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Endocrinology, doi:10.1210/en.2007-0939
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Endocrinology Vol. 148, No. 12 5734-5745
Copyright © 2007 by The Endocrine Society

Expression of Transgenic FLIP on Thyroid Epithelial Cells Inhibits Induction and Promotes Resolution of Granulomatous Experimental Autoimmune Thyroiditis in CBA/J Mice

Yujiang Fang, Vincent G. DeMarco, Gordon C. Sharp and Helen Braley-Mullen

Departments of Internal Medicine (Y.F., G.C.S., H.B.-M.), of Child Health (V.G.D.), of Pathology (G.C.S.), and of Molecular Microbiology and Immunology (H.B.-M.), School of Medicine, University of Missouri, and Veterans Affairs Research Service (H.B.-M.), Department of Veterans Affairs Medical Center, Columbia, Missouri 65212

Address all correspondence and requests for reprints to: Dr. Helen Braley-Mullen, Division of Immunology and Rheumatology, Department of Medicine, University of Missouri, NE307 Medical Sciences, Columbia, Missouri 65212. E-mail: mullenh{at}health.missouri.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by transfer of thyroglobulin-primed in vitro activated splenocytes. Thyroid lesions reach maximal severity 20 d later, and inflammation resolves or progresses to fibrosis by d 60, depending on the extent of thyroid damage at d 20. Depletion of CD8+ T cells inhibits G-EAT resolution. We showed that expression of Fas-associated death domain-like IL-1ß-converting enzyme inhibitory protein (FLIP) transgene (Tg) on thyroid epithelial cells (TECs) of DBA/1 mice had no effect on G-EAT induction but promoted earlier resolution of G-EAT. However, when CBA/J wild-type donor cells were transferred to transgenic CBA/J mice expressing FLIP on TECs, they developed less severe G-EAT than FLIP Tg– littermates. Both strains expressed similar levels of the FLIP Tg, but endogenous FLIP was up-regulated to a greater extent on infiltrating T cells during G-EAT development in DBA/1 compared with CBA/J mice. After transient depletion of CD8+ T cells, FLIP Tg+ and Tg– CBA/J recipients both developed severe G-EAT at d 20. Thyroid lesions in CD8-depleted Tg+ recipients were resolving by d 60, whereas lesions in Tg– littermates did not resolve, and most were fibrotic. FLIP Tg+ recipients had increased apoptosis of CD3+ T cells compared with Tg– recipients. The results indicate that transgenic FLIP expressed on TECs in CBA/J mice promotes G-EAT resolution, but induction of G-EAT is inhibited unless CD8+ T cells are transiently depleted.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXPERIMENTAL autoimmune thyroiditis (EAT) is an organ-specific autoimmune disease that can be induced in genetically susceptible strains of mice by injection of mouse thyroglobulin (MTG) and adjuvant (1, 2), or by transfer of MTG-primed donor spleen cells activated with MTG in vitro (2, 3, 4). A severe and histologically distinct granulomatous form of EAT (G-EAT) is induced when MTG-sensitized donor spleen cells are activated in vitro with MTG and IL-12 (5, 6). Thyroid lesions in G-EAT are characterized by infiltration of inflammatory cells (ICs), including T cells, plasma cells, and neutrophils, and destruction of thyroid epithelial cells (TECs) (5, 6, 7, 8). DBA/1 and CBA/J mice, used in most G-EAT experiments in our laboratory, develop severe G-EAT when donor cells are activated with MTG and IL-12 (3, 5, 6, 7, 8, 9). In both DBA/1 and CBA/J mice, thyroid lesions reach maximal severity 20 d after cell transfer, and inflammation either resolves or progresses to fibrosis at d 60, depending on the extent of damage at d 20 (7, 9). In general, DBA/1 recipients develop very severe (5+ severity scores) thyroid lesions at d 20, with few or no intact follicles remaining, and there is ongoing inflammation and fibrosis 60 d after cell transfer (3, 5, 6, 7, 8, 9). CBA/J recipients also develop very severe G-EAT at d 20, but there are usually some intact thyroid follicles, less neutrophil infiltration, and less fibrosis compared with lesions in DBA/1 mice, and thyroid lesions in CBA/J mice usually spontaneously resolve by 60 d after cell transfer (10). CD4+ T cells are the primary effector cells for G-EAT (5). Depletion of CD8+ T cells inhibits G-EAT resolution (5), and thyroids of CD8-depleted CBA/J recipients, like DBA/1 recipients, have fibrosis and ongoing inflammation at d 60 (7, 10, 11).

The Fas/Fas ligand (FasL) apoptotic pathway plays an important role in many human and murine autoimmune diseases, including Graves’ disease, Hashimoto’s thyroiditis, and EAT or G-EAT in mice (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31). Our previous studies showed that resolution of G-EAT involved apoptosis of CD4+ effector cells mediated, at least in part, through the Fas/FasL pathway by FasL expressing thyrocytes (25, 26, 32). The antiapoptotic molecule Fas-associated death domain-like IL-1ß-converting enzyme inhibitory protein (FLIP) inhibits Fas-mediated apoptosis by blocking activation of caspase-8 (28, 29). Therefore, cellular FLIP (cFLIP) is an important inhibitor of the initial upstream steps of Fas-mediated apoptosis (28). cFLIP expression was increased in lymphocytes of patients with active multiple sclerosis (15, 29), in Graves’ disease (13, 14), in T cells in animal models of inflammatory arthritis (29), and in G-EAT (26). The Fas/FasL pathway can function to induce autoimmune damage (19, 20, 21, 22) and also to reduce autoimmune responses (17, 18, 25). Recently, the long form of cFLIP (cFLIPL) was found to divert Fas signals toward pathways leading to cell growth and differentiation (31). These studies indicate that the function of cFLIP is complex, and more studies are required to elucidate its role in regulating autoimmune inflammatory responses.

We previously reported that FLIP was expressed primarily by FasL+ thyrocytes in thyroids of mice with G-EAT before resolution of inflammation (26). We postulated that expression of FLIP on thyrocytes "protected" them from apoptosis, so that the FasL-expressing thyrocytes could induce apoptosis of inflammatory CD4+ T cells, leading to G-EAT resolution. We hypothesized that transgenic (Tg) overexpression of FLIP by TECs would promote earlier resolution of G-EAT (26, 32). In support of this hypothesis, recipient DBA/1 mice expressing Tg FLIP on TECs and their non-Tg littermates both developed very severe (5+) G-EAT 20 d after cell transfer, but G-EAT resolved earlier in FLIP Tg+ compared with non-Tg littermate recipients (32).

