The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, K.
Right arrow Articles by Braley-Mullen, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, K.
Right arrow Articles by Braley-Mullen, H.
The Journal of Immunology, 2003, 171: 6236-6243.
Copyright © 2003 by The American Association of Immunologists

Mechanisms of Spontaneous Resolution versus Fibrosis in Granulomatous Experimental Autoimmune Thyroiditis 1

Kemin Chen*, Yongzhong Wei*, Gordon C. Sharp* and Helen Braley-Mullen2,*,{dagger},{ddagger}

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
When granulomatous experimental autoimmune thyroiditis (G-EAT) was induced in CBA/J or DBA/1 mice, thyroid lesions resolved in less severe (3+) G-EAT in wild-type mice or severe (5+) G-EAT in IFN-{gamma}-/- mice, but progressed to fibrosis in 5+ G-EAT in wild-type mice. To define the mechanisms leading to these distinct outcomes, the expression of inflammatory and apoptotic molecules and infiltrating cells was evaluated using immunohistochemistry, RT-PCR, and confocal microscopy. The ratio of CD4+/CD8+ T cells in thyroid infiltrates was one factor that predicted G-EAT outcome. CD4+ T cells outnumbered CD8+ T cells when lesions progressed to fibrosis, while CD8+ T cells outnumbered CD4+ T cells in thyroids that resolved. Fas, Fas ligand, FLIP, TNF-{alpha}, inducible NO synthase, TGF-{beta}, and IFN-{gamma} were highly expressed by infiltrating cells when G-EAT progressed to fibrosis. The expression of active caspase-3 was low, possibly contributing to the persistence of CD4+ T cells in fibrosis. In contrast, FLIP was mainly expressed by thyrocytes in resolving G-EAT, the expression of active caspase-3 was high, and resolution correlated with apoptosis of infiltrating cells. There was also relatively less expression of TGF-{beta}, IFN-{gamma}, TNF-{alpha}, and inducible NO synthase and higher expression of IL-10 in resolving G-EAT than in G-EAT that progressed to fibrosis. These differences were particularly striking when comparing IFN-{gamma}-/- vs wild-type mice. These results suggest that several opposing biological mechanisms contribute to the outcome of an ongoing autoimmune response. These include differential expression of pro- and antiapoptotic molecules, cytokines, and the ratio of CD4+ vs CD8+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The adoptive transfer model of granulomatous experimental autoimmune thyroiditis (G-EAT)3 studied by our laboratory has facilitated a detailed analysis of the mechanisms involved in induction of autoimmunity. Following transfer of mouse thyroglobulin (MTg)-sensitized donor spleen cells activated with MTg and IL-12, recipient mice develop thyroid lesions with granulomatous histopathology. These granulomatous inflammatory lesions evolve over time to either of two distinct outcomes, resolution or fibrosis (1). When animals have granulomatous thyroid lesions with severity scores of 3–4+ (indicating significant numbers of uninvolved follicles) on day 21, thyroid lesions spontaneously resolve by 35–60 days after cell transfer (2, 3). In contrast, when G-EAT severity scores are 5+ on day 21 with few or no remaining uninvolved thyroid follicles, thyroids become fibrotic and atrophic on days 35–60, and serum thyroxine (T4) levels are low (4). On day 21, these thyroids have, in addition to granulomatous features, many polymorphonuclear neutrophils (PMN), microabscesses, and necrosis, and inflammation extends beyond the thyroid to involve adjacent connective tissue and muscle (4). CD8+ T cells are required for G-EAT resolution, but not for the development of fibrosis (1, 2, 3). The distinct outcomes of G-EAT are also evident when comparing IFN-{gamma}+/+ and IFN-{gamma}-/- DBA/1 mice. Severe G-EAT develops in both IFN-{gamma}+/+ and IFN-{gamma}-/- DBA/1 mice (5), and as shown here, fibrosis is reduced, and G-EAT lesions resolve by day 35 in thyroids of IFN-{gamma}-/- mice. CD4+, but not CD8+, T cells are required for the development of fibrosis, and overproduction of TGF-{beta} contributes to fibrosis (1, 4, 6, 7). However, the detailed mechanisms that dictate whether an autoimmune inflammatory lesion will resolve or progress to fibrosis is unknown.

Fibrosis is an end-stage feature of certain human diseases, such as pulmonary fibrosis, systemic sclerosis, myocardial infarction, and liver cirrhosis (8). Fibrosis is characterized by increased deposition of extracellular matrix, resulting in tissue destruction and loss of organ function (4, 7, 8). It is therefore important to identify mechanisms that might promote resolution and prevent fibrosis in an autoimmune inflammatory response. The present study was undertaken to determine whether the relative expression of various cells or molecules in thyroid infiltrates might differ in lesions that resolved vs those that progressed to fibrosis. The results indicate that the relative expression of CD4+ vs CD8+ T cells and of Th1 vs Th2 cytokines, and the site of expression of some apoptotic pathway molecules at the peak of disease correlate with a particular outcome. This is therefore a useful model for studying the mechanisms that can determine the outcome of an autoimmune inflammatory response and how an inflammatory response in a particular target organ can be influenced by particular inflammatory cells and mediators. Insights gained from such studies may suggest novel therapeutic strategies for human autoimmune diseases.


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

The original breeding stocks of DBA/1 and IFN-{gamma}-/- DBA/1 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Both strains have since been maintained through homozygous breeding in our colonies at University of Missouri. CBA/J mice were obtained through C. Reeder at the National Institutes of Health (Bethesda, MD). Both male and female mice were used, and mice were 7- to 14-wk-old when used as donors or recipients.

