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The Journal of Immunology, 2008, 181, 2238 -2245
Copyright © 2008 by The American Association of Immunologists, Inc.
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TGF-β Promotes Thyroid Epithelial Cell Hyperplasia and Fibrosis in IFN-{gamma}-Deficient NOD.H-2h4 Mice1

Shiguang Yu*,{dagger}, Gordon C. Sharp{dagger},{ddagger} and Helen Braley-Mullen2,*,{dagger},§

* Department of Veterans Affairs Research Service, Columbia, MO 65212; and {dagger} Department of Internal Medicine, {ddagger} Department of Pathology, and § Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IFN-{gamma}–/–NOD.H-2h4 mice given 0.05% NaI in their water develop severe thyroid epithelial cell (thyrocyte) hyperplasia and proliferation (TEC H/P) and fibrosis. Proliferating thyrocytes of IFN-{gamma}–/– mice with TEC H/P produce TGF-β as demonstrated by immunohistochemical staining and in situ hybridization. Strong expression of activating phosphorylated Smad-2/3 and weak expression of inhibitory Smad-7 by proliferating thyrocytes correlate with the severity of TEC H/P. Splenocytes from IFN-{gamma}–/– mice with severe TEC H/P transfer severe TEC H/P to IFN-{gamma}–/–NOD.H-2h4.SCID mice. Mice given anti-TGF-β had markedly reduced thyrocyte proliferation and decreased fibrosis compared with mouse Ig-treated controls, suggesting that TGF-β plays an important role in development of TEC H/P induced by activated splenocytes. Moreover, transgenic IFN-{gamma}–/–NOD.H-2h4 mice expressing TGF-β on thyrocytes all develop fibrosis and moderate to severe TEC H/P with accelerated kinetics, directly demonstrating a role for TGF-β in severe TEC H/P and fibrosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The NOD.H-2h4 mouse develops spontaneous autoimmune thyroiditis (SAT)3 characterized by lymphocyte infiltration of the thyroid (lymphocytic SAT (L-SAT)) (1, 2, 3, 4, 5, 6). L-SAT in NOD.H-2h4 mice is accelerated by addition of NaI to the drinking water (1, 2, 3, 5, 6). IFN-{gamma}–/–NOD.H-2h4 mice do not develop L-SAT but develop severe thyroid epithelial cell (TEC or thyrocyte) hyperplasia and proliferation (TEC H/P) with fibrosis and low serum T4 levels (7, 8). Lymphocytes are required for development of TEC H/P, and mice with TEC H/P produce anti-thyroglobulin Abs, indicating that TEC H/P has an autoimmune basis (8). Splenocytes from IFN-{gamma}–/– mice with severe TEC H/P transfer severe TEC H/P to IFN-{gamma}–/– NOD.H-2h4.SCID mice (8).

Abnormal proliferation of thyrocytes resulting in thyroid nodules is common in humans, and the incidence of thyroid nodules and thyroid carcinoma is increased in patients with Hashimoto’s thyroiditis (9, 10). Thyroid carcinomas and thyroids of humans and rats with thyrocyte hyperplasia highly express TGF-β (11, 12, 13). Increased expression of the TGF-βR or loss of signaling through the TGF-βR can promote tumor growth (14, 15, 16, 17), whereas loss of TGF-β signaling in T cells can increase tumor immunity (18, 19). Resistance of hyperplastic thyrocytes and thyroid tumors to the growth-inhibiting effects of TGF-β resulting in increased proliferation of epithelial cells can be due to up-regulation of molecules such as NF-{kappa}B, increased expression of the TGF-βR, or disruption of normal signaling through TGF-β (18, 19, 20, 21, 22). TGF-β plays a role in many other pathological and physiological processes. It functions in embryonic development, tissue morphogenesis, fibrosis, and wound healing (15, 18, 19). TGF-β can promote cell proliferation, e.g., in the case of many tumor cells (20, 21), and it has antiproliferative or apoptotic effects on cells, including epithelial cells (17, 22). TGF-β also has both promoting and inhibitory effects on immune responses and autoimmune diseases. For example, TGF-β is required for development of proinflammatory CD4+ T cells that produce IL-17 (Th17 cells) (23, 24), and it promotes fibrosis and tissue repair in some target organs in autoimmune diseases (15, 25, 26). The anti-inflammatory role of TGF-β is exemplified by the fact that regulatory T cells that suppress inflammation and autoimmune diseases frequently produce TGF-β, and TGF-β is generally required for regulatory T cell development (27). TGF-β also inhibits T cell proliferation and production of IFN-{gamma} (19). IFN-{gamma} and TGF-β reciprocally regulate one another. IFN-{gamma} inhibits TGF-β production and inhibits proliferation and collagen synthesis induced by TGF-β (28, 29, 30). These effects are due, at least in part, to induction by IFN-{gamma} of Smad-7, a protein in the TGF-β-signaling pathway that inhibits TGF-β signaling by an autocrine feedback mechanism (18, 28, 29, 30). In the absence of IFN-{gamma}, Smad-7 is decreased and the activating phosphorylated Smad (p-Smad)-2 and 3 proteins are increased (18, 28, 29, 30). Thus, the activities of TGF-β are complex, and its role when it is overexpressed in tissues is not easily predicted.

Because IFN-{gamma} and TGF-β reciprocally regulate each other and TGF-β can promote hyperplasia and fibrosis in other models, we hypothesized that TGF-β might be overexpressed on proliferating TEC of IFN-{gamma}–/–NOD.H-2h4 mice with severe TEC H/P and fibrosis. This study was undertaken to test this hypothesis and to determine whether TGF-β is functionally relevant for promoting thyrocyte proliferation and fibrosis. Using IFN-{gamma}–/– mice that develop TEC H/P and fibrosis, and SCID mice as recipients for adoptive cell transfer and TGF-β neutralization, the results indicate that TGF-β is overexpressed on proliferating TEC and is important for development of severe TEC H/P and fibrosis. Moreover, all transgenic (Tg) IFN-{gamma}–/–NOD.H-2h4 mice expressing TGF-β on thyrocytes develop fibrosis and moderate to severe TEC H/P with markedly accelerated kinetics compared with non-Tg IFN-{gamma}–/–NOD.H-2h4 mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

NOD.H-2h4 mice express H-2Kk, I-Ak, and Dd on the NOD background (31). NOD.H-2h4 wild-type (WT), IFN-{gamma}–/–, and IFN-{gamma}–/–SCID mice were generated in our animal facility as previously described (7, 32). All mice were given 0.05% NaI water for 2–7 mo beginning at 7–8 wk of age (7). Both male and female mice were used, but all mice in an individual experiment were the same sex. All animal protocols were approved by the University of Missouri and Veterans Affairs Animal Care and Use Committee.