CBA/J mice are another G-EAT susceptible strain extensively used in our adoptive transfer G-EAT model. They differ from DBA/1 in that thyroid lesions 20 d after cell transfer are generally slightly less severe (4+), and lesions resolve earlier than in DBA/1 mice (7, 10). Therefore, CBA/J mice expressing Tg FLIP on TECs were generated to define further the role of FLIP on TECs in the development and resolution of G-EAT. Surprisingly, when Tg CBA/J mice expressing FLIP on TECs were used as recipients, FLIP Tg+ CBA/J mice developed less severe G-EAT at d 20 compared with their Tg– littermates. The resistance of FLIP Tg+ CBA/J mice to the development of G-EAT was reversed after transient depletion of CD8+ T cells, with similar G-EAT severity scores in Tg+ and Tg– recipients at d 20. As in DBA/1 mice, lesions resolved at d 60 in FLIP Tg+ but not in Tg– recipients. A major goal of this study was to determine if differences in expression of endogenous or Tg FLIP on TECs or ICs in CBA/J vs. DBA/1 mice might explain the different effect of Tg FLIP on G-EAT induction in the two strains. Comparison of endogenous FLIP expression on ICs in the thyroid suggests that relatively higher expression of FLIP on thyroid infiltrating ICs in FLIP Tg+ DBA/1 mice compared with Tg+ CBA/J mice may contribute, at least in part, to the different susceptibility to G-EAT induction in the two FLIP Tg mouse strains.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of cFLIPL Tg CBA/J mice
CBA/J FLIP Tg+ mice were generated using the recombinant construct containing rat thyroglobulin promoter and Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG)-tagged mouse cFLIPL cDNA described previously (32). The construct was microinjected into fertilized CBA/J oocytes (Transgenic Core Facility, University of Missouri, Columbia, MO). A Tg male founder was crossed with CBA/J females, and offspring were screened by PCR of tail DNA using primers specific to the rat thyroglobulin promoter and cFLIPL recombined construct as previously described (32). Tg+ and Tg– littermates were further crossed to generate the Tg+ and Tg– littermates used for these experiments. Immunohistochemistry (IHC) using rabbit anti-FLIP polyclonal Ab (Abcam) and anti-FLAG polyclonal Ab (Abcam), and Western blot using anti-FLIP mAb (Alexis) and anti-FLAG Ab were performed to confirm expression of the transgene on TECs, but not other cell types (32).

Induction of G-EAT
Wild-type CBA/J and DBA/1 mice, generated in our breeding colony, were used as donors for all experiments. They were injected iv twice at 10-d intervals with 150 µg MTG prepared as previously described (3) and 15 µg lipopolysaccharide (Sigma-Aldrich, St. Louis, MO). One week later, donor spleen cells were activated in vitro with 25 µg/ml MTG and 5 ng/ml IL-12 (PeproTech, Inc., Rocky Hill, NJ), as previously described (6). After 72 h, cells were harvested, and 3 x 107 cells were transferred iv to 500-Rad irradiated syngeneic FLIP Tg+ or Tg– littermate recipients. Recipient thyroids were evaluated 20 and 60 d after cell transfer, corresponding to the time of maximal disease and its resolution, respectively (6, 25). All mice were bred and maintained in accordance with University of Missouri institutional guidelines for animal care. In most experiments, CBA/J recipients were given 300 µg anti-CD8 mAb (HB-129; American Type Culture Collection, Manassas, VA), ip 1 d after cell transfer (10).

Evaluation of G-EAT histopathology and fibrosis
Thyroids were harvested from groups of six to eight FLIP Tg+ or Tg– recipients 20 or 60 d after cell transfer. One lobe of each thyroid was fixed in formalin, and paraffin sections were stained with hematoxylin and eosin. Thyroids were scored quantitatively for G-EAT severity, defined as the extent of thyroid follicle destruction, using a scale of 1+ to 5+, as previously described in detail (5, 6). Briefly, 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 more than 50% of the gland is destroyed, and 5+ indicates virtually complete destruction of the gland, with few or no remaining follicles. Thyroids had numerous histocytes, and increased neutrophils in addition to mononuclear cells (primarily T cells and macrophages). For evaluation of collagen deposition, thyroid sections from some FLIP Tg– and Tg+ recipients were stained using Masson trichrome (33). All slides were evaluated by at least two investigators, one of whom had no knowledge of the experimental groups. Differences in interpretation were very rare.

IHC
IHC staining for FLIP, FLAG, CD4, and CD8 was described previously (11, 32). Briefly, frozen thyroid sections were fixed in acetone for 10 min at 4 C. After treatment with 0.1% saponin in 1% BSA (FLIP, FLAG) or 1% BSA alone (CD4, CD8) for 30 min, slides were incubated with anti-FLIP, anti-FLAG, rat anti-CD4 (GK1.5; American Type Culture Collection), or rat anti-CD8 mAb (YTS156; American Type Culture Collection) 60 min at room temperature. After incubation with a secondary biotinylated Ab (Jackson ImmunoResearch, West Grove, PA), immunoreactivity was demonstrated using the avidin-biotin complex immunoperoxidase system (Vector Laboratories, Burlingame, CA) and developed using NovaRED (Vector). Slides were counterstained with hematoxylin. As a negative control, primary Ab was replaced with an equal amount of normal rabbit, goat, or rat IgG, and controls were always negative.

Confocal laser scanning double-immunofluorescence microscopy
To detect differential expression of FLIP, FasL, active caspase-8, and active caspase-3 by CD4+, CD8+ T cells, or TECs, dual-color immunofluorescence and confocal laser scanning microscopy was performed as previously described (8, 32) using polyclonal anti-FLIP (described previously), anti-FasL (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-active caspase-8 (Imgenex Corp., San Diego, CA), or anti-active caspase-3 (BD PharMingen, San Diego, CA), together with rat anti-CD4, anti-CD8 mAb, anti-CD3 (DakoCytomation, Glostrup, Denmark) for T cells, or pan-cytokeratin (PCK)-26 (Sigma-Aldrich) for TECs. Frozen sections of thyroids were used except for dual staining of FasL and CD3 or PCK, which used formalin-fixed paraffin sections. FasL, FLIP, active caspase-8, and active caspase-3 were visualized with Alexa 568 (red; Molecular Probes, Inc., Eugene, OR). Cytokeratin (TECs), CD3, CD4, and CD8 molecules were visualized by Alexa 488 (green; Molecular Probes). Apoptosis was detected using an in situ cell death kit (red; Roche, Indianapolis, IN) with formalin-fixed paraffin sections of thyroids as previously described (32). Slides were observed with a Bio-Rad Radiance 2000 confocal system (Bio-Rad Laboratories, Hercules, CA) coupled to an Olympus IX70 inverted microscope (Hamburg, Germany). To quantify the number of double-positive cells, double-positive cells (yellow, overlay, or red circled by green) in five to six randomly selected high-power fields were manually counted, and the average number of cells per field for three individual animals per group was determined.