Induction of G-EAT

Donor CBA/J or DBA/1 mice were immunized twice with 150 µg of MTg and 15 µg of LPS (Escherichia coli 0111:B4; Sigma-Aldrich, St. Louis, MO) i.v. at 10-day intervals (3, 4, 5, 6). Seven days after the second injection of MTg and LPS, donor spleen cells were cultured at 107 cells/ml in RPMI 1640 containing 25 mM HEPES buffer (Cell and Immunobiology Core Facility, University of Missouri), 5% FCS (Sigma-Aldrich), 2 mM glutamine, 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 together with 5 ng/ml IL-12 (PeproTech, Rocky Hill, NJ) as previously described (6). After 72 h cells were harvested, washed, and injected i.v. into syngeneic irradiated (500 rad) recipient mice. Recipient thyroids were evaluated 19–21 days later, the time of maximal severity of G-EAT in this adoptive transfer model, or at 35–60 days following cell transfer when lesions have resolved or become fibrotic (2, 3, 4, 5, 6, 7).

Evaluation of G-EAT histopathology and fibrosis

Thyroids were removed from four to six mice per group at various times after cell transfer, and one lobe of each thyroid was fixed in formalin. For histologic analysis, paraffin tissues were sectioned and stained with H&E. Thyroids were scored quantitatively for G-EAT severity using a scale of 1+ to 5+ according to previously established criteria (6). 1+ thyroiditis 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 also evaluated qualitatively. In general, thyroids with 1–2+ severity scores have infiltrates consisting mainly of lymphocytes with few neutrophils. The more severely destroyed thyroids (4–5+ severity scores) have extensive granulomatous changes with follicular cell proliferation, multinucleated giant cells, large numbers of histiocytes, numerous lymphocytes, and neutrophils with microabscess formation, necrosis, and fibrosis. Thyroids of IFN-{gamma}-/- mice had extensive infiltration by eosinophils rather than neutrophils (5). The granulomatous inflammation in thyroids graded 4+ to 5+ characteristically extended beyond the thyroid to involve the adjacent connective tissue and muscle. For evaluation of collagen deposition, some thyroid sections were stained using Masson’s Trichrome (4, 7). Serum T4 levels were determined using a T4 enzyme immunoassay kit (Biotecx, Houston, TX) according to the manufacturer’s instructions (7).

Immunohistochemistry

Immunostaining of CD4+ and CD8+ T cells, TGF-{beta}1, FLIP, and active caspase-3 was performed as previously described (3, 4). Cytokine expression was detected on frozen thyroid sections. After fixation and blocking, sections were incubated with rat anti-IFN-{gamma} (XMG1.2), rabbit anti-inducible NO synthase (anti-iNOS; M19; Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-TNF-{alpha} (M18; Santa Cruz Biotechnology), rat anti-IL-4 (11B11), or rat anti-IL-10 (JESS-2A5). Following incubation with a secondary biotinylated goat anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA), goat anti-rat (Caltag Laboratories, Burlingame, CA), or rabbit anti-goat Ab (Jackson ImmunoResearch Laboratories), endogenous peroxidase was quenched with 0.3% hydrogen peroxide. Immunoreactivity was demonstrated using a Vector ABC peroxidase kit (Vector Laboratories, Burlingame, CA) with 3,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) or VIP (very intense purple) (Vector Laboratories) as the chromogen. The intensity of immunostaining was graded semiquantitatively. Controls using nonimmune rat, rabbit, or goat Ig instead of the respective primary Abs were always negative.

Confocal laser scanning double-immunofluorescence microscopy

To detect the differential expression of FLIP by CD4+ T cells or thyrocytes, dual-color immunofluorescence and confocal laser scanning microscopy was performed using established markers for CD4 and thyroid follicular cells (pan-cytokeratin). After fixation and blocking, thyroid frozen sections were incubated with rabbit anti-FLIP (1/200) for 45 min at room temperature and visualized with Alexa 488 conjugated anti-rabbit Ab (Molecular Probes, Eugene, OR; 1/500). For CD4+ T cell staining, slides were incubated with monoclonal rat IgG anti-CD4 (YTS191) for 30 min. This was followed by incubation with biotin-conjugated anti-rat Ab (1/500) for 30 min, and fluorescence was visualized by streptavidin-conjugated Alexa 568 (Molecular Probes) for 30 min. For cytokeratin and FLIP dual staining, microwave irradiation was used for cytokeratin retrieval (4). Cryosections were incubated with FITC-conjugated rat anti-cytokeratin (Sigma-Aldrich) overnight, then stained for FLIP as described above. Slides were observed with a Bio-Rad Radiance 2000 confocal system (Hercules, CA) coupled to an Olympus IX70 inverted microscope (New Hyde Park, NY).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distinct outcomes of G-EAT in CBA/J and IFN-{gamma}-/- vs IFN-{gamma}+/+ DBA/1 mice: resolution or fibrosis

G-EAT was induced by adoptive transfer of spleen cells from MTg-sensitized donors activated with MTg and IL-12 in vitro. After G-EAT lesions reached maximal severity 19–21 days after cell transfer, thyroid lesions either resolved or the thyroids became atrophic and fibrotic by days 35–50 (Table I and Fig. 1). This was true for both DBA/1 (Table I and Fig. 1) and CBA/J (Table I) mice. Specifically, when G-EAT severity scores on days 19–21 were 3–4+ in DBA/1 (Fig. 1A) or CBA/J (not shown) mice, inflammation resolved by days 35–40 (Fig. 1B). Collagen deposition in the thyroid, an indicator of fibrosis, was assessed by Masson’s Trichrome staining. As shown in Fig. 1, C and D, there was minimal, if any, deposition of collagen on days 19–21 in 3–4+ G-EAT thyroids or on day 35 in resolving G-EAT thyroids. However, when G-EAT lesions were very severe (5+) on days 19–21, most thyroid follicles were destroyed, and there was extensive infiltration by neutrophils, lymphocytes, and histiocytes, with microabscesses, necrosis, and focal fibrosis in both DBA/1 (Fig. 1E) and CBA/J (not shown) mice. By days 35–40, these extensively destroyed thyroids became atrophic and fibrotic in both strains (Fig. 1F; not shown). Extensive deposition of collagen was apparent on day 19 (Fig. 1G), and collagen persisted and became more extensive by day 35 (Fig. 1H). Mice with very severe fibrotic lesions had low serum T4, especially on day 35 (4, 7). Thus, in both DBA/1 and CBA/J mice, thyroid lesions reached maximal severity 19–21 days after cell transfer, and inflammation either resolved or progressed to fibrosis depending on the extent of damage on days 19–21.