Generation of TGF-β-Tg IFN-{gamma}–/–NOD.H-2h4 mice

The TGF-β-rat thyroglobulin (RTg) promoter construct was provided by Dr. L. Kohn (Edison Biotechnology Institute, Ohio University, Athens, OH). The SalI sites of the TGF-β RTg promoter construct were used to excise the cassette for microinjection. This construct contains two G to C point mutations in the TGF-β coding sequence that result in Cys to Ser amino acid substitutions at residues 223 and 225 of TGF-β1, resulting in a bioactive TGF-β1 (33). The construct was directly injected into superovulated NOD.H-2h4 females (University of Missouri Transgenic Core), resulting in two founder TGF-β-Tg NOD.H-2h4 females. The founders were mated with IFN-{gamma}–/–NOD.H-2h4 males, Tg+ heterozygotes were further crossed, and IFN-{gamma}–/– offspring were selected by PCR analysis of tail DNA as previously described (7). The TGF-β-Tg founders and progeny were genotyped by PCR analysis of mouse tail DNA using primers specific for the RTg promoter 5'-AGA GCA CTG CTT GCC ACT GTG C-3' (forward), and 5'-GCT GTT GTA CAA AGC GAG CAC C-3' (reverse) located in the mouse TGF-β genomic sequence and the Tg vector. These primers amplify a 340-bp band in TGF-β transgene-positive mice.

Scoring of TEC H/P and L-SAT severity

Thyroids were removed and one thyroid lobe was fixed in formalin, sectioned, and stained with H&E as previously described (2, 7, 8). All slides were scored by two individuals, one of whom had no knowledge of the experimental groups. The other thyroid lobe was snap-frozen in liquid nitrogen, and stored at –70°C for later use, e.g., immunohistochemical staining. Thyroid histopathology in IFN-{gamma}–/– mice and SCID recipients was scored for the extent of thyroid follicle replacement and hyperplasia/proliferation using a scale of 0 to 5+ as previously described (2, 7, 8). Briefly, a score of 0 indicates a normal thyroid, and 0+ indicates mild enlargement of TEC and/or a few inflammatory cells infiltrating the thyroids. A 1+ score is defined as hyperplastic changes sufficient to cause replacement of several follicles. A 2+ score represents hyperplastic changes causing replacement of up to one-fourth of the gland by abnormal thyroid follicles; 3+ indicates that one-fourth to one-half of the gland is replaced by hyperplastic TEC; and 4+ indicates that greater than one-half of the gland is replaced by hyperplastic proliferating TEC. Thyroids given a score of 5+ had few or no remaining normal follicles and the areas of proliferating thyrocytes were generally surrounded by collagen (fibrosis) (8). All thyroids with hyperplasia had many fewer infiltrating lymphocytes compared with thyroids of WT mice with L-SAT (8). For WT mice, L-SAT severity scores are based on the percentage of thyroid follicles replaced by infiltrating lymphocytes as previously described in detail (3, 6, 7).

Masson’s trichrome staining

Fibrosis was evaluated using Masson’s trichrome as previously described (34).

Immunohistochemical staining

Thyroid sections were deparaffinized in xylene, rehydrated through sequential ethanol, and rinsed in PBS. TGF-β staining was done as previously described (34). Anti-p-Smad-2/3, -Smad-7, and -TGF-βR II (all obtained from Santa Cruz Biotechnology) were used as primary Ab for p-Smad-2/3, Smad-7, and TGF-βR II staining, respectively. Biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) was used as secondary Ab, followed by incubation with the Vectastain Elite avidin-biotin complex (Vector Laboratories). Peroxidase activity was visualized using the Nova Red substrate (Vector Laboratories).

Confocal laser-scanning double immunofluorescence microscopy

Cytokeratin and TGF-β or TGF-βR II double immunofluorescence staining was done using paraffin sections of thyroids as previously described (8). After deparaffinization and microwave retrieval, sections were incubated with anti-TGF-β (R&D Systems) for 40 min at room temperature and visualized with 1/500 diluted Alexa 488-conjugated anti-chicken IgY Ab (Molecular Probes/Invitrogen). TGF-βR II staining was visualized with 1/500 diluted Alexa 488-conjugated anti-rabbit IgG (Invitrogen). For cytokeratin staining, slides were incubated with anti-cytokeratin (PCK-26; Sigma-Aldrich) followed by biotin-conjugated goat anti-mouse IgG Fab (Kirkegaard & Perry) and visualized with streptavidin-conjugated Alexa 568 (Invitrogen) for 30 min. Slides were observed using a Bio-Rad Radiance 2000 confocal system coupled to an Olympus IX70 inverted microscope.

Western blot

Thyroids from WT and IFN-{gamma}–/– mice with L-SAT or TEC H/P of varying severity were homogenized with lysing buffer containing 250 mM NaCl, 50 mM HEPES (pH 7.9), 5 mM EDTA, 0.1% Triton X-100 as previously described (35). Proteins were separated on 10% SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were incubated overnight at 4°C in TBS containing 5% nonfat dry milk. After blocking, membranes were incubated with rabbit anti-TGF-βR II (Santa Cruz Biotechnology) followed by detection with peroxidase-conjugated goat-anti-rabbit IgG (The Jackson Laboratory) and ECL substrate (Bio-Rad). For normalization of signals, the membranes were stripped by incubating at 55°C for 30 min in stripping buffer (62.5 mM/L Tris-HCl, 100 mM/L 2-ME, and 2% SDS), washed in TBS/Tween 20, and reprobed with rabbit anti-actin primary Ab (Santa Cruz Biotechnology) and HRP-conjugated anti-rabbit IgG. Bands were scanned and quantitated using Quality One 4.41 software. Each lane in the figures represents 30 µg of protein extracted from an individual thyroid.

Adoptive transfer and administration of anti-TGF-β

Splenocytes from IFN-{gamma}–/– mice with severe (4–5+) TEC H/P (identified by the large size of their thyroids, and later confirmed by histology) were injected i.v. (3 x 107 cells) into IFN-{gamma}–/–NOD.H-2h4 SCID mice (8). All recipients were given 0.05% NaI water. One group of SCID recipients was given 0.5 mg of anti-TGF-β1 1D11.16.8 (mouse IgG1) (ATCC HB 9849; American Type Culture Collection) every 4–5 days for 30 days, and the other group received the same amount of mouse IgG. After 30 days, thyroids were removed and thyroid histology was scored as described above.