Isolation of infiltrating cells from inflammatory thyroids
Immediately after dissection, thyroids were incubated overnight at 4 C in Eagle’s MEM, containing 1.2 U/ml dispase II (Roche) and 0.25 U/ml collagenase I (Sigma-Aldrich). After digestion at 37 C for 30 min and shaking every 5 min, cells were washed, and ICs were isolated from the thyroid cell suspensions using phycoerythrin-labeled anti-CD45 mAb (eBioscience, San Diego, CA) and a positive selection EasySep PE Selection Kit (Stem Cell Technologies, Inc., Tukwila, WA) according to the manufacturer’s instructions. TRIZOL (Invitrogen Corp., Carlsbad, CA) was used to extract RNA from the isolated ICs and the unbound fraction (mainly TECs) for RT-PCR or real-time PCR analysis as described below. Thyroglobulin primers were used to assess the extent of contamination of IC fractions by TECs. Primer sequences for thyroglobulin are: sense, 5'-CCTGGTCTTGTGGGTCTCTA-3'; and antisense, 5'-GAGGAAGGTAGAGAGCATCG-3'.

RT-PCR
Thyroid lobes from individual mice were harvested 20 d after adoptive transfer, and one lobe was stored at –80 C before processing. Frozen thyroids were homogenized in TRIZOL, and RNA was extracted and reverse transcribed as previously described (32, 34, 35). To determine the relative initial amounts of target cDNA, each cDNA sample was serially diluted one fifth and one twenty-fifth. Hypoxanthine phosphoribosyltransferase (HPRT) was used as a housekeeping gene to verify that the same amount of RNA was amplified. PCR products were electrophoresed in 2% agarose gel, visualized by UV light after staining with ethidium bromide, and normalized between samples relative to levels of HPRT using an IS-1000 Digital Imaging System (Life Sciences, St. Louis, MO). Primer sequences were described previously (32, 34, 35).

Real-time PCR
FLIP mRNA was also quantified by real-time PCR using ABI 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Amplification was performed for 40 cycles in a total volume of 30 µl, and products were detected using SYBR Green (Abgene, Epsom, UK). Each sample was run in triplicate, and relative expression levels were determined by normalizing expression of each target to HPRT. Expression levels of normalized samples are expressed as relative expression units. Primers for FLIP are: sense, 5'-AGCAACCGTGGAGGACCA-3'; and antisense, 5'-CCATCAGCAGGACCCTATAATCA-3'.

Western blot
Western blot was performed as previously described (32) using 30 µg protein loaded to a 10% SDS-PAGE gel.

Dot blot analysis
To compare the level of FLIP transgene in FLIP Tg+ CBA/J and DBA/1 mice, dot blot analysis was performed as previously described (32).

Statistical analysis
All experiments were repeated at least twice. Statistical analysis of data was performed using an unpaired two-tailed Student’s t test or the Mann-Whitney U rank sum test. A P value less than or equal to 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of Tg CBA/J mice expressing murine cFLIPL on TECs
Our recent work showed that Tg FLIP expressed on TECs promoted early resolution of G-EAT but had no effect on the development of G-EAT in DBA/1 mice (32). Tg CBA/J mice expressing mouse cFLIPL on TECs were generated using the same construct. FLIP and FLAG were both highly expressed on TECs in the founder and in all CBA/J Tg+ mice, but not in other tissues such as spleen and cervical lymph nodes (data not shown). Some mice were typed for FLIP transgene expression by both PCR analysis of tail DNA and dot blot analysis, and results using both assays were consistent. Dot blot indicated that the FLIP transgene level in naive CBA/J FLIP Tg+ mice was over five times higher than in Tg– littermates (see below). Neither FLIP nor FLAG was detected in thyroids of naive Tg– mice by immunohistochemical staining (data not shown).

Tg FLIP expressed on TECs inhibits the development of G-EAT in CBA/J FLIP Tg+ recipients
When G-EAT was induced by adoptive transfer of MTG-sensitized DBA/1 splenocytes activated with MTG and IL-12 in vitro, thyroid lesions in both FLIP Tg+ and Tg– littermate recipients reached maximal severity 20 d after cell transfer, and G-EAT severity scores were similar (4–5+) for both Tg+ and Tg– recipients (32). Unexpectedly, when similar experiments were done using CBA/J mice as donors, most CBA/J Tg+ recipients developed less severe G-EAT than CBA/J Tg– littermates (average severity score 1.4 ± 0.4 vs. 3.5 ± 0.2; P < 0.05; Fig. 1Go), suggesting that expression of Tg FLIP on TECs inhibits the development of G-EAT in Tg+ CBA/J mice, but not in DBA/1 mice. Although most Tg+ recipients did not develop G-EAT, a few Tg+ recipients developed moderately severe G-EAT (Fig. 1Go). This was not apparently due to differences in expression of the FLIP transgene in different mice because FLAG staining was of comparable intensity in Tg+ thyroids with different severity scores (data not shown).


Figure 1
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  		FIG. 1. Expression of Tg FLIP on TECs inhibits development of G-EAT in CBA/J FLIP Tg+ recipients. G-EAT severity scores of individual Tg+ and Tg– recipients 20 d after transfer of MTG-activated splenocytes was significantly decreased compared with those in Tg– recipients (average severity score 1.4 ± 0.4 vs. 3.5 ± 0.2). *, P < 0.05. Results are pooled from two experiments and are representative of four independent experiments.

 
Comparison of Tg FLIP expression in thyroids of naive CBA/J and DBA/1 FLIP Tg+ and Tg– mice
Differences in expression levels of the FLIP transgene could explain why FLIP Tg+ DBA/1 mice develop G-EAT similar to that of Tg– mice, whereas FLIP Tg+ CBA/J mice are relatively more resistant to G-EAT compared with their Tg– littermates. To address this possibility, FLIP genomic DNA and FLIP mRNA and protein levels in thyroids of naive CBA/J and DBA/1 FLIP Tg+ mice were compared. Dot blot analysis of tail DNA showed that FLIP genomic DNA levels in Tg+ CBA/J and DBA/1 mice were similar (P > 0.05), and were 5- to 6-fold higher than in their respective Tg– littermates (P < 0.05; Fig. 2AGo). FLIP mRNA was undetectable in thyroids of naive CBA/J and DBA/1 Tg– mice, and was highly and comparably expressed in thyroids of naive CBA/J and DBA/1 Tg+ mice by both semiquantitative RT-PCR (Fig. 2BGo; P > 0.05), and quantitative real-time PCR (Fig. 2CGo; P > 0.05). FLIP protein was undetectable in thyroids of naive Tg– CBA/J (data not shown) and DBA/1 Tg– mice (32), and was comparably expressed in thyroids of naive CBA/J and DBA/1 Tg+ mice (Fig. 2DGo). Therefore, differences in susceptibility to G-EAT induction in Tg+ CBA/J and DBA/1 mice are not explained by differences in expression of the FLIP transgene on TECs.