View this table:
[in this window]
[in a new window]
  		Table I. Resolution of G-EAT or development of fibrosis in CBA/J and DBA/1 mice

 


View larger version (185K):
[in this window]
[in a new window]
  		FIGURE 1. G-EAT (3–4+) in DBA/1 WT mice with remaining follicles on day 19 (A) almost completely resolves by day 35 (<1+ severity; B). Masson’s Trichrome staining showed no deposition of collagen in these thyroids on day 19 (C) or day 35 (D). Thyroids from WT DBA/1 mice with 5+ severity scores on day 19 (E) with destruction of nearly all thyroid follicles became very atrophic and fibrotic on day 35 (F). An atrophic thyroid surrounded by muscle and connective tissue is indicated by the arrows (F). Masson’s Trichrome staining shows deposition of collagen (blue) on both day 19 (G) and day 35 (H). I–L, Thyroids from IFN-{gamma}-/- DBA/1 mice with 5+ severity scores on day 19 (I) show substantial resolution (1+ severity) by day 35 (J). Collagen deposition was minimal in IFN-{gamma}-/- thyroids with 5+ severity scores on day 19 (K), and negative in resolving thyroids on day 19 (L). Magnification: A, B, E, F, I, and J, x100; C, D, G, H, K, and L, x400.

 
We previously showed that IFN-{gamma}-/- DBA/1 mice developed very severe G-EAT 19–21 days after cell transfer (5). Although thyroids of IFN-{gamma}-/- mice had extensive inflammation with severity scores of 4–5+ on days 19–21 (Table II and Fig. 1I), lesions consistently resolved almost completely or diminished in severity without fibrosis by days 35–40 after cell transfer (Fig. 1J and Table II). Compared with wild-type (WT) mice (Fig. 1, G and H), there was minimal deposition of collagen in IFN-{gamma}-/- thyroids on day 19 (Fig. 1K), and collagen was not present on day 35 (Fig. 1L) when lesions had largely resolved. The changes leading to each respective outcome in both WT and IFN-{gamma}-/- mice began soon after day 21 and were nearly complete in most mice 35–40 days after cell transfer (Fig. 1 and Tables I and II).


View this table:
[in this window]
[in a new window]
  		Table II. Resolution of severe G-EAT in IFN-{gamma}-/- mice and progression to fibrosis in IFN-{gamma}+/+ DBA/1 mice

 
Different expression pattern of apoptotic molecules in G-EAT thyroids undergoing resolution or fibrosis

To begin to understand why inflammatory cells were rapidly cleared in less severe G-EAT and in more severe G-EAT that developed in IFN-{gamma}-/- DBA/1 mice, but persisted and led to fibrosis in very severely destroyed (5+) thyroids of WT DBA/1 and CBA/J mice (Fig 1, G and H, and Tables I and II), the expressions of pro- and antiapoptotic molecules in G-EAT thyroids were compared.

The Fas pathway of apoptosis is important in many autoimmune diseases, including thyroiditis (3, 9). Fas is expressed primarily by inflammatory cells, whereas Fas ligand (FasL) is expressed primarily by thyroid follicular cells (TFC) on day 19 in CBA/J and NOD.H-2h4 mice with 3–4+ G-EAT that resolves by days 35–40 (3). This was also true for DBA/1 mice, whose lesions resolved by days 35–40 (Table III). In contrast, inflammatory cells and TFC both expressed Fas and FasL on day 19 in DBA/1 or CBA/J thyroids that progressed to fibrosis (Table III). Because it is difficult to distinguish inflammatory cells from proliferating TFC in very severely destroyed thyroids, thyroids were stained for Fas or FasL and for cytokeratin, which stains TFC. Staining of adjacent sections showed that cytokeratin-positive TFC were positive for Fas and FasL (not shown).


View this table:
[in this window]
[in a new window]
  		Table III. Summary of Fas, FasL, Flip and active caspase3 staining in G-EAT thyroids that will resolve or progress to fibrosisa

 
FLIP is an important inhibitor of Fas-mediated apoptosis (10). The expression of FLIP in DBA/1 or CBA/J thyroids with 3+ severity scores was predominant in TFC both on day 19 (Fig. 2A and Table III) and day 28 (not shown). When inflammation was resolving, FLIP expression diminished on day 35, but it was still expressed predominantly by TFC (Fig. 2B). FLIP was also expressed by TFC in IFN-{gamma}-/- DBA/1 G-EAT thyroids on day 19 (Fig. 2C). Up-regulation of FLIP by TFC was still evident on day 28 in thyroids with 4+ severity scores (not shown) and on day 35 in IFN-{gamma}-/- mice (Fig. 2D). In contrast, FLIP was expressed primarily by some inflammatory cells on days 19 and 28 in thyroids of WT DBA/1 mice with 5+ G-EAT (Fig. 2E and not shown). FLIP was also up-regulated on myofibroblasts in severely destroyed thyroids on day 19 (data not shown). By day 35, when thyroids were fibrotic and there were few or no remaining thyroid follicles, FLIP was still expressed by many inflammatory cells (Fig. 2F). Confocal analysis confirmed that effector CD4+ T cells expressed FLIP (Fig. 2G), but cytokeratin-positive TFC did not express FLIP (Fig. 2H) in thyroids that progressed to fibrosis. In contrast, FLIP was predominantly expressed by TFC in thyroids (3+) that would resolve (Fig. 2I). In conclusion, FLIP was predominantly expressed by TFC in thyroids with less severe (3+) G-EAT severity scores or in more severe G-EAT (4–5+) in thyroids of IFN-{gamma}-/- mice in which inflammation would resolve, while FLIP was primarily expressed by inflammatory cells in thyroids with severe (5+) G-EAT that progressed to fibrosis. Thus, the site of expression of FLIP may be predictive of the distinct outcome of thyroid inflammation in G-EAT.