In situ hybridization (ISH)

An ISH detection kit (Maxim Biotech) was used for TGF-β ISH on formalin-fixed, paraffin-embedded thyroid sections. Biotinylated anti-mouse TGF-β1 probe (Maxim Biotech) was used for hybridization at 37°C overnight according to the manufacturer’s instructions. Following hybridization, the sections were treated with RNase to remove unbound probe and slides were washed extensively. The hybridized probe was detected by addition of alkaline phosphatase-conjugated streptavidin followed by 5-bromo-4-chloro-3-indolyl phosphate/NBT substrates (Maxim Biotech).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Thyrocytes of IFN-{gamma}–/– mice with severe TEC H/P strongly express TGF-β

Sixty to 70% of IFN-{gamma}–/– mice given 0.05% NaI water for 6–7 mo develop very severe (4–5+) TEC H/P with minimal lymphocytes infiltrating the thyroids (8). Thyroids of mice with severe TEC H/P have few or no remaining normal thyroid follicles, proliferation of TEC, and extensive fibrosis (Fig. 1A). All IFN-{gamma}–/– mice develop mild (Fig. 1B) or severe (Fig. 1A) TEC H/P and none of them develop L-SAT. In contrast, all WT NOD.H-2h4 mice develop 2–3+ L-SAT (Fig. 1C). Severe TEC H/P is always associated with thyroid fibrosis (8). Because TGF-β promotes fibrosis in many tissues and organs (19, 26, 27), we asked whether the proliferating thyrocytes in IFN-{gamma}–/– mice express TGF-β. TEC from IFN-{gamma}–/– mice with severe TEC H/P strongly express TGF-β (brown color, Fig. 1D), and TEC from IFN-{gamma}–/– mice with mild (0–1+) TEC H/P express much less TGF-β (Fig. 1E). In contrast, TGF-β is expressed by thyroid-infiltrating inflammatory cells in WT mice, and thyrocytes in WT mice do not express TGF-β (Fig. 1F, arrows). TEC express cytokeratin (7, 8). To determine whether proliferating TEC were the cells that strongly expressed TGF-β, anti-cytokeratin and -TGF-β dual immunofluorescence staining was done on thyroid sections from WT and IFN-{gamma}–/– mice with various TEC H/P severity scores. Confocal microscopy demonstrated that TGF-β (green) and cytokeratin (red) colocalized (yellow) in thyroids of IFN-{gamma}–/– mice with TEC H/P, confirming that proliferating TEC in IFN-{gamma}–/– mice with severe TEC H/P strongly express TGF-β (Fig. 1G). TEC of IFN-{gamma}–/– mice with mild TEC H/P also express TGF-β (Fig. 1H), but at much lower levels. In contrast, anti-cytokeratin and -TGF-β staining did not colocalize in thyroids of WT mice with 2–3+ L-SAT severity scores (Fig. 1I) because TGF-β in WT mice was produced primarily by infiltrating lymphocytes.


Figure 1
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  		FIGURE 1. Thyroids of IFN-{gamma}–/– mice with severe TEC H/P strongly express TGF-β. Representative histology of IFN-{gamma}–/– mice with severe (A) or mild (B) TEC H/P and WT mice with 2–3+ L-SAT (C). Insets, Lower power views of the same slides. Thyrocytes of IFN-{gamma}–/– mice with severe TEC H/P strongly express TGF-β (D, *) and thyrocytes with mild thyrocyte hyperplasia in IFN-{gamma}–/– mice express minimal TGF-β (E). Thyrocytes of WT mice with 2–3+ L-SAT express little or no TGF-β (F, arrows), and thyroid-infiltrating inflammatory cells express moderate TGF-β (F). TGF-β (green) and cytokeratin (red) dual staining confirmed that thyrocytes of IFN-{gamma}–/– mice with severe TEC H/P strongly express TGF-β (overlay; yellow) (G), and thyrocytes with mild TEC H/P express little TGF-β (H). Thyrocytes of WT mice with L-SAT express minimal TGF-β (I). p-Smad2/3 (red nuclear staining) is highly expressed in IFN-{gamma}–/– thyroids with 5+ TEC H/P (J, arrows) or mild thyrocyte hyperplasia (K, arrows). A few inflammatory cells in WT mice also express p-Smad2/3 (L). Inhibitory Smad7 (red cytoplasmic staining) is minimally expressed on hyperplastic thyrocytes in IFN-{gamma}–/– mice (M and N) and strongly expressed by thyrocytes and inflammatory cells of WT mice (O, arrow). Representative photos are shown. Magnification, x400. Insets in A–C, x100.

 
p-Smad-2/3 expression on thyrocytes correlates with TEC H/P severity

TGF-β and IFN-{gamma} reciprocally regulate one another (28, 29, 30). IFN-{gamma} induces expression of Smad-7 which down-regulates TGF-β signaling by inhibiting phosphorylation of Smad-2 and -3 and their interaction with the TGF-βR (30). p-Smad-2/3 associates with Smad-4 to transfer to the nucleus and activate TGF-β-induced gene expression (36). Immunohistochemical staining was used to determine whether thyroids of IFN-{gamma}–/– mice with severe TEC H/P and WT mice with L-SAT differentially express the inhibitory Smad-7 and stimulatory p-Smad-2/3 proteins. Staining for p-Smad-2/3 (red nuclear staining) was strong and distributed in areas of proliferating TEC in thyroids of IFN-{gamma}–/– mice with 5+ TEC H/P (red color, Fig. 1J, arrows), suggestive of activation of TGF-β signaling. This contrasts with the weak cytoplasmic staining of Smad-7 (red) in thyrocytes of IFN-{gamma}–/– mice with TEC H/P (Fig. 1M). A few p-Smad-2/3 positive cells were also detected in thyroids of IFN-{gamma}–/– mice with mild (0–1+) TEC H/P (Fig. 1K, arrows) and Smad-7 was undetectable (Fig. 1N). In marked contrast, Smad-7 was strongly expressed on both thyroid inflammatory cells and thyrocytes of WT mice with L-SAT, and only a few inflammatory cells weakly expressed p-Smad-2/3 (red color, Fig. 1, L and O). Thyrocytes of WT mice without SAT expressed no detectable p-Smad-2/3 or Smad-7 (data not shown).

We previously showed that transfer of WT splenocytes as a source of IFN-{gamma} to IFN-{gamma}–/– mice inhibits TEC H/P and the mice develop L-SAT, whereas WT splenocytes do not inhibit TEC H/P or induce L-SAT in IFN-{gamma}R–/– recipients (32). When thyroids of IFN-{gamma}–/– or IFN-{gamma}R–/– recipients of WT splenocytes were examined for expression of TGF-β, p-Smad-2/3, and Smad-7, IFN-{gamma}–/– recipients of WT splenocytes had lower expression of TGF-β and p-Smad-2/3, and increased Smad-7 expression, and the pattern of expression of these molecules was similar to that in thyroids of WT mice with L-SAT (data not shown). In marked contrast, TGF-β and p-Smad-2/3 expression was not reduced on thyrocytes of IFN-{gamma}R–/– recipients of WT splenocytes (data not shown). These results indicate that the site and extent of expression of inhibitory Smad-7 and stimulatory p-Smad-2/3 is largely dictated by the presence or absence of IFN-{gamma} and the ability of thyrocytes to respond to IFN-{gamma}.

