12): 7111 - 7118. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wei, K. Chen, G. C. Sharp, H. Yagita, and H. Braley-Mullen Expression and Regulation of Fas and Fas Ligand on Thyrocytes and Infiltrating Cells During Induction and Resolution of Granulomatous Experimental Autoimmune Thyroiditis J. Immunol., December 1, 2001; 167(11): 6678 - 6686. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen Induction of Experimental Autoimmune Thyroiditis in IL-12-/- Mice J. Immunol., August 1, 2001; 167(3): 1720 - 1727. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