View larger version (74K):
[in this window]
[in a new window]
  		FIGURE 2. Comparison of FLIP and active caspase-3 expression in thyroids with G-EAT that will resolve or progress to fibrosis. FLIP was expressed primarily by TFC on day 19 in thyroids of DBA/1 mice with 3+ G-EAT (A) or in IFN-{gamma}-/- DBA/1 mice with 5+ G-EAT (C) and on day 35 in both groups when inflammation was resolving (1+; B and D). In contrast, FLIP was primarily expressed by inflammatory cells in thyroids of DBA/1 mice with 5+ G-EAT on day 19 (E) and after the lesions progressed to fibrosis on day 35 (F). Dual staining and confocal microscopy demonstrate that CD4 (green) and FLIP (red) are coexpressed (yellow, overlay) by some cells (G) in thyroids of DBA/1 mice with 5+ G-EAT on day 19 (thyroids that would progress to fibrosis). Cytokeratin-positive thyrocytes (green; H) in these thyroids did not express FLIP (red). In contrast, cytokeratin (I, green) and FLIP (I, red) were coexpressed (I, yellow, overlay) on day 19 in DBA/1 thyroids with 3+ G-EAT that would resolve. Active caspase-3 (brown) in 3+ G-EAT (J and M) or 5+ G-EAT of IFN-{gamma}-/- mice (K and N) or in 5+ G-EAT of wild-type DBA/1 mice (L and O) on day 19 (J–L) and day 28 (M–O). Lesions in M and N were resolving on day 28, while that in O had progressed to fibrosis. Magnification: A–F, x1000; G–I, x600; J–O, x400.

 
Up-regulation of FLIP (Fig. 2, A and C, and Table III) and FasL (3, 11) on TFC in G-EAT thyroids may protect TFC from further destruction, enabling them to survive to induce apoptosis of Fas+ CD4+ effectors (3). In contrast, the expression of FLIP by inflammatory cells in very severe G-EAT (Fig. 2, E and F) may contribute to their resistance to apoptosis. Results obtained using staining for active caspase-3, which is downstream of the Fas and TNF-{alpha} apoptosis cascade (10), support this hypothesis. Active caspase-3 was highly expressed by inflammatory cells in thyroids with 3–4+ G-EAT severity scores on day 19 and during resolution on day 28 (Fig. 2, J and M). This is true for both CBA/J (11), and DBA/1 WT mice (Fig. 2, J and M) and for DBA/1 IFN-{gamma}-/- mice with early resolution (Table II and Fig. 2, K and N). In contrast, thyroids of CBA/J (not shown) and DBA/1 mice with very severe 5+ G-EAT on days 19 and 28 (Fig. 2, L and O) that would progress to fibrosis, had few active caspase-3 positive cells. Similar results were obtained when apoptotic cells were examined by TUNEL staining, i.e., thyroids of IFN-{gamma}-/- mice with 4–5+ severity scores and thyroids of DBA/1 and CBA/J mice with 3–4+ severity scores (thyroids in which G-EAT would resolve) had many TUNEL+ infiltrating cells, whereas thyroids in which G-EAT progressed to fibrosis had few TUNEL+ infiltrating cells (data not shown). Thus, the different expression patterns of FLIP, FasL, and apoptotic cells in thyroids of CBA/J, DBA/1, and IFN-{gamma}-/- DBA/1 mice 19 days after cell transfer can be predictive of the outcome of the thyroid inflammatory response.

Ratios of CD4+ vs CD8+ T cells differ in G-EAT thyroids that will resolve or progress to fibrosis

CD4+ and CD8+ T cells play different roles in G-EAT, i.e., CD4+ T cells are the primary effector cells, while CD8+ T cells have no apparent effector function and promote early resolution of inflammation (2, 3). We previously showed that CD8+ T cells outnumbered CD4+ T cells on day 21 in thyroids of CBA/J mice with early resolution, whereas CD4+ T cells were predominant in thyroids of CBA/J mice with chronic inflammation (e.g., depleted of CD8+ T cells or given anti-FasL mAb) (3, 12). To determine whether the ratio of CD4+/CD8+ T cells might differ in thyroids where G-EAT will resolve or progress to fibrosis, infiltration of CD4+ and CD8+ T cells was monitored over time in thyroids of CBA/J and DBA/1 mice with the two outcomes. As previously shown for CBA/J mice (3), thyroids of DBA/1 mice with 3+ G-EAT that will resolve had more CD8+ T cells than CD4+ T cells on both day 19 (Fig. 3, A and B) and day 35 (Fig. 3, C and D). Although the numbers of CD4+ T cells and CD8+ T cells were similar in thyroids of IFN-{gamma}-/- DBA/1 mice with 4–5+ resolving G-EAT on day 19 (Fig. 3, E and F), CD8+ T cells distinctly outnumbered CD4+ T cells on day 28 (not shown) and day 35 (Fig. 3, G and H). In contrast, CD4+ T cells outnumbered CD8+ T cells on both day 19 (Fig. 3, I and J) and day 35 (Fig. 3, K and L) in DBA/1 or CBA/J thyroids with 5+ G-EAT that progresses to fibrosis. These results, summarized in Table IV, suggest that the ratio of CD4+/CD8+ T cells in G-EAT thyroids is independent of the mouse strain and can be a good indicator of the outcome of the autoimmune inflammatory response, i.e., resolution vs fibrosis.