TGF-βR II is highly expressed on thyrocytes of IFN-{gamma}–/– mice with severe TEC H/P

Increased expression of TGF-β on proliferating TEC of IFN-{gamma}–/– mice with severe TEC H/P could be due to increased expression of TGF-βR II compared with thyrocytes of WT mice. This could lead to increased binding of TGF-β by proliferating TEC, resulting in increased TGF-β signaling. To address this possibility, TGF-βR II immunohistochemical staining was done on thyroid sections from IFN-{gamma}–/– mice with mild or severe TEC H/P or thyroids from WT mice with L-SAT. Thyrocytes of IFN-{gamma}–/– mice with mild TEC H/P (Fig. 2A) and thyrocytes of naive WT mice (Fig. 2C) expressed minimal TGF-β RII, whereas thyrocytes of IFN-{gamma}–/– mice with 4–5+ TEC H/P and thyrocytes and inflammatory cells in thyroids of WT mice with 2–3+ L-SAT had strong TGF-βR II staining (red color, Fig. 2, B and D). When TGF-βR II protein was quantitated using Western blot, thyroids of WT mice with 2–3+ L-SAT severity scores and thyroids of IFN-{gamma}–/– mice with 4–5+ TEC H/P severity scores had similar levels of TGF-βR II (Fig. 2, E and F). Many thyroid inflammatory cells in WT mice express TGF-βR II, and this contributes to the TGF-βR II detected in Western blots. Therefore, this method could not be used to determine whether thyrocytes of IFN-{gamma}–/– and WT mice differ in their expression of TGF-βR II. To compare TGF-βR II expressed by thyrocytes of IFN-{gamma}–/– mice with severe TEC H/P with TGF-βR II expressed by TEC of WT mice with L-SAT, TGF-βR II, and cytokeratin dual immunofluorescence staining was done on thyroid sections of IFN-{gamma}–/– mice with 0–1+ (Fig. 2G) and 4–5+ TEC H/P (Fig. 2H), and on thyroids of WT mice with no L-SAT (Fig. 2I) and 2–3+ L-SAT (Fig. 2J). The cells that expressed TGF-βR II in IFN-{gamma}–/– mice with severe TEC H/P were proliferating TEC (yellow, overlay) (Fig. 2H). In contrast, TEC in WT mice with L-SAT (Fig. 2J) express less TGF-βR II and TGF-βR II was expressed primarily by thyroid inflammatory cells (Fig. 2J). These results indicate that proliferating thyrocytes in IFN-{gamma}–/– mice express more TGF-βR II compared with thyrocytes of WT mice with L-SAT.


Figure 2
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  		FIGURE 2. TGF-βR II (TβRII) immunohistochemical staining and Western blots. TβR II is highly expressed on thyrocytes from IFN-{gamma}–/– mice with severe TEC H/P (B) and in thyroids of WT mice with 2–3+ L-SAT (D), but not in thyroids with mild TEC H/P (A) or thyroids of naive WT mice (C). Western blots also confirmed the results of TβR II immunohistochemical staining (E). Results are expressed as the mean ratio of densitometric U/β-actin ± SEM (x100) of four to five thyroids and are representative of two independent experiments (F). Anti-TGF-β RII (green) and anti-cytokeratin (red) dual staining on thyroid sections of IFN-{gamma}–/– mice with mild (G) 4–5+ (H) TEC H/P, and WT mice with no (I) or 2–3+ (J) L-SAT. Proliferating thyrocytes in IFN-{gamma}–/– mice with severe TEC H/P strongly express TGF-β (H, yellow), but thyrocytes of WT mice do not (I and J). ISH showed that TGF-β mRNA was predominantly detected in the area of severe TEC H/P in IFN-{gamma}–/– mice (K, arrows), and by thyroid inflammatory cells, but not by thyrocytes, in WT mice with 2–3+ SAT (M, shorter arrow). A blue color visible by light microscopy is seen at the specific site of the hybridized probe. TGF-β mRNA was weakly detected in thyroids of IFN-{gamma}–/– mice with mild TEC H/P (L) and normal thyroids of naive WT mice (N). Magnification: A–D, G–N, x400.

 
TGF-β is produced by proliferating TEC in IFN-{gamma}–/– mice

TGF-β expressed on proliferating thyrocytes in IFN-{gamma}–/– mice could be produced by the thyrocytes themselves or by other cells. For example, lymphocytes or macrophages infiltrating the thyroids could produce TGF-β that could bind to the TGF-βR on proliferating TEC. To determine whether proliferating thyrocytes produce TGF-β, the cellular localization of TGF-β1 mRNA was determined using ISH (blue color, Fig. 2, K–N). TGF-β mRNA was primarily expressed in the areas of proliferating thyrocytes in thyroids of IFN-{gamma}–/– mice (blue, Fig. 2K, arrows). Inflammatory cells in WT mice express high levels of TGF-β1 mRNA, but WT thyrocytes express little TGF-β mRNA (Fig. 2M, shorter arrow). TGF-β mRNA was barely detectable in thyroids of IFN-{gamma}–/– mice with 0+ TEC H/P (Fig. 2L) or in thyroids of naive WT mice (Fig. 2N). These results suggest that at least some of the TGF-β expressed by the proliferating TEC in IFN-{gamma}–/– mice is produced by the TEC.

TEC H/P and fibrosis induced in SCID recipients of IFN-{gamma}–/– splenocytes is inhibited by anti-TGF-β

Splenocytes of IFN-{gamma}–/– mice with severe TEC H/P transfer severe TEC H/P to IFN-{gamma}–/–NOD.H-2h4.SCID mice (8). To determine whether TGF-β, shown above to be overexpressed by proliferating thyrocytes in thyroids of IFN-{gamma}–/– mice with TEC H/P, was functionally relevant for transfer of severe TEC H/P, pooled splenocytes from IFN-{gamma}–/–NOD.H-2h4 mice with severe TEC H/P were transferred to IFN-{gamma}–/–NOD.H-2h4 SCID recipients. Half of the mice were given anti-TGF-β and the other half received mouse IgG every 4–5 days. After 30 days, thyroids were removed for evaluation of thyroid histology (Fig. 3). Twelve of 14 recipients given mouse Ig developed moderate to severe (3–5+ severity) TEC H/P (Fig. 3A), with proliferation of TEC and collagen (fibrosis) surrounding the areas of proliferating TEC. The thyrocytes highly expressed stimulatory p-Smad-2/3 (Fig. 3E). In contrast, only 3 of 12 recipients given anti-TGF-β developed moderate to severe (3–5+) TEC H/P (p < 0.002) (Fig. 3, A and F). Most mice given anti-TGF-β had only mild TEC hyperplasia and minimal fibrosis (Fig. 3F). Expression of TGF-β (brown color) and p-Smad-2/3 (red color) by thyrocytes was also reduced (Fig. 3, G–I), suggesting that TGF-β is important for transfer of severe TEC H/P to SCID recipients.


Figure 3
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  		FIGURE 3. Anti-TGF-β decreases TEC H/P severity and fibrosis in IFN-{gamma}–/–NOD.H-2h4.SCID recipients of IFN-{gamma}–/– splenocytes. Recipient mice were given NaI water, and 0.5 mg of mouse IgG or anti-TGF-β every 4–5 days. Thyroids were removed 30 days after cell transfer. TEC H/P severity scores of individual mice are shown. Anti-TGF-β significantly reduced thyrocyte proliferation and development of fibrosis in recipient mice (p < 0.002). One of two representative experiments is shown (A). Representative H&E (B and F), TGF-β (C, D, G, and H), p-Smad2/3 (E and I) stained thyroids from anti-TGF-β-treated (2+ TEC H/P), and mouse IgG-treated (control) recipients (5+ TEC H/P severity) are shown. Arrows in B indicate collagen (fibrosis) surrounding proliferating TEC (*). Thyroids of mice given anti-TGF-β (F) had minimal or no fibrosis. Magnification: C, F, x100; A, D, E, F, H, I, x400.