Figure 2
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  		FIG. 2. Comparison of Tg FLIP expression in thyroids of naive CBA/J and DBA/1 FLIP Tg+ and Tg– mice. A, Dot blot analysis of tail DNA of naive CBA/J and DBA/1 Tg+ mice. Twenty micrograms of DNA were loaded, and 1.5 kb murine FLIP Klenow were used as probe template. ß-Actin was used for data normalization. Bars are means of results from four to six individual naive CBA/J or DBA/1 Tg– mice and 15–17 individual naive CBA/J or DBA/1 Tg+ mice ± SEM. Results are expressed as the mean ratio of densitometric U/ß-actin ± SEM. Dot intensities were expressed in arbitrary relative units to naive Tg– mice. B and C, FLIP mRNA was undetectable in thyroids of naive CBA/J and DBA/1 Tg– mice, and was highly expressed in thyroids of naive CBA/J and DBA/1 Tg+ mice. FLIP mRNA levels of naive CBA/J and DBA/1 Tg+ mice were not significantly different by either RT-PCR (B) or real-time PCR (C). Bars are means of data for thyroids of five to six individual mice ± SEM. Results are expressed as the mean ratio of FLIP densitometric U/HPRT ± SEM (x100) (B) or relative expression levels normalized to HPRT (C). D, FLIP protein levels in thyroids of naive CBA/J and DBA/1 Tg+ mice. Thirty micrograms of protein from thyroids of the CBA/J (four individuals) and DBA/1 Tg+ mice (four individuals) were loaded in each lane, and results for FLIP and ß-actin are shown at the top. Results are expressed as the mean ratio of densitometric U/ß-actin ± SEM (x100) and are representative of three independent experiments.

 
Development of severe G-EAT in CBA/J FLIP Tg+ and Tg– recipients after depletion of CD8+ T cells
CBA/J FLIP Tg+ recipients develop less severe G-EAT than their Tg– littermates (Fig. 1Go). Our previous studies showed that depletion of recipient CD8+ T cells resulted in increased G-EAT severity, and G-EAT resolution was inhibited in both CBA/J and DBA/1 mice (7, 10, 25). To determine if CD8+ T cells might directly or indirectly inhibit G-EAT development in CBA/J Tg+ mice, Tg+ and Tg– recipients of CBA/J donor cells were given a single injection of anti-CD8 mAb to transiently deplete CD8+ T cells in recipient mice during the period of G-EAT development. As shown in Fig. 3Go, G-EAT lesions in both Tg+ and Tg– recipients of CBA/J spleen cells reached maximal severity 20 d after a single injection of anti-CD8 Ab, with an average disease severity score of 4.4 in both Tg+ and Tg– recipients. By d 60, splenic CD8+ T cells in spleens of both Tg+ and Tg– recipients were approximately 50% of those in recipients not given anti-CD8, as determined by flow cytometry (data not shown). Thyroid lesions in most Tg+ recipients resolved (Fig. 3AGo) or had begun to resolve by d 60 (Fig. 3BGo), whereas thyroids in most Tg– littermates had ongoing inflammation and fibrosis, with an average severity score of 4.0–4.5 (Fig. 3Go, A and B). The difference in G-EAT severity scores in Tg+ vs. Tg– CBA/J recipients at d 60 was highly significant (P < 0.001, Fig. 3AGo; P < 0.05, Fig. 3BGo). Collagen deposition in the thyroid, an indicator of fibrosis, was assessed by Masson trichrome staining (Fig. 4Go). Thyroids of Tg+ and Tg– CBA/J recipients had minimal (Tg+) or moderate (Tg–) fibrosis at d 20 (Fig. 4Go, A–D). Fibrosis was more extensive in thyroids of Tg– mice, with extensive collagen deposition (blue) at d 60 (Fig. 4Go, E and G), whereas thyroids of Tg+ recipients had minimal fibrosis (Fig. 4Go, F1 and H1), even when severity scores at d 60 were comparable (4+) to those in Tg– recipients (Fig. 4Go, F2 and H2). Serum T4 was low in all mice, with 4–5+ severity scores at d 60, but was normal in Tg+ recipients, with 0–2+ severity scores at d 60 (data not shown). These results are consistent with those shown for DBA/1 mice in our earlier study (32), i.e. thyroid lesions resolved earlier, and there was less fibrosis in Tg+ compared with Tg– recipients of the same donor cells. However, results in Tg+ CBA/J and DBA/1 mice differed in that the initial development of G-EAT was similar in both Tg+ and Tg– DBA/1 recipients, whereas Tg+ CBA/J recipients developed minimal G-EAT compared with Tg– littermates unless CD8+ T cells were transiently depleted.


Figure 3
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  		FIG. 3. Development of severe G-EAT in CBA/J FLIP Tg+ and Tg– recipients after depletion of CD8+ T cells. G-EAT severity scores at d 20 and 60 of individual Tg+ and Tg– recipients given anti-CD8 1 d after cell transfer. A, G-EAT lesions in Tg+ and Tg– recipients reached similar maximal severity at d 20 (average severity score 4.4). At d 60, thyroid lesions in Tg+ recipients were significantly decreased compared with those in Tg– recipients (average severity score 0.4 vs. 4.0; P < 0.001). B, In another experiment, G-EAT lesions in Tg+ and Tg– recipients also had similar maximal severity scores at d 20 (average severity score 4.6). By d 60, thyroid lesions in most Tg+ recipients had resolved or started to resolve, whereas thyroids in Tg– recipients had ongoing inflammation and fibrosis (average severity score 2.9 vs. 4.5, respectively; P < 0.05). A significant difference is indicated by the asterisk. Results are representative of four independent experiments.

 

Figure 4
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  		FIG. 4. Collagen deposition (fibrosis) in thyroids of FLIP Tg+ and Tg– recipients. A–D, Masson trichrome staining for collagen (blue) in thyroids of FLIP Tg+ and Tg– recipients at d 20 (A–D) and 60 (E–H). There was moderate fibrosis in thyroids of Tg– recipients (A and C) and minimal fibrosis in thyroids of Tg+ CBA/J recipients (B and D) at d 20. By d 60, there was extensive collagen deposition (blue) in thyroids of Tg– mice (E and G), but not in thyroids of Tg+ recipients (F1 and H1), even when severity scores at d 60 were comparable (4–5+) to those in Tg– recipients (F2 and H2). A, B, E, and F magnification, x100. C, D, G, and H magnification, x400.