View larger version (110K):
[in this window]
[in a new window]
  		FIGURE 3. CD8+ T cells outnumber CD4+ T cells in resolving G-EAT thyroids, and CD4+ T cells are predominant in thyroids that will progress to fibrosis. CD4+ T cells and CD8+ T cells in thyroids of DBA/1 mice (A–D) with 3+ G-EAT on day 19 (A and B) and thyroids of IFN-{gamma}-/- mice (E–H) with 5+ G-EAT on day 19 (E and F) that resolve by day 35 (C, D, G, and H) or thyroids of WT DBA/1 mice with 5+ G-EAT on day 19 (I and J) that progress to fibrosis on day 35 (K and L). A, B, E, F, I, and J, Day 19; C, D, G, H, K, and L, day 35. Magnification: A–J, x400; K and L, x100. Figures are representative of at least five experiments.

 

View this table:
[in this window]
[in a new window]
  		Table IV. Ratio of CD4+/CD8+ T cells infiltrating thyroids in which G-EAT will resolve or progress to fibrosisa

 
Expression of cytokines in G-EAT thyroids undergoing resolution or fibrosis

Thyroid infiltrating cells produce cytokines that could directly contribute to the outcome of G-EAT or regulate the expression of apoptotic or antiapoptotic molecules (10). Thyroids of DBA/1 mice that would resolve (Fig. 4, A–L) or progress to fibrosis (Fig. 4, M–R) expressed both Th1 (IFN-{gamma}, TNF-{alpha}) and Th2 (IL-4, IL-10) cytokines as well as TGF-{beta} and iNOS, except no IFN-{gamma} was detected in thyroids of IFN-{gamma}-/- recipients of IFN-{gamma}-/- spleen cells (Fig. 4G). The staining intensity for IFN-{gamma}, iNOS, TNF-{alpha}, and TGF-{beta} was greater in 5+ G-EAT thyroids that progressed to fibrosis (Fig. 4, M–P) compared with thyroids of WT mice with 3+ G-EAT (Fig. 4, A–D) or thyroids of IFN-{gamma}-/- mice with 5+ G-EAT that would resolve (Fig. 4, G–J). Thyroids that would resolve had less intense staining for IFN-{gamma}, TNF-{alpha}, iNOS, and TGF-{beta} on day 19 (Fig. 4, A–D and G–J) than those that would progress to fibrosis (Fig. 4, M–P). IL-10 and, to a lesser extent, IL-4 expressions were generally more intense in thyroids that would resolve (Fig. 4, E, F, K, and L) than in those that would progress to fibrosis (Fig. 4, Q and R). Although immunohistochemical staining is not quantitative, these results suggest that the balance of cytokines produced during the course of the thyroid inflammatory response may contribute to its magnitude and duration, thus influencing the distinct outcomes.



View larger version (120K):
[in this window]
[in a new window]
  		FIGURE 4. Th1 and Th2 cytokine expression on day 19 in thyroids of DBA/1 mice with 3+ G-EAT (A–F), IFN-{gamma}-/- DBA/1 mice with 5+ G-EAT (G–L), and thyroids of WT DBA/1 mice with 5+ G-EAT (M–R). IFN-{gamma} (A, G, and M), iNOS (B, H, and N), TNF-{alpha} (C, I, and O), TGF-{beta} (D, J, and P), IL-4 (E, K, and Q), and IL-10 (F, L, and R). Although cytokine expression was not quantitative, staining intensity was graded using a scale of - to +++ (-, not detectable; +, weak; ++, moderate; +++, strong) to compare the relative levels of cytokine expression between groups. Magnification of A–R, x400. Figures are representative of at least three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The G-EAT model established in our laboratory is an excellent model for studying mechanisms that determine whether an autoimmune response will resolve or progress to fibrosis (1, 2, 3, 4). G-EAT lesions reach maximal severity 19–21 days after cell transfer. Lesions resolve when G-EAT is less severe (3+) or when IFN-{gamma} is absent, but progress to fibrosis in WT mice when G-EAT is very severe (5+). These results are consistent with the concept that opposing biological mechanisms can regulate the outcome of an autoimmune response, some promoting progressive inflammation, tissue injury, and fibrosis, and others promoting resolution of inflammation. One level of control is mediated through regulation of pro- and antiapoptotic molecules, thus determining the fate of thyroid-infiltrating cells and TFC (11) (Fig. 2). A second level of control, mediated by regulation of cytokines (Fig. 4), increases or reduces the autoimmune inflammation, while a third level of control is mediated through CD8+ T cells (1, 2, 3).

Resolution of inflammation requires the removal of inflammatory cells that were recruited and expanded during the response. In this study, one characteristic that distinguished resolution of inflammation from progression to fibrosis was the extent of apoptosis of inflammatory cells. Many TUNEL+ inflammatory cells were detected (not shown), and the expression of active capase-3 was predominant in thyroids with G-EAT that would resolve (Fig. 2). These results are consistent with our earlier observation that the apoptosis of infiltrating cells is increased during G-EAT resolution (3, 11). Apoptosis results in the clearance of inflammatory cells and limits the influx of collagen-producing myofibroblasts through reduction of TGF-{beta} (4, 13). Not surprisingly, the incidence of apoptotic infiltrating cells was associated with the differential expression of apoptotic and antiapoptotic molecules. For example, FLIP and FasL were highly expressed by TFC before resolution. Since FLIP can protect cells from apoptosis (10, 11, 14, 15, 16), FLIP may be up-regulated to protect TFC from destruction, while Fas+ inflammatory cells that do not express FLIP are cleared by Fas-mediated apoptosis. The expression of antiapoptotic molecules, including FLIP (Fig. 2), Bcl-2, and Bcl-xL (not shown), in inflammatory cells was higher in thyroids that progressed to fibrosis than in those that resolved. Up-regulation of FLIP can protect pancreatic {beta} cells from apoptosis (17), and overexpression of Bcl-xL or FLIP in T cells increased the severity of experimental autoimmune encephalomyelitis and decreased apoptosis of inflammatory cells in the CNS (14, 16, 18). FLIP and FasL up-regulation by TFC may protect TFC from further destruction, enabling them to survive and induce the apoptosis of Fas+ CD4+ effectors, leading to resolution. In contrast, FLIP, Bcl-xL, and Bcl-2 are expressed primarily in infiltrating cells in thyroids that progress to fibrosis, thus conferring resistance to apoptosis. The finding that the differential expression of pro- and antiapoptotic molecules by TFC and infiltrating cells can lead to different manifestations of autoimmune thyroid disease is also observed in comparisons of Grave’s disease and Hashimoto’s thyroiditis in man (15, 19).