 
All IFN-{gamma}–/–TGF-β NOD.H-2h4 mice expressing Tg TGF-β on thyrocytes develop severe TEC H/P and fibrosis

Tg IFN-{gamma}–/–NOD.H-2h4 mice expressing TGF-β on thyrocytes were generated to directly test the hypothesis that overexpression of TGF-β by thyrocytes results in severe TEC H/P and fibrosis. Thyroids of TGF-β Tg IFN-{gamma}–/– mice strongly expressed TGF-β (brown color, Fig. 4A), whereas thyrocytes of transgene-negative IFN-{gamma}–/– littermates had only mild TEC H/P and expressed minimal TGF-β (Fig. 4B). Other tissues such as cervical lymph nodes did not express TGF-β (data not shown). Thyrocytes of TGF-β Tg IFN-{gamma}–/– mice given NaI water for 8 wk (16 wk of age) and thyrocytes of non-Tg IFN-{gamma}–/– mice given NaI water for 7 mo (brown, Fig. 4C) both strongly expressed TGF-β. Thyroids of non-Tg IFN-{gamma}–/– mice with severe TEC H/P were greatly enlarged (Fig. 4F), whereas thyroids of transgene-negative IFN-{gamma}–/– mice with mild TEC H/P (Fig. 4E) and those of TGF-β-Tg mice with severe TEC H/P (Fig. 4D) were not enlarged. Quantitation of TGF-β by real-time PCR indicated that thyroids of IFN-{gamma}–/–TGF-β-Tg mice expressed 3- to 5-fold more TGF-β mRNA than thyroids of non-Tg IFN-{gamma}–/– mice without TEC H/P (data not shown). If overexpression of TGF-β on thyrocytes is important for promoting TEC H/P and fibrosis, TEC H/P should develop earlier and be more severe in thyroids of Tg+ mice. Consistent with this hypothesis, 100% of TGF-β-Tg IFN-{gamma}–/– mice given NaI water for 8 wk developed moderate to severe TEC H/P and extensive fibrosis (blue color) with minimal lymphocyte infiltration (Fig. 4, D, G, and J). The severity of TEC H/P and fibrosis was similar to that seen in non-Tg IFN-{gamma}–/– mice with 4–5+ TEC H/P given NaI water for 7 mo (Fig. 4, F, I, and L). In contrast, as reported previously (8), non-Tg IFN-{gamma}–/– NOD.H-2h4 mice given NaI water for 8 wk had only mild (0+ to 1+) TEC H/P and minimal fibrosis (Fig. 4, E, H, and K). These results indicate that overexpression of TGF-β on thyrocytes promotes earlier development of TEC H/P and more extensive fibrosis compared with transgene-negative IFN-{gamma}–/– littermates.


Figure 4
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  		FIGURE 4. All Tg IFN-{gamma}–/–NOD.H-2h4 mice expressing TGF-β on thyrocytes develop moderate to severe TEC H/P and extensive fibrosis. TGF-β expression by thyrocytes from Tg IFN-{gamma}–/– mice (A), non-Tg IFN-{gamma}–/– NOD.H-2h4 mice with minimal (0+) TEC H/P (B), and non-Tg IFN-{gamma}–/– mice with severe TEC H/P (5+) (C). All IFN-{gamma}–/– Tg mice developed TEC H/P (D and G, H&E staining) and fibrosis (J, trichrome staining (blue)) after 2 mo on NaI water. Most IFN-{gamma}–/– non-Tg mice developed mild TEC H/P (E and H, H&E staining) and little fibrosis (K, blue trichrome staining) after 2 mo on NaI water. Most IFN-{gamma}–/– mice given NaI water for 7 mo developed severe TEC H/P (F and I, H&E staining) and fibrosis (L, blue trichrome staining). Representative slides are shown. Magnification: A–C, G–J, L, x400; D–F, K, x100.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results presented here show that thyrocytes of IFN-{gamma}–/– mice with severe TEC H/P and fibrosis strongly express TGF-β. These thyrocytes also strongly express p-Smad-2/3 indicative of TGF-β activation, and inhibitory Smad-7 is weakly expressed (Fig. 1). TGF-β and p-Smad-2/3 expression decreased and Smad-7 increased on thyrocytes of IFN-{gamma}–/– mice following transfer of WT splenocytes, shown previously (32) to inhibit TEC H/P and induce L-SAT in IFN-{gamma}–/– recipients (data not shown). These results indicate that IFN-{gamma} may directly or indirectly inhibit TEC H/P by blocking TGF-β signaling. TEC H/P and fibrosis induced in SCID mice by transfer of splenocytes from IFN-{gamma}–/– mice with severe TEC H/P was markedly reduced by anti-TGF-β (Fig. 3). These results are consistent with the hypothesis that TGF-β plays an important role in the development of severe TEC H/P and fibrosis. The increased expression of stimulatory p-Smad-2/3 and decreased expression of inhibitory Smad-7 may increase TGF-β signaling, contributing to TEC H/P and fibrosis in IFN-{gamma}–/– mice. Experiments showing that all IFN-{gamma}–/–TGF-β Tg NOD.H-2h4 mice constitutively expressing TGF-β on thyrocytes develop moderate to severe TEC H/P and fibrosis directly demonstrates that overexpression of TGF-β on thyrocytes results in TEC H/P and fibrosis.

TGF-β signaling is initiated when TGF-β induces assembly of the complex of TGF-βR II and TGF-βR I. The activated TGF-βR II and TGF-βR I complex binds to and phosphorylates members of the intracellular Smad-signaling pathway (36). TGF-βR II is expressed by both TEC and infiltrating inflammatory cells in thyroids of WT NOD.H-2h4 mice. Proliferating TEC in IFN-{gamma}–/– mice with severe TEC H/P and fibrosis express more TGF-βR II than TEC of WT mice with L-SAT (Fig. 2, H vs J). Proliferating thyrocytes strongly express TGF-β and its signal pathway mediator p-Smad-2/3, suggesting that TGF-βRs on proliferating thyrocytes should be functional. Hence, TGF-β and increased TGF-βR II on TEC of IFN-{gamma}–/– mice may both contribute to the histopathology of TEC H/P and fibrosis.