 
DBA/1 and CBA/J mice differ in their ability to up-regulate endogenous FLIP on ICs during the development of G-EAT
Endogenous FLIP is up-regulated on both TECs and thyroid-infiltrating ICs during G-EAT development (25, 26). Although expression of the FLIP transgene was comparable in thyroids of naive CBA/J and DBA/1 mice (Fig. 2Go), the greater resistance of CBA/J FLIP Tg+ mice to the development of G-EAT could be explained, at least in part, if endogenous FLIP was differentially up-regulated in thyroids of Tg+ DBA/1 and CBA/J mice during G-EAT development. To address this possibility, FLIP expression levels in thyroids of CBA/J and DBA/1 Tg+ or Tg– recipients of activated effector cells were compared 20 d after cell transfer. FLIP mRNA expression increased in thyroids of both strains 20 d after cell transfer (Fig. 5AGo). However, FLIP mRNA expression was always lower in thyroids of both Tg+ and Tg– CBA/J recipients with G-EAT compared with Tg+ and Tg– DBA/1 recipients with G-EAT (P < 0.05). This difference was not due solely to the fact that CD8+ T cells were depleted in thyroids of Tg+ CBA/J mice because thyroids of both CD8-depleted and untreated Tg+ CBA/J mice with 4+ severity scores (Fig. 1Go) had comparable levels of FLIP, and both were lower than in thyroids of Tg+ DBA/1 mice (Fig. 5AGo). FLIP protein was also lower in thyroids of CBA/J Tg+ or Tg– recipients with G-EAT than in DBA/1 Tg+ or Tg– recipients, respectively (P < 0.05; Fig. 5BGo). Therefore, even though expression of the FLIP transgene was comparable in both strains, endogenous FLIP was up-regulated to a greater extent in thyroids of both Tg+ and Tg– DBA/1 mice compared with CBA/J mice.


Figure 5
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  		FIG. 5. Thyroids of DBA/1 and CBA/J mice differ in their ability to up-regulate endogenous FLIP after G-EAT induction. FLIP expression levels in thyroids of CBA/J and DBA/1 Tg+ or Tg– recipients 20 d after cell transfer. A, By real-time PCR, FLIP mRNA expression in thyroids with G-EAT increased in both strains (Fig. 5AGo vs. Fig. 2CGo). FLIP mRNA expression was always lower in thyroids of both Tg+ and Tg– CBA/J recipients with G-EAT than in Tg+ or Tg– DBA/1 recipients with G-EAT (P < 0.05). FLIP expression levels were not altered after depletion of CD8+ T cells because Tg+ CBA/J thyroids with 4+ severity scores and CD8-depleted Tg+ thyroids with 4+ severity scores both had comparable FLIP expression. B, By Western blot, FLIP protein expression was also lower in thyroids of CBA/J Tg+ or Tg– recipients with G-EAT than in DBA/1 Tg+ or Tg– recipients (P < 0.05). Thirty micrograms of protein from thyroids of the CBA/J Tg+ or Tg– (three individuals) and DBA/1 Tg+ or Tg– mice (four individuals) were loaded in each lane, and results for FLIP and ß-actin are shown at the top. Results are expressed as the mean ratio of densitometric U/ß-actin ± SEM (x100), and are representative of three independent experiments. A significant difference between Tg+ CBA/J (with or without CD8-depletion) and DBA/1 is indicated by the asterisk, and a significant difference between Tg– CBA/J and DBA/1 is indicated by the club.

 
FLIP is expressed by both TECs and ICs in thyroids of recipient mice with G-EAT (25, 26, 32), whereas in naive FLIP Tg+ mice, FLIP is expressed only on TECs. Therefore, the relatively lower expression of FLIP in thyroids of CBA/J Tg+ compared with DBA/1 Tg+ recipients could be due to decreased expression of endogenous FLIP on infiltrating ICs in CBA/J compared with DBA/1 mice. To address this possibility, thyroid-infiltrating ICs were isolated from thyroids of Tg+ DBA/1 and CD8-depleted Tg+ CBA/J mice with 4–5+ G-EAT severity scores, as described in Materials and Methods. ICs were isolated using magnetic beads, cells were frozen, and mRNA was isolated to assess the relative levels of FLIP mRNA in IC vs. TEC fractions. Cells positively selected by CD45+ magnetic beads (mainly ICs) highly expressed CD4 message (Fig. 6AGo), and expression of CD4 message was similar for both Tg+ CD8-depleted CBA/J and DBA/1 groups. Both IC fractions expressed minimal thyroglobulin message (Fig. 6BGo), suggesting minimal contamination of ICs by TECs. In contrast, cells not selected by CD45+ magnetic beads (mainly TECs) highly expressed thyroglobulin message (Fig. 6BGo), but little CD4 message (Fig. 6AGo). By both RT-PCR (Fig. 6CGo) and real-time PCR (Fig. 6DGo), ICs isolated from thyroids of DBA/1 Tg+ mice expressed significantly more FLIP than those from CD8-depleted CBA/J Tg+ mice (P < 0.05). When FLIP message was expressed as a ratio of FLIP to CD4, DBA/1 ICs also expressed more FLIP compared with CBA/J ICs (Fig. 6EGo). These results are consistent with the idea that decreased expression of FLIP by ICs in Tg+ CBA/J thyroids may lead to greater sensitivity to apoptosis compared with ICs in Tg+ DBA/1 thyroids, and this might, at least in part, explain the greater resistance of CBA/J mice to G-EAT induction.


Figure 6
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  		FIG. 6. Thyroid infiltrating ICs in DBA/1 and CBA/J mice differ in their ability to up-regulate FLIP. mRNA analysis of ICs and TECs isolated from thyroids of Tg+ DBA/1 and CBA/J mice with 4–5+ G-EAT severity scores at d 20 as described in Materials and Methods (Tg+ CBA/J mice were CD8-depleted to induce 4–5+ disease). A and B, Cells that were positively selected by CD45+ magnetic beads (mainly ICs) highly expressed CD4 but not thyroglobulin message, and expression of CD4 and thyroglobulin was comparable for CD8-depleted Tg+ CBA/J and DBA/1 groups. The unbound cells (mainly TECs) highly expressed thyroglobulin but not CD4 message, and expression of CD4 and thyroglobulin was comparable in both groups. C and D, RT-PCR (C) and real-time PCR (D) showed that ICs isolated from thyroids of DBA/1 Tg+ mice expressed more FLIP than those from thyroids of CD8-depleted CBA/J Tg+ mice, with comparable G-EAT severity scores, whereas TEC fractions from both strains expressed comparable levels of FLIP. E, When FLIP message was expressed as a ratio of FLIP to CD4, Tg+ DBA/1 ICs also expressed more FLIP compared with Tg+ CBA/J ICs. Results are representative of two independent experiments. A significant difference between G-EAT CD8-depleted Tg+ CBA/J and Tg+ DBA/1 is indicated by the asterisk.