Inflammation is associated with the production of proinflammatory cytokines that recruit and activate inflammatory cells to amplify the response (20, 21), and G-EAT lesions that resolve or progress to fibrosis had different cytokine profiles (Fig. 4). Although both Th1 and Th2 cytokines were present in G-EAT thyroids (5, 6, 22, 23), the expression of the profibrotic cytokine TGF-{beta} and the proinflammatory mediators IFN-{gamma}, TNF-{alpha}, and iNOS was generally higher in G-EAT thyroids progressing to fibrosis, while IL-10 and IL-4 were generally higher in thyroids that would resolve (Fig. 4). TGF-{beta} is important in fibrosis (8), and inhibition of TGF-{beta} by anti-TGF-{beta} or linsinopril inhibited the development of fibrosis and promoted G-EAT resolution (7). IL-4 may not play an important role in thyroid fibrosis, since G-EAT severity and fibrosis were comparable in both WT and IL-4-/- mice (23) (our unpublished observations). TNF-{alpha}, IFN-{gamma}, and iNOS can cause necrosis (24, 25, 26) or cooperate to induce tissue damage by apoptosis of epithelial cells (27), and apoptosis of TFC (not shown) and necrosis (7) are evident in G-EAT thyroids that progress to fibrosis. Although some studies suggest that IFN-{gamma} can inhibit fibrosis (28, 29, 30), fibrosis was always more extensive in WT compared with IFN-{gamma}-/- mice in this study. Thyroids of WT mice had a stronger expression of TGF-{beta}, a greater expression of TNF-{alpha} and iNOS (Fig. 4), and a higher expression of certain chemokines, such as monocyte chemoattractant protein-1, which can up-regulate TGF-{beta} and are implicated in fibrosis (7, 31, 32) (K. Chen, unpublished observations). Very preliminary results suggest that neutralization of TNF-{alpha} may also inhibit fibrosis and promote resolution in this model. The effect of iNOS inhibitors is currently under investigation.

Proinflammatory cytokines can also suppress the apoptosis of PMN (33, 34). In G-EAT, infiltration of PMN is associated with necrosis and development of fibrosis (4, 7). Exaggerated neutrophil activation can cause severe tissue damage (35), and tissue necrosis can prolong inflammatory responses (35, 36). Since maximal apoptosis of inflammatory cells occurred before resolution, apoptosis of inflammatory cells during resolution may result in a change in cytokine profile and suppression of ongoing inflammatory responses (14, 36, 37, 38). Removal of apoptotic cells can actively suppress the production of proinflammatory growth factors, cytokines, and chemokines (39).

This study also demonstrated that effector CD4+ T cells always outnumbered CD8+ T cells when G-EAT progressed to fibrosis, whereas CD8+ T cells always outnumbered CD4+ T cells in resolving G-EAT (Fig. 3). CD4+ T cells were also predominant in G-EAT thyroids when anti-CD8 or anti-FasL was used to inhibit resolution (3). Since CD8+ T cells always outnumbered CD4+ T cells when G-EAT would resolve, CD8+ T cells may control the expansion of activated CD4+ T cells. After depletion of CD8+ T cells, apoptosis of inflammatory cells decreased, CD4+ T cells persisted in thyroids (3), and FLIP was expressed primarily by inflammatory cells (11), as shown here for G-EAT thyroids that progressed to fibrosis (Fig. 2). Thus, CD8+ T cells in this model may induce the apoptosis of effector cells via regulation of pro- or antiapoptotic molecules. CD8+ T cells also play a role in T cell apoptosis in experimental autoimmune encephalomyelitis (40, 41, 42), and they can produce inhibitory cytokines, such as TGF-{beta} and IL-10 (42). Whether CD8+ T cells produce inhibitory cytokines that promote G-EAT resolution is an important issue that requires further clarification.

In this study we demonstrated several unique features of the autoimmune inflammatory response in thyroids of mice with inflammation that resolves or progresses to fibrosis. The site of expression of particular pro- and antiapoptotic molecules, the ratio of CD4+/CD8+ T cells in thyroid infiltrates, and the relative expression of particular cytokines can all be important predictors of whether inflammation will resolve or lead to continuing injury and fibrosis. Our findings provide further insight into the biological control of inflammation, an issue that must be better understood for successful repair of tissue injury. Insights gained from these studies may suggest novel therapeutic strategies to promote protective mechanisms and inhibit disease-potentiating mechanisms, such as fibrosis. They may also increase our understanding of how the body limits inflammation, and how successful repair of tissue injury results in the resolution of inflammation.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant DK35527 and the University of Missouri Research Board. K.C. was supported by an Arthritis Foundation Postdoctoral Fellowship. Back

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

3 Abbreviations used in this paper: G-EAT, granulomatous experimental autoimmune thyroiditis; FasL, Fas ligand; iNOS, inducible NO synthase; MTg, mouse thyroglobulin; PMN, polymorphonuclear neutrophil; T4, thyroxine; TFC, thyroid follicular cells; WT, wild type. Back