Increased expression of TGF-β by TEC of IFN-{gamma}–/– mice with severe TEC H/P is apparently due to production of TGF-β by the proliferating TEC, because ISH indicated that TGF-β1 mRNA was predominantly localized on TEC in areas of severe TEC H/P of IFN-{gamma}–/– mice (Fig. 2K). In contrast, most thyroid inflammatory cells in WT mice with 2–3+ L-SAT express TGF-β1 mRNA, while thyrocytes of WT mice are negative for TGF-β mRNA (Fig. 2M). These experiments do not exclude the possibility that some of the TGF-β expressed on proliferating TEC in IFN-{gamma}–/– thyroids could be provided by T cells or macrophages that infiltrate thyroids of IFN-{gamma}–/– mice. Because infiltration of the thyroid by inflammatory cells (primarily T cells) is required for development of severe TEC H/P in IFN-{gamma}–/– mice (8), the infiltrating inflammatory cells could produce TGF-β and/or another cytokine that induces thyrocytes to produce TGF-β, resulting in TEC proliferation and fibrosis. Alternatively, TGF-β produced by inflammatory cells or other cells could simply bind to the TGF-βR on TEC.

The results of this study indicate that TGF-β is important for development of severe TEC H/P induced by activated splenocytes. We hypothesize that in the absence of IFN-{gamma}, some T lymphocytes become activated and migrate to the thyroid. These thyroid-infiltrating T lymphocytes produce one or more cytokines that act on TEC to initiate TEC hyperplasia and increase production of TGF-β by the thyrocytes. Infiltrating T cells in thyroids of IFN-{gamma}–/– mice with severe TEC H/P produce several cytokines including IL-4, IL-13, IL-17, and IL-22 (data not shown). Development of T cells that produce IL-17 and IL-22 (Th17 cells) is promoted when IFN-{gamma} is absent, and TGF-β is required for development of Th17 cells (23, 24). Th17 cells are important for development of several autoimmune diseases, and receptors for IL-17 and IL-22 are expressed on many tissue epithelial cells (37, 38). IL-22 reportedly acts on epithelial cells to promote hyperplasia (38, 39, 40). Thyroid-infiltrating T cells in thyroids of IFN-{gamma}–/– mice with severe TEC H/P also produce IL-4 and IL-13, both of which can promote development of fibrosis in other models (41, 42), and thyrocytes express receptors for IL-4 (43). It is not yet known which, if any, of these cytokines is most important for development of severe TEC H/P. However, the results are consistent with the idea that activated inflammatory cells migrate to the thyroid where they produce a cytokine or cytokines that act on TEC to promote TEC proliferation, resulting in increased production of TGF-β by the proliferating TEC. TGF-β produced by proliferating TEC might then promote further activation and cytokine production by the inflammatory cells, ultimately leading to severe TEC H/P and fibrosis. Further studies using Abs to neutralize specific cytokines are needed to address the relationship of TGF-β and cytokines produced by thyroid-infiltrating inflammatory cells in development of severe TEC H/P and fibrosis.

Transgenic overexpression of TGF-β on thyrocytes of IFN- {gamma}–/–NOD.H-2h4 mice leads to moderate to severe thyrocyte hyperplasia and fibrosis in all mice after 8 wk on NaI water. In contrast, even after 6–7 mo on NaI water, only 60–70% of non-Tg IFN-{gamma}–/–NOD.H-2h4 mice develop severe TEC H/P and fibrosis, and most have only mild TEC H/P after 2 mo on NaI water (Fig. 4) (8, 32). These results directly demonstrate that overexpression of TGF-β on TEC promotes development of TEC H/P and fibrosis.

TGF-β has a wide range of biological effects on various cell types. Smad-3 is important in signal transduction pathways leading to fibrosis, and ablation of Smad-3 is associated with decreased fibrosis in a mouse model of systemic sclerosis and in pulmonary fibrosis (44, 45, 46). Transgenic expression of TGF-β in the vascular wall causes fibroproliferative thickening and hyperplasia (47). However, disruption of TGF-β-Smad-3 signaling can also lead to increased hyperplasia in response to vascular injury (48). These observations highlight the complex and dual functions of TGF-β. Neutralization of TGF-β resulted in decreased TEC proliferation and fibrosis in SCID recipients of IFN-{gamma}–/– splenocytes (Fig. 3). Neutralization of TGF-β also decreased p-Smad-2/3 expression, suggesting that p-Smad-2/3 signaling is involved in TGF-β-mediated TEC H/P and fibrosis. Others have shown that anti-TGF-β decreased skin and lung fibrosis in a murine scleroderma model and soluble TGF-βR II inhibited both bleomycin-induced and injury-induced lung and liver fibrosis (49, 50).

TGF-β can regulate epithelial cell growth and is overexpressed in thyroid follicular tumors and thyroid hyperplasia (11, 12, 13). Interventions that decrease TGF-β production, activation, and/or TGF-β effector functions might have beneficial effects in diseases associated with abnormal cell proliferation and fibrosis (49, 50, 51, 52). Studies using this animal model may help elucidate the mechanisms by which particular cytokines function to promote abnormal cellular proliferation and fibrosis that develop in some patients with autoimmune thyroid diseases as well as in other autoimmune diseases associated with hyperplasia and fibrosis.


   Acknowledgments
 
We thank Alicia Duren and Patti Mierzwa for technical assistance, and Dr. Leonard Kohn (Ohio University) for providing the RTg promoter-linked TGF-β construct.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Merit Review Grant from the Department of Veterans Affairs, the A. P. Green Foundation, the University of Missouri Research Council, and the University of Missouri Research Board. Back

2 Address correspondence and reprint requests to Dr. Helen Braley-Mullen, Division of Immunology and Rheumatology, Department of Internal Medicine, University of Missouri, M307 Health Science Center, One Hospital Drive, Columbia, MO 65212. E-mail address: mullenh{at}health.missouri.edu Back

3 Abbreviations used in this paper: SAT, spontaneous autoimmune thyroiditis; L-SAT, lymphocytic SAT; TEC H/P, thyroid epithelial cell hyperplasia and proliferation; p-Smad, phosphorylated Smad; Tg, transgenic; WT, wild type; RTg, rat thyroglobulin; ISH, in situ hybridization; TβR II, TGF-β receptor II. Back