 
CD8+ T cells that repopulate recipients after transient depletion of CD8+ T cells are comparable in spleens of both Tg+ and Tg– recipients, but increased in thyroids of Tg+ recipients at d 60
Our previous studies indicated that earlier resolution of G-EAT was associated with decreased numbers of CD4+ T cells and increased numbers of CD8+ T cells in thyroids of DBA/1 FLIP Tg+ vs. Tg– recipients (32). Because it was necessary to transiently deplete CD8+ T cells for severe G-EAT to develop in FLIP Tg+ CBA/J recipients, it was important to compare the numbers of CD4+ and CD8+ T cells in spleens and thyroids of Tg+ vs. Tg– recipients at d 20 and 60. CD4+ T cells (red) in thyroids of Tg– recipients outnumbered those in Tg+ recipients at d 20 (Fig. 7Go, A and B) and 60 (Fig. 7Go, G and H), whereas CD4+ T cells in spleens of Tg– and Tg+ recipients were comparable both by IHC (Fig. 7Go, E and K) and flow cytometry (data not shown). Because all mice were given anti-CD8 the day after cell transfer, there were very few CD8+ T cells in thyroids or spleens of either group at d 20 (Fig. 7Go, C, D, and F). At d 60, CD8+ T cells were partially repopulated, and splenic CD8+ T cells were comparable in both Tg+ and Tg– recipients both by IHC (Fig. 7LGo) and flow cytometry (data not shown). However, many more CD8+ T cells were present in thyroids of Tg+ recipients (Fig. 7Go, I and J, right panels) than in thyroids of Tg– recipients (Fig. 7Go, I and J, left panels), indicating that CD8+ T cells were preferentially recruited to the thyroid in Tg+ compared with Tg– recipients. CD4+ and CD8+ T cells in thyroids and spleens were assessed semiquantitively by counting five to six randomly selected high-power fields on slides of three mice in each group with comparable 4–5+ G-EAT severity scores (Fig. 7Go, M and N). The results indicate that the ratio of CD4+ vs. CD8+ T cells in G-EAT thyroids was a good predictor of the outcome of the autoimmune inflammatory response, i.e. thyroids of Tg+ recipients with earlier resolution had fewer CD4+ T cells and more CD8+ T cells compared with thyroids of Tg– recipients.


Figure 7
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  		FIG. 7. CD8+ T cells that repopulate recipients after transient depletion of CD8+ T cells are comparable in spleens of both Tg+ and Tg– recipients but increased in thyroids of Tg+ recipients at d 60. A–F, CD4+ and CD8+ T cells in thyroids (A–D) and spleens (E and F) at d 20. CD4+ T cells (red) in thyroids of Tg– (A and B, left panels) outnumbered those in Tg+ recipients (A and B, right panels), and there were few CD8+ T cells (red) in thyroids due to anti-CD8 treatment (C and D). CD4+ T cells were comparable in spleens of Tg– (E, left panel) and Tg+ recipients (E, right panel), and there were very few CD8+ T cells (F). G–L, CD4+ and CD8+ T cells in thyroids (G–J) and spleens (K and L) at d 60. CD4+ T cells in thyroids of Tg– (G and H, left panels) outnumbered those in Tg+ recipients (G and H, right panels), and CD8+ T cells in thyroids of Tg+ recipients (I and J, right panels) outnumbered those in Tg– recipients (I and J, left panels). Comparable numbers of both CD4+ (K) and CD8+ (L) T cells were present in spleens of both groups. B, D, H, and J magnification, x400. Other magnifications, x100. Mice in both groups had comparable severity scores (4–5+). M and N, Numbers of CD4+ and CD8+ T cells in thyroids or spleens at d 20 (M) or 60 (N) were assessed semiquantitively by counting five to six randomly selected high-power fields on slides of three mice in each group. A significant difference between G-EAT Tg– and Tg+ recipients is indicated by the asterisk (P < 0.05).

 
CD3+ T cells in CBA/J FLIP Tg+ recipients undergo increased apoptosis compared with CD3+ T cells in Tg– littermates
Resolution of G-EAT is due, at least in part, to increased apoptosis of CD4+ effector T cells by a FasL-dependent mechanism (9, 25, 26). Tg FLIP on TECs in DBA/1 mice promotes earlier resolution of G-EAT by protecting TECs from apoptosis and inducing increased apoptosis of effector CD4+ T cells through the caspase cascade pathway (32). A fluorescence apoptosis kit was used to determine if apoptosis of T cells was increased in thyroids of CBA/J Tg+ vs. Tg– recipients (Fig. 8Go, A and B). TECs were identified by PCK (green), T cells were identified by CD3 (green), and apoptosis was detected as red nuclear staining. Many apoptotic cells were detected in thyroids of both Tg+ and Tg– recipients with G-EAT 20 d after cell transfer (Fig. 8Go, A and B), whereas only a few apoptotic cells were detected 60 d after cell transfer (data not shown). Apoptosis (red) was mainly in ICs in Tg+ recipients (Fig. 8AGo, right panel) and mainly in TECs in Tg– recipients (Fig. 8BGo, left panel) at d 20. Double-positive cells (red circled by greenin Fig. 8Go, A and B) in four to five randomly selected high-power fields of three individual mice per group were manually counted, and the results are summarized in Fig. 8CGo (bars correspond to Fig. 8Go, A and B). These results indicate that apoptosis of T cells is increased in thyroids of Tg+ compared with Tg– mice.


Figure 8
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  		FIG. 8. More apoptotic CD3+ T cells are present in thyroids of FLIP Tg+ compared with Tg– littermates. A and B, Apoptosis in thyroids of Tg+ and Tg– recipients at d 20. T cells were identified by CD3 (A, green), and TECs by PCK (B, green). Apoptosis was detected as red nuclear staining on paraffin sections. Apoptosis (red) was mainly in ICs in Tg+ recipients (A, right panel, arrows) and mainly in TECs in Tg– recipients (B, left panel, arrows). C, Double-positive cells (red circled by green in A and B) in four to five randomly selected high-power fields of slides of three individual mice per group were manually counted, and the results are summarized (bars correspond to A and B). A significant difference between G-EAT Tg– and Tg+ recipients is indicated by the asterisk (P < 0.05). Magnification, x800.

 
Our previous studies indicate that the outcome of G-EAT correlates with the site of expression of FLIP and FasL (26, 32). In thyroid lesions that do not resolve, e.g. G-EAT thyroids in DBA/1 Tg– mice or CD8-depleted CBA/J Tg– mice, up-regulation of FLIP by ICs contributes to chronic inflammation by blocking apoptosis of FasL+ ICs. In contrast, when FLIP is expressed mainly by FasL+ TECs, there is less apoptosis of TECs and increased apoptosis of CD4+ T cells, resulting in G-EAT resolution (26, 32). In this study confocal microscopy indicated that CBA/J Tg+ and Tg– recipients exhibited a differential expression pattern of FasL and FLIP, i.e. FasL and FLIP were primarily expressed on TECs in CD8-depleted Tg+ recipients with early resolution and on CD4+ ICs in CD8-depleted Tg–recipients with ongoing inflammation (data not shown). These results are consistent with previous results suggesting that expression of FLIP on TECs can promote G-EAT resolution by protecting TECs from Fas/FasL-mediated apoptosis.

Cytokine and Foxp3 mRNA expression in thyroids of CBA/J FLIP Tg+ and Tg– mice
The balance between proinflammatory cytokines such as IFN (interferon)-{gamma} and TNF-{alpha}, and antiinflammatory cytokines such as IL-10 and IL-13 is important for the development and progression of inflammation in autoimmune diseases, including G-EAT (9, 32, 33, 36, 37, 38, 39, 40, 41). Because regulatory T cells can reduce inflammation in many autoimmune diseases, expression of Foxp3, a marker of regulatory T cells (40), was also investigated. As shown previously (32), 20 d after cell transfer, cytokine and Foxp3 mRNA was undetectable in thyroids of normal Tg+ or Tg– mice (Fig. 9Go, A–E). mRNA expression of proinflammatory cytokines such as IFN-{gamma} and TNF-{alpha} was lower (Fig. 9Go, A and B), and mRNA expression of antiinflammatory cytokines such as IL-10 and IL-13 was higher (Fig. 9Go, C and D) in thyroids of CBA/J Tg+ compared with Tg– recipients with similar G-EAT severity scores (4–5+). Foxp3 mRNA was similar in thyroids of Tg+ and Tg– recipients with G-EAT (Fig. 9EGo). These results indicate that expression of major proinflammatory and antiinflammatory cytokines in recipient thyroids correlates with differences in the outcome of G-EAT seen at d 60 in Tg+ vs. Tg– recipients.


Figure 9
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  		FIG. 9. Analysis of cytokine and Foxp3 mRNA expression in thyroids of CBA/J FLIP Tg+ and Tg– recipients at d 20. A–E, mRNA was isolated from thyroid lobes of individual recipient mice 20 d after cell transfer and amplified as described in Materials and Methods. All thyroids had comparable 4–5+ severity scores. IFN-{gamma}, TNF-{alpha}, IL-10, IL-13, and Foxp3 mRNA (A–E) was undetectable in thyroids of normal Tg+ or Tg– mice. IFN-{gamma} and TNF-{alpha} mRNA expression (A and B) was lower, IL-10 and IL-13 mRNA expression (C and D) was higher, and Foxp3 mRNA expression (E) was similar in thyroids of FLIP Tg+ compared with Tg– littermate recipients. Results are expressed as the mean ratio of cytokine densitometric U/HPRT ± SEM (x100) of five to six mice per group, and are representative of three independent experiments. A significant difference between G-EAT FLIP Tg– recipients and Tg+ recipients is indicated by the asterisk (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
FLIP is an important inhibitor of caspase-8 that plays an important antiapoptotic role in many autoimmune diseases (13, 15, 29, 42, 43, 44). Previous studies indicate that Tg expression of FLIP on murine TECs has no effect on thyroid histology or function (32, 45). Expression of Tg FLIP on TECs had no effect on G-EAT induction in DBA/1 recipients of sensitized effector cells, but Tg FLIP promoted earlier resolution of G-EAT compared with that in non-Tg littermates (32). Unexpectedly, when Tg FLIP was expressed on TECs in CBA/J recipients, G-EAT induction was inhibited (Fig. 1Go). Transient depletion of CD8+ T cells resulted in comparable G-EAT severity scores in both Tg+ and Tg– CBA/J recipients, and lesions resolved earlier in Tg+ recipients compared with their Tg– littermates (Fig. 3Go).

The major goal of this study was to determine the basis for the differences in the development of G-EAT in Tg+ CBA/J and DBA/1 mice to define further the role of FLIP in regulation of G-EAT. In general, FLIP Tg– DBA/1 recipients develop very severe (5+ severity scores) thyroid lesions at d 20, with few or no intact follicles remaining, and there is ongoing inflammation and fibrosis at d 60 (3, 5, 6, 7, 8, 9). Although FLIP Tg– CBA/J recipients also develop severe G-EAT at d 20, there are usually some intact thyroid follicles, less neutrophil infiltration compared with lesions in DBA/1 mice, and lesions usually spontaneously resolve by 60 d (9, 10, 25, 26). Thus, the different genetic background of the two strains contributes in part to the difference in G-EAT induction in FLIP Tg+ CBA/J and DBA/1 recipients. The results of this study indicate that expression of the FLIP transgene on TECs was comparable in the two strains (Figs. 2Go and 5Go). Tg FLIP in the two strains was also comparable functionally because cultured TECs from Tg+ CBA/J and DBA/1 mice did not differ in their ability to protect TECs from apoptosis induced in vitro by agonist anti-Fas Ab when TECs were cultured in the presence of IFN-{gamma} and TNF-{alpha} (Fang, Y., and H. Braley-Mullen, unpublished results). However, CBA/J and DBA/1 mice differed in the extent to which endogenous FLIP was up-regulated on ICs during G-EAT induction (Fig. 6Go). Lower expression of FLIP on ICs of CBA/J mice could result in increased sensitivity to apoptosis, resulting in fewer ICs in thyroids. Conversely, increased expression of endogenous FLIP on ICs of DBA/1 mice would tend to favor survival of infiltrating ICs, resulting in more severe G-EAT (Table 1Go). Although other factors may also contribute, the differential up-regulation of endogenous FLIP on ICs in CBA/J vs. DBA/1 mice is consistent with the differences in G-EAT susceptibility of Tg+ CBA/J and DBA/1 mice. The resistance to G-EAT of Tg+ CBA/J mice compared with their Tg– littermates suggests that overexpression of FLIP on TECs of Tg+ mice favors survival of TECs, but not of ICs.


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  		TABLE 1. Summary of characteristics of CBA/J FLIP Tg–/Tg+ vs. DBA/1 FLIP Tg–/Tg+ mice to G-EAT induction, resolution, and fibrosis

 
As shown previously, depletion of CD8+ T cells in CBA/J recipients resulted in slightly higher G-EAT severity scores 20 d after cell transfer, although the primary effect of CD8 depletion in this model is to inhibit G-EAT resolution (25, 26, 46). In this study transient depletion of CD8+ T cells in recipients was sufficient to overcome the relative resistance to G-EAT induction of FLIP Tg+ CBA/J mice (Fig. 3Go vs. Fig. 1Go). When CD8+ T cells repopulated the recipients, as seen 60 d after cell transfer, thyroid lesions in Tg+ recipients resolved or had begun to resolve, whereas those in Tg– recipients had ongoing inflammation and fibrosis. Of particular interest, there were more CD8+ T cells in thyroids of Tg+ vs. Tg– recipients (Fig. 7Go, I and J), even though comparable numbers of CD8+ T cells had reappeared in the spleens of Tg+ and Tg– mice at d 60. Apparently, CD8+ T cells in FLIP Tg+ mice preferentially migrate to the thyroid where they function directly or indirectly to promote apoptosis of CD4+ T cells, leading to resolution of inflammation. These results are consistent with the finding that apoptotic CD3+ T cells (mainly CD4+ T cells since CD8 cells were depleted) were increased in thyroids of Tg+ recipients compared with Tg– recipients (Fig. 8AGo). The increased apoptosis of CD4+ effector T cells (9, 25) presumably contributes to the different outcomes of G-EAT in CBA/J Tg+ and Tg– recipients at d 60 (Fig. 3Go).

CD8+ T cells promote G-EAT induction and resolution, at least in part, by increasing FasL and FLIP expression on TECs and decreasing FasL and FLIP expression on ICs (25, 26, 27, 46). CD8+ T cells also produce IL-10, and G-EAT resolution is inhibited in IL-10 deficient mice (Fang, Y., and H. Braley-Mullen, unpublished results). IL-10 can increase FasL expression on nonlymphoid cells, resulting in apoptosis of T cells and resolution of inflammation (47, 48, 49), and IL-10 has protected TECs from apoptosis (13). During induction of G-EAT, FasL expression on TECs is decreased after transient depletion of CD8+ T cells, there is decreased production of IL-10 in the thyroid (data not shown), and increased expression of FLIP on ICs (27). CD8+ T cells repopulating the thyroid in FLIP Tg+ mice produce IL-10 (Fang, Y., and H. Braley-Mullen, unpublished results), which may facilitate resolution of inflammation (Table 1Go).

In this study the different outcomes of G-EAT in CBA/J Tg+ and Tg– recipients correlated, at least in part, with modulation of Fas-mediated apoptosis by overexpressing the antiapoptotic molecule FLIP on TECs. The site of expression of FLIP and FasL, and active caspase-8 and active caspase-3 proteins differed in thyroids of Tg+ and Tg– recipients (32) (data not shown). In Tg– recipients with chronic inflammation and fibrosis, FasL and FLIP were mainly expressed by ICs, whereas in Tg+ recipients with resolving G-EAT, FLIP and FasL were primarily expressed by TECs. Active caspase-8 and active caspase-3 were also mainly expressed by CD4+ ICs in Tg+ recipients with resolving G-EAT, whereas active caspases were primarily expressed by TECs in Tg– recipients with chronic inflammation and fibrosis (data not shown). The constitutive overexpression of FLIP by TECs in Tg+ recipients inhibits TEC apoptosis. TECs in Tg+ recipients also expressed FasL (32), which may induce apoptosis of ICs, contributing to resolution in Tg+ recipients (25, 26, 27).

The development and resolution of inflammation are regulated by cytokines (9, 32, 33, 36, 37, 38, 39, 40, 41). Proinflammatory cytokines such as IFN-{gamma} and TNF-{alpha} promote inflammation (38, 39, 40), and can sensitize TECs to undergo Fas-mediated apoptosis (13, 38, 39, 50), whereas antiinflammatory cytokines such as IL-10 and IL-13 inhibit inflammation (9, 32, 36, 41) and promote apoptosis of ICs (12, 13, 14). Consistent with our previous observations in DBA/1 FLIP Tg+ mice, IFN-{gamma} and TNF-{alpha} mRNA was decreased, and IL-10 and IL-13 mRNA was increased in thyroids of CBA/J FLIP Tg+ compared with Tg– recipients at d 20 (Fig. 9Go). The decreased proinflammatory cytokines in Tg+ recipients might, at least in part, occur as a result of increased apoptosis of CD4+ T cells secreting these cytokines. Because most active caspase-8+ and active caspase-3+ cells in FLIP Tg+ recipients are CD4+ T cells (data not shown) and Th1 cells are more sensitive than Th2 cells to caspase-mediated cell death (29, 51, 52), this may contribute to the relatively higher expression of Th2 cytokines in FLIP Tg+ recipients. The fact that Foxp3 mRNA and protein expression was similar in thyroids of FLIP Tg+ compared with Tg– recipients at d 20 (Fig. 9EGo) is consistent with our previous study (32), and suggests that naturally occurring regulatory T cells do not contribute to G-EAT resolution in Tg+ mice, although Foxp3- IL-10-producing Tr1 or CD8+ T cells could be important (53, 54). Thus, the balance between proinflammatory and antiinflammatory cytokines in recipient thyroids that may function together with pro-apoptotic or antiapoptotic molecules correlates with and contributes to the different outcome of G-EAT in CBA/J FLIP Tg+ vs. Tg– recipients.

In summary, Tg FLIP expressed on TECs promotes G-EAT resolution in both CBA/J and DBA/1 mice. Decreased up-regulation of endogenous FLIP on ICs of CBA/J mice inhibits the development of G-EAT, and this can be reversed after transient depletion of CD8+ T cells. As summarized in Table 1Go, modulation of FLIP expression in the target organ and on ICs plays a pivotal role in regulating the outcome of an autoimmune inflammatory response. Therefore, modulation of FLIP could provide a new therapeutic target for some autoimmune diseases.


   Acknowledgments
 
We thank Dr. George Smith and Mr. Mark Foecking (University of Missouri, Columbia, MO) for assistance with FLAG-tagged protein detection. We also thank Drs. Yongzhong Wei and Kemin Chen for generating the transgene construct.


   Footnotes
 
This work was supported by National Institutes of Health Grant DK35527.

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 6, 2007

Abbreviations: cFLIP, Cellular Fas-associated death domain-like inhibitory protein; cFLIPL, long form of cellular Fas-associated death domain-like inhibitory protein; EAT, experimental autoimmune thyroiditis; FasL, Fas ligand; FLAG, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys; FLIP, Fas-associated death domain-like inhibitory protein; G-EAT, granulomatous experimental autoimmune thyroiditis; HPRT, hypoxanthine phosphoribosyltransferase; IC, inflammatory cell; IFN, interferon; IHC, immunohistochemistry; MTG, mouse thyroglobulin; PCK, pan-cytokeratin; TEC, thyroid epithelial cell; Tg, transgene or transgenic.

Received July 11, 2007.

Accepted for publication August 24, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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