Received for publication May 28, 2003. Accepted for publication September 25, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Braley-Mullen, H., G. C. Sharp. 2000. Adoptive transfer murine model of granulomatous experimental autoimmune thyroiditis. Int. Rev. Immunol. 19:535.[Medline]
  2. Braley-Mullen, H., R. W. McMurray, G. C. Sharp, M. Kyriakos. 1994. Regulation of the induction and resolution of granulomatous experimental autoimmune thyroiditis in mice by CD8+ T cells. Cell. Immunol. 153:492.[Medline]
  3. Wei, Y., K. Chen, G. C. Sharp, H. Yagita, H. Braley-Mullen. 2001. Expression and regulation of Fas and Fas ligand on thyrocytes and infiltrating cells during induction and resolution of granulomatous experimental autoimmune thyroiditis. J. Immunol. 167:6678.[Abstract/Free Full Text]
  4. 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.[Abstract/Free Full Text]
  5. Tang, H., G. C. Sharp, K. E. Peterson, H. Braley-Mullen. 1998. IFN-{gamma}-deficient mice develop severe granulomatous experimental autoimmune thyroiditis with eosinophil infiltration in thyroids. J. Immunol. 160:5105.[Abstract/Free Full Text]
  6. Braley-Mullen, H., G. C. Sharp, H. Tang, K. Chen, M. Kyriakos, J. T. Bickel. 1998. Interleukin-12 promotes activation of effector cells that induce a severe destructive granulomatous form of experimental autoimmune thyroiditis. Am. J. Pathol. 152:1347.[Abstract]
  7. Chen, K., Y. Z. Wei, G. C. Sharp, H. Braley-Mullen. 2002. Inhibition of TGF{beta}1 by anti-TGF{beta}1 antibody or lisinopril reduced thyroid fibrosis in granulomatous experimental autoimmune thyroiditis. J. Immunol. 169:6530.[Abstract/Free Full Text]
  8. Border, R. W. A., N. A. Noble. 1994. Transforming growth factor-{beta} in tissue fibrosis. N. Engl. J. Med. 331:1286.[Free Full Text]
  9. De Maria, R., R. Testi. 1998. Fas-FasL interactions: a common pathogenetic mechanism in organ-specific autoimmunity. Immunol. Today 19:121.[Medline]
  10. Thome, M., J. Tschopp. 2001. Regulation of lymphocyte proliferation and death by FLIP. Nat. Rev. Immunol. 1:50.[Medline]
  11. Wei, Y., K. Chen, G. C. Sharp, H. Braley-Mullen. 2003. Expression of FLIP and FasL in inflammatory cells vs. thyrocytes can be predictive of chronic inflammation vs resolution of autoimmune thyroiditis. Clin. Immunol. 108:221.[Medline]
  12. McMurray, R. W., G. C. Sharp, H. Braley-Mullen. 1994. Intrathyroidal cell phenotype in murine lymphocytic and granulomatous experimental autoimmune thyroiditis. Autoimmunity 18:93.[Medline]
  13. Iredale, J. P., R. C. Benyon, J. Pickering, M. McCullen, M. Northrop, S. Pawley, C. Hovell, M. J. P. Arthur. 1998. Mechanisms of spontaneous resolution of rat liver fibrosis, hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J. Clin. Invest. 102:538.[Medline]
  14. Schlapbach, R., K. S. Spanaus, U. Malipiero, S. Lens, A. Tasinato, J. Tschopp, A. Fontana. 2000. TGF-{beta} induces the expression of the FLICE-inhibitory protein and inhibits Fas-mediated apoptosis of microglia. Eur. J. Immunol. 30:3680.[Medline]
  15. Stassi, G., D. Di Liberto, M. Todaro, A. Zeuner, L. Ricci-Vitiani, A. Stoppacciaro, L. Ruco, F. Farina, G. Zummo, R. De Maria. 2000. Control of target cell survival in thyroid autoimmunity by T helper cytokines via regulation of apoptotic proteins. Nat. Immunol. 1:483.[Medline]
  16. Djerbi, M., K.-B. Abdul-Majid, M. Abedi-Valugerdi, T. Olsson, R. A. Harris, A. Grandien. 2003. Expression of the long form of human FLIP by retroviral gene transfer of hemopoietic stem cells exacerbates experimental autoimmune encephalomyelitis. J. Immunol. 170:2064.[Abstract/Free Full Text]
  17. Maedler, K., A. Fontana, F. Ris, P. Sergeev, C. Toso, J. Oberholzer, R. Lehmann, F. Bachmann, A. Tasinato, G. A. Spinas, et al 2002. FLIP switches Fas-mediated glucose signaling in human pancreatic {beta} cells from apoptosis to cell replication. Proc. Natl. Acad. Sci. USA 99:8236.[Abstract/Free Full Text]
  18. Issazadeh, S., K. Abdallah, T. Chitnis, A. Chandraker, A. D. Wells, L. A. Turka, M. H. Sayegh, S. J. Khoury. 2000. Role of passive T-cell death in chronic experimental autoimmune encephalomyelitis. J. Clin. Invest. 105:1109.[Medline]
  19. Giordano, C., P. Richiusa, M. Bagnasco, G. Pizzolanti, F. Di Blasi, M. S. Sbriglia, A. Mattina, G. Pesce, P. Montagna, F. Capone, et al 2001. Differential regulation of Fas-mediated apoptosis in both thyrocyte and lymphocyte cellular compartments correlates with opposite phenotypic manifestations of autoimmune thyroid disease. Thyroid 11:233.[Medline]
  20. Huang, J., F. J. DeGraves, S. D. Lenz, D. Gao, P. Feng, D. Li, T. Schlapp, B. Kaltenboeck. 2002. The quantity of nitric oxide released by macrophages regulates chlamydia-induced disease. Proc. Natl. Acad. Sci. USA 99:3914.[Abstract/Free Full Text]
  21. Nathan, C.. 1997. Inducible nitric oxide synthase: what difference does it make?. J. Clin. Invest. 100:2417.[Medline]
  22. Tang, H., G. C. Sharp, K. Chen, H. Braley-Mullen. 1998. The kinetics of cytokine gene expression in the thyroids of mice developing granulomatous experimental autoimmune thyroiditis. J. Autoimmun. 11:581.[Medline]
  23. Tang, H., G. C. Sharp, K. E. Peterson, H. Braley-Mullen. 1998. Induction of granulomatous experimental autoimmune thyroiditis in IL-4 gene-disrupted mice. J. Immunol. 160:155.[Abstract/Free Full Text]