Received for publication November 7, 2007. Accepted for publication May 26, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Rasooly, L., C. L. Burek, N. R. Rose. 1996. Iodine-induced autoimmune thyroiditis in NOD.H-2h4 mice. Clin. Immunol. Immunopathol. 81: 287-292. [Medline]
  2. Braley-Mullen, H., G. C. Sharp, B. Medling, H. Tang. 1999. Spontaneous autoimmune thyroiditis in NOD.H-2h4 mice. J. Autoimmun. 12: 157-165. [Medline]
  3. Yu, S., B. Medling, H. Yagita, H. Braley-Mullen. 2001. Characteristics of inflammatory cells in spontaneous autoimmune thyroiditis of NOD.H-2h4 mice. J. Autoimmun. 16: 37-46. [Medline]
  4. Verma, S., P. Hutchings, J. Guo, S. McLachlan, B. Rapoport, A. Cooke. 2000. Role of MHC class I expression and CD8+ T cells in the evolution of iodine-induced thyroiditis in NOD-H2(h4) and NOD mice. Eur. J. Immunol. 30: 1191-1202. [Medline]
  5. Hutchings, P., S. Verma, J. M. Phillips, S. Z. Harach, S. Howlett, A. Cooke. 1999. Both CD4+ and CD8+ T cells are required for iodine accelerated thyroiditis in NOD mice. Cell. Immunol. 192: 113-121. [Medline]
  6. Braley-Mullen, H., S. Yu. 2000. Early requirements for B cells for development of spontaneous autoimmune thyroiditis in NOD.H-2h4 mice. J. Immunol. 165: 7262-7269. [Abstract/Free Full Text]
  7. Yu, S., G. C. Sharp, H. Braley-Mullen. 2002. Dual role for IFN-{gamma}, but not for IL-4, in spontaneous autoimmune thyroiditis in NOD.H-2h4 mice. J. Immunol. 169: 3999-4007. [Abstract/Free Full Text]
  8. Yu, S., G. C. Sharp, H. Braley-Mullen. 2006. Thyroid epithelial cell hyperplasia in IFN-{gamma} deficient NOD.H-2h4 mice. Clin. Immunol. 118: 92-100. [Medline]
  9. Ross, D. S.. 2007. Editorial: predicting thyroid malignancy: thyroid nodules and thyroid cancer: an epidemic. J. Clin. Endocrinol. Metab. 91: 4253-4255.
  10. Larson, S. D., L. N. Jackson, T. S. Riall, T. Uchida, R. P. Thomas, S. Qiu, B. M. Evers. 2007. Increased incidence of well-differentiated thyroid cancer associated with Hashimoto’s thyroiditis and the role of the PI3k/Akt pathway. J. Am. Coll. Surg. 204: 764-775. [Medline]
  11. Jasani, B., F. S. Wyllie, P. A. Wright, N. R. Lemoine, E. D. Williams, D. Wynford-Thomas. 1990. Immunocytochemically detectable TGF-β associated with malignancy in thyroid epithelial neoplasia. Growth Factors 2: 149-155. [Medline]
  12. Kimura, E. T., P. Kopp, J. Zbaeren, L. M. Asmis, C. Ruchti, R. M. Maciel, H. Studer. 1999. Expression of transforming growth factor β1, β2, and β3 in multinodular goiters and differentially thyroid carcinomas: a comparative study. Thyroid 9: 119-125. [Medline]
  13. Logan, A., C. Smith, G. P. Becks, A. M. Gonzale, I. D. Phillips, D. J. Hill. 1994. Enhanced expression of transforming growth factor-β1 during thyroid hyperplasia in rats. J. Endocrinol. 141: 45-57. [Abstract/Free Full Text]
  14. Blaydes, J. P., D. Wynford-Thomas. 1996. Loss of responsiveness to TGF-β is tightly linked to tumorigenicity in a model of thyroid tumor progression. Int. J. Cancer 65: 525-530. [Medline]
  15. Massague, J., S. W. Blain, R. S. Lo. 2000. TGF-β signaling in growth control, cancer, and heritable disorders. Cell 103: 295-309. [Medline]
  16. Turco, A., A. Coppa, S. Aloe, G. Baccheschi, S. Morrone, G. Zupi, G. Colletta. 1999. Overexpression of TGF-β-type II receptor reduces tumorigenicity and metastatic potential of K-ras-transformed thyroid cells. Int. J. Cancer 80: 85-91. [Medline]
  17. Coppa, A., G. Mincione, D. Lazzereschi, A. Ranieri, A. Turco, B. Lucignano, S. Scarpa, M. Ragano-Caracciolo, G. Colletta. 1997. Restored expression of TGF-β type II receptor in K-ras-transformed thyroid cells: TGF-β resistant, reverses their malignant phenotype. J. Cell. Physiol. 172: 200-208. [Medline]
  18. Massague, J., J. Seoane, D. Wotton. 2005. Smad transcription factors. Genes Dev. 19: 2783-2810. [Abstract/Free Full Text]
  19. Li, M., Y. Y. Wan, S. Sanjabi, A. K. Robertson, R. A. Flavell. 2006. Transforming growth factor-β regulation of immune response. Annu. Rev. Immunol. 24: 99-146. [Medline]
  20. Tang, B., M. Vu, T. Booker, S. J. Santner, F. R. Miller, M. R. Anver, L. M. Wakefield. 2003. TGF-β switches from tumor suppressor to prometastatic factor in a model of breast cancer progression. J. Clin. Invest. 112: 1116-1124. [Medline]
  21. Lu, S. L., D. Reh, A. G. Li, J. Woods, C. L. Corless, M. Kulesz-Martin, X. J. Wang. 2004. Overexpression of transforming growth factor β1 in head and neck epithelia results in inflammation, angiogenesis, and epithelial hyperproliferation. Cancer Res. 64: 4405-4410. [Abstract/Free Full Text]
  22. Bravo, S. B., S. Pampin, J. Cameselle-Teijeiro, C. Carneiro, F. Dominguez, F. Barreiro, C. V. Alvarez. 2003. TGF-β induced apoptosis in human thyrocytes is mediated by p27kip1 reduction and is overridden in neoplastic thyrocytes by NF-{kappa}B activation. Oncogene 22: 7819-7830. [Medline]
  23. Mangan, P. R., L. E. Harrington, D. B. O'Quinn, W. S. Helms, D. C. Bullard, C. O. Elson, R. D. Hatton, S. M. Wahl, T. R. Schoeb, C. T. Weaver. 2006. Transforming growth factor-β induces development of the Th17 lineage. Nature 441: 231-234. [Medline]
  24. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, B. Stockinger. 2006. TGF-β in the context of an inflammatory cytokines milieu supports de novo differentiation of IL-17-producing T cells. Immunity 24: 179-189. [Medline]
  25. Contempre, B., O. Le Moine, J. E. Dumont, J. F. Denef, M. C. Many. 1996. Selenium deficiency and thyroid fibrosis: a key role for macrophages and TGF-β. Mol. Cell. Endocrinol. 124: 7-15. [Medline]
  26. Saxena, V., D. W. Lienesch, M. Zhou, R. Bommireddy, M. Azhar, T. Doetschman, R. R. Singh. 2008. Dual roles of immunoregulatory cytokines TGF-β in the pathogenesis of autoimmune-mediated organ damage. J. Immunol. 180: 1903-1912. [Abstract/Free Full Text]
  27. Wan, Y. Y., R. A. Flavell. 2007. "Yin-Yang" functions of transforming growth factor-β and T regulatory cells in immune regulation. Immunol. Rev. 220: 199-213. [Medline]
  28. Ishida, Y., T Kondo, T. Takyasu, Y. Iwakura, N. Mukaida. 2004. The essential involvement of cross-talk between IFN-{gamma} and TGF-β in the skin wound-healing process. J. Immunol. 172: 1848-1855. [Abstract/Free Full Text]
  29. Asano, Y., H. Ihn, K. Yamane, M. Kubo, K. Tamaki. 2004. Impaired Smad-7-Smurf-mediated negative regulation of TGF-β signaling in scleroderma fibroblasts. J. Clin. Invest. 113: 253-264. [Medline]
  30. Ulloa, L., J. Doody, J. Massague. 1999. Inhibition of transforming growth factor-β/SMAD signaling by interferon-{gamma}/STAT pathway. Nature 387: 710-713.
  31. Podolin, P. L., A. Pressey, N. H. DeLarato, P. A. Fischer, L. B. Peterson, L. S. Wicker. 1993. I-E+ nonobese diabetic mice develop insulitis and diabetes. J. Exp. Med. 178: 793-803. [Abstract/Free Full Text]
  32. Yu, S., G. C. Sharp, H. Braley-Mullen. 2006. Thyrocytes responding to IFN-{gamma} are essential for development of lymphocytic spontaneous autoimmune thyroiditis and for inhibition of thyrocyte hyperplasia. J. Immunol. 176: 1259-1265. [Abstract/Free Full Text]
  33. Sanderson, N., V. Factor, P. Nagy, J. Kopp, P. Kondaiah, L. Wakefield, A. B. Roberts, M. B. Sporn, S. S. Thorgeirsson. 1995. Hepatic expression of mature transforming growth factor β1 in transgenic mice results in multiple tissue lesions. Proc. Natl. Acad. Sci. USA 92: 2572-2576. [Abstract/Free Full Text]
  34. Chen, K., Y. Z. Wei, G. C. Sharp, H. Braley-Mullen. 2000. Characterization of thyroid fibrosis in a murine model of granulomatous experimental autoimmune thyroiditis. J. Leukocyte Biol. 68: 828-835. [Abstract/Free Full Text]
  35. Fang, Y., Y. Wei, V. Demarco, K. Chen, G.C. Sharp, H. Braley-Mullen. 2007. Murine FLIP transgene expressed on thyroid epithelial cells promotes resolution of granulomatous experimental autoimmune thyroiditis in DBA/1 mice. Am. J. Pathol. 170: 875-887. [Abstract/Free Full Text]
  36. Derynck, R., Y. E. Zhang. 2003. Smad-dependent and Smad-independent pathways in TGF-β family signaling. Nature 425: 577-584. [Medline]
  37. Ouyang, W., J. K. Kolls, V. Zheng. 2008. The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity 28: 454-467. [Medline]
  38. Zeng, Y., D. M. Danilenko, P. Valdez, I. Kasman, J. Eastham-Anderson, J. Wu, W. Ouyang. 2007. Interleukin-22, a Th17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature 445: 648-651. [Medline]
  39. Zaba, L. C., I. Cardinale, P. Gilleaudeau, M. Sullivan-Whalen, M. S. Farinas, J. Fuentes-Duculan, I. Novitskaya, A. Khatcherian, M. J. Bluth, M. A. Lowes, J. G. Krueger. 2007. Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 response. J. Exp. Med. 204: 3183-3194. [Abstract/Free Full Text]
  40. Ma, H., S. Liang, J. Li, L. Napierata, T. Brown, S. Benoit, M. Senices, D. Gill, K. Dunussi-Joannopoulos, M. Collins, et al 2008. IL-22 is required for Th17-mediated pathology in a mouse model of psoriasis-like skin inflammation. J. Clin. Invest. 118: 597-607. [Medline]
  41. Fichtner-Feigl, S., W. Strober, K. Kawakami, R. K. Puri, A. Kitani. 2006. IL-13-signaling through the IL-13R{alpha}2 is involved in induction of TGF-β1 production and fibrosis. Nat. Med. 12: 99-106. [Medline]
  42. Ma, B., M. R. Blackburn, C. G. Lee, R. J. Homer, W. Liu, R. A. Flavell, L. Boyden, R. P. Lifton, C. X. Sun, H. W. Young, J. A. Elias. 2006. Adenosine metabolism and murine strain-specific IL-4 induced inflammation, emphysema, and fibrosis. J. Clin. Invest. 116: 1274-1283. [Medline]
  43. Todaro, M., M. Zerilli, L. Ricci-Vitiani, M. Bini, M. Perez-Alea, A. Maria Florena, L. Miceli, G. Condorelli, S. Bonventre, G. Di Gesu, et al 2006. Autocrine production of interleukin-4 and interleukin-10 is required for survival and growth of thyroid cancer cells. Cancer Res. 66: 1491-1499. [Abstract/Free Full Text]
  44. Lakos, G, S. Takagawa, S. J. Chen, A. M. Ferreira, G. Han, K. Masuda, X. J. Wang, L. A. DiPietro, J. Varga. 2004. Targeted disruption of TGF-β/Smad3 signaling modulates skin fibrosis in a mouse model of scleroderma. Am. J. Pathol. 165: 203-217. [Abstract/Free Full Text]
  45. Bonniaud, P., P. J. Margetts, K. Ask, K. Flanders, J. Gauldie, M. Kolb. 2005. TGF-β and Smad-3 signaling link inflammation to chronic fibrogenesis. J. Immunol. 175: 5390-5395. [Abstract/Free Full Text]
  46. Bonniaud, P., M. Kolb, T. Galt, J. Robertson, C. Robbins, M. Stampfli, C. Lavery, P. J. Margrets, A. B. Roberts, J. Gauldie. 2004. Smad-3 null mice develop airspace enlargement and are resistant to TGF-β-mediated pulmonary fibrosis. J. Immunol. 173: 2099-2108. [Abstract/Free Full Text]
  47. Schulick, A. H., A. J. Taylor, W. Zuo, C.-B. Qiu, G. Dong, R. N. Woodward, R. Agah, A. B. Roberts, R. Virmani, D. A. Dichek. 1998. Overexpression of transforming growth factor β1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia. Proc. Natl. Acad. Sci. USA 95: 6983-6988. [Abstract/Free Full Text]
  48. Kobayashi, K., K. Koutaro Yokote, M. Fujimoto, K. Yamashita, A. Sakamoto, M. Kitahara, H. Kawamura, Y. Maezawa, S. Asaumi, T. Tokuhisa, et al 2005. Targeted disruption of TGF-β-smad-3 signaling leads to enhanced neointimal hyperplasia with diminished matrix deposition in response to vascular injury. Circ. Res. 96: 904-912. [Abstract/Free Full Text]
  49. Yamamoto, T., S. Takagawa, I. Katayama, K. Nishioka. 1999. Anti-sclerotic effect of transforming growth factor-β antibody in a mouse model of bleomycin-induced scleroderma. Clin. Immunol. 92: 6-13. [Medline]
  50. Yata, Y., P. Gotwals, V. Koteliansky, D. C. Rockey. 2002. Dose-dependent inhibition of hepatic fibrosis in mice by a TGF-β soluble receptor: implications for antifibrotic therapy. Hepatology 35: 1022-1030. [Medline]
  51. Derynck, R., R. J. Akhurst, A. Balmain. 2001. TGF-β signaling in tumor suppression and cancer progression. Nat. Genet. 29: 117-129. [Medline]
  52. Wynn, T. A.. 2007. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J. Clin. Invest. 117: 524-529. [Medline]




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