  24. Barbu, A., N. Welsh, J. Saldeen. 2002. Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential ({Delta}{psi}m) in pancreatic RINm5F cells: prevention by Bcl-2. Mol. Cell. Endocrinol. 190:75.[Medline]
  25. Raza, S. M., F. F. Lang, B. B. Aggarwal, G. N. Fuller, D. M. Wildrick, R. Sawaya. 2002. Necrosis and glioblastoma: a friend or a foe: a review and a hypothesis. Neurosurgery 51:2.[Medline]
  26. Suk, K., I. Chang, Y. H. Kim, S. Kim, J. Y. Kim, H. Kim, M. S. Lee. 2001. Interferon {gamma} and tumor necrosis factor {alpha} synergism in ME-180 cervical cancer cell apoptosis and necrosis: IFN{gamma} inhibits cytoprotective NF-{kappa}B through STAT1/IRF-1 pathways. J. Biol. Chem. 276:13153.[Abstract/Free Full Text]
  27. Wang, S. H., J. D. Bretz, E. Phelps, E. Mezosi, P. L. Arscott, S. Utsugi, J. R. Baker, Jr. 2002. A unique combination of inflammatory cytokines enhances apoptosis of thyroid follicular cells and transforms nondestructive to destructive thyroiditis in experimental autoimmune thyroiditis. J. Immunol. 168:2470.[Abstract/Free Full Text]
  28. Jimenez, S. A., B. Freundlich, J. Rosenbloom. 1984. Selective inhibition of human diploid fibroblast collagen synthesis by interferons. J. Clin. Invest. 74:1112.
  29. Duncan, M. R., B. Berman. 1985. {gamma} interferon is the lymphokine and {beta} interferon the monokine responsible for inhibition of fibroblast collagen production and late but not early fibroblast proliferation. J. Exp. Med. 162:516.[Abstract/Free Full Text]
  30. Oldroyd, S. D., G. L. Thomas, G. Gabbiani, A. M. El Nahas. 1999. Interferon-{gamma} inhibits experimental renal fibrosis. Kidney Int. 56:2116.[Medline]
  31. Hogaboam, C., C. Bone-Larson, S. Lipinski, N. Lukacs, S. Chensue, R. Strieter, S. Kunkel. 1999. Differential monocyte chemoattractant protein-1 and chemokine receptor 2 expression by murine lung fibroblasts derived from Th-1 and Th-2 type pulmonary granuloma models. J. Immunol. 163:2193.[Abstract/Free Full Text]
  32. Lloyd, C. M., A. W. Minto, M. E. Dorf, A. Proudfoot, T. N. Wells, D. J. Salant, J. C. Gutierrez-Ramos. 1997. RANTES and monocyte chemoattractant protein-1 (MCP-1) play an important role in the inflammatory phase of crescentic nephritis, but only MCP-1 is involved in crescent formation and interstitial fibrosis. J. Exp. Med. 185:1371.[Abstract/Free Full Text]
  33. Dibbert, B., M. Weber, W. H. Nikolaizik, P. Vogt, M. H. Schoni, K. Blaser, H. U. Simon. 1999. Cytokine-mediated Bax deficiency and consequent delayed neutrophil apoptosis: a general mechanism to accumulate effector cells in inflammation. Proc. Natl. Acad. Sci. USA 96:13330.[Abstract/Free Full Text]
  34. Liles, W., P. Kiener, J. Ledbetter, A. Aruffo, S. Klebanoff. 1996. Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: implications for the regulation of apoptosis in neutrophils. J. Exp. Med. 184:429.[Abstract/Free Full Text]
  35. Henson, P. M., R. B. Johnston. 1988. Tissue injury in inflammation: oxidants, proteinases and cationic proteins. J. Clin. Invest. 79:669.
  36. Fadok, V. A., D. L. Bratton, L. Guthrie, P. M. Henson. 2001. Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases. J. Immunol. 166:6847.[Abstract/Free Full Text]
  37. Gao, Y., J. M. Herndon, H. Zhang, T. S. Griffith, T. A. Ferguson. 1998. Antiinflammatory effects of CD95 ligand (FasL)-induced apoptosis. J. Exp. Med. 188:887.[Abstract/Free Full Text]
  38. Bzowska, M., K. Guzik, K. Barczyk, M. Ernst, H. D. Flad, J. Pryjma. 2002. Increased IL-10 production during spontaneous apoptosis of monocytes. Eur. J. Immunol. 32:2011.[Medline]
  39. Savill, J., V. Fadok. 2000. Corpse clearance defines the meaning of cell death. Nature 407:784.[Medline]
  40. Sun, D., Y. Qin, J. Chluba, J. T. Epplen, H. Wekerle. 1988. Suppression of experimentally induced autoimmune encephalomyelitis by cytolytic T-T cell interactions. Nature 332:843.[Medline]
  41. Jiang, H., L. Chess. 2000. The specific regulation of immune responses by CD8+ T cells restricted by the MHC class Ib molecule, Qa-1. Annu. Rev. Immunol. 18:185.[Medline]
  42. Jiang, H., N. S. Braunstein, B. Yu, R. Winchester, L. Chess. 2001. CD8+ T cells control the TH phenotype of MBP-reactive CD4+ T cells in EAE mice. Proc. Natl. Acad. Sci. USA 98:6301.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
Y. Fang, G. C. Sharp, and H. Braley-Mullen
Interleukin-10 Promotes Resolution of Granulomatous Experimental Autoimmune Thyroiditis
Am. J. Pathol., June 1, 2008; 172(6): 1591 - 1602.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
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]

Home page
 Am. J. Pathol.Home page
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]

Home page
 J. Leukoc. Biol.Home page
K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen
Decreasing TNF-{alpha} results in less fibrosis and earlier resolution of granulomatous experimental autoimmune thyroiditis
J. Leukoc. Biol., January 1, 2007; 81(1): 306 - 314.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
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]

Home page
 J. Leukoc. Biol.Home page
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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, K.
Right arrow Articles by Braley-Mullen, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, K.
Right arrow Articles by Braley-Mullen, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS