Interleukin (IL)-12-Driven Primary Hypothyroidism: the Contrasting Roles of Two Th1 Cytokines (IL-12 and Interferon-)
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内分泌学杂志 2005年第8期
Department of Pathology (H.K., S.-C.T., R.R., M.K., N.R.R., P.C.), The Johns Hopkins School of Medicine, Baltimore, Maryland 21205; Department of Microbiology (K.S.), Leprosy Research Center, National Institute of Infectious Diseases, Tokyo 189-0002, Japan; National Hormone and Peptide Program (A.F.P.), Harbor-University of California at Los Angeles Medical Center, Torrance, California 90509; and Feinstone Department of Molecular Microbiology and Immunology (N.R.R., P.C.), The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205
Address all correspondence and requests for reprints to: Patrizio Caturegli, Johns Hopkins Pathology, Ross Building, Room 656, 720 Rutland Avenue, Baltimore, Maryland 21205. E-mail: pcat@jhmi.edu.
Abstract
IL-12, a prototypic T helper 1 cytokine, has been implicated in the pathogenesis of organ-specific autoimmune diseases, such as Hashimoto’s thyroiditis, but reported to give conflicting results in murine models of lymphocytic thyroiditis. To determine the effects of chronic, local production of IL-12 within the thyroid gland, we created transgenic mice that express IL-12 p70 under the transcriptional control of the thyroglobulin promoter. Transgenics developed growth retardation, moderate primary hypothyroidism, and mild lymphocytic infiltration of the thyroid gland. The hypothyroidism was associated with increased mRNA levels of the sodium-iodide symporter, an increase partly due to a direct effect of IL-12 on the thyrocyte. Upon immunization with a suboptimal dose of mouse thyroglobulin, IL-12 transgenic mice developed a lymphocytic thyroiditis that was more frequent and severe than that observed in wild-type littermates. The disease-promoting effect of IL-12 was independent of interferon-, as shown by the similar interferon- levels in transgenics and controls. These findings highlight the contrasting roles of two T helper 1 cytokines and report a novel role of IL-12 on thyroid hormonogenesis.
Introduction
IL-12 IS A PROINFLAMMATORY cytokine mainly produced by activated macrophages, dendritic cells, and granulocytes (1, 2, 3). It acts upon T, B, and NK lymphocytes, although it is best known for inducing the differentiation of CD4+ T lymphocytes from a Th0 to a Th1 phenotype (4). IL-12 is a heterodimer (p70) composed of a p40 subunit that is expressed predominantly on antigen-presenting cells and a p35 subunit that is present constitutively in numerous cells. Both subunits, which are encoded by separate chromosomes, have to be secreted by the same cell for production of a bioactive molecule. IL-12 binds to a specific plasma membrane receptor, composed of a ?1- and a ?2-subunit; the p40 subunit binds to ?1, whose intracellular domain associates with Tyk2; and p35 binds to ?2, associated with Jak2. Ligation of the IL-12 receptor activates Tyk2 and Jak2 kinases, which, in turn, induce phosphorylation, dimerization, and nuclear translocation of STAT4 (5), ultimately resulting in transcription of the genes involved in prototypic Th1 responses, such as interferon (IFN)-.
In light of its ability to stimulate Th1 responses, IL-12 has been invoked as a key cytokine in the pathogenesis of organ-specific autoimmune diseases, which are often mediated by cellular immunity (6). In a murine model of autoimmune diabetes in the NOD mouse, IL-12 is up-regulated in the initial phases of the disease and accelerates disease (7) via the generation of pathogenic autoreactive Th1 cells (8). In addition, in the NOD mouse, a susceptibility locus for type 1 diabetes (Idd4) is located near the IL-12 p40 gene (9). Similarly, IL-12 is up-regulated in patients suffering from multiple sclerosis attacks (10) and in experimental autoimmune encephalomyelitis (11, 12). Pagenstecher et al. (13) expressed IL-12 via transgenesis specifically in the cerebellum. With age, transgenic mice developed a neurological disorder characterized by modest mononuclear cell infiltration, up-regulation of proinflammatory cytokines, hypomyelination, and calcification at the site of transgene expression. Immunization with a neural protein induced an earlier and more frequent disease, which could also be induced by complete Freund’s adjuvant lacking neural proteins (14) or by infecting newborn mice with the Borna disease virus (15). By crossing the IL-12 transgenic to RAG2-deficient mice, the authors subsequently showed that mature T and B lymphocytes and IFN are required for disease induction (16). A similar disease-promoting role for IL-12 has been shown in experimental autoimmune myocarditis, where this cytokine acted independently of IFN (17) and through induction of pathogenic CD8+ effector T cells (18).
Relatively few studies are available on the role of IL-12 in autoimmune (lymphocytic) thyroiditis, either in patients with Hashimoto’s thyroiditis or in mice. In patients, Weetman’s laboratory reported that IL-12 p40 mRNA was detected in three of four thyroid specimens removed at surgery from Hashimoto’s patients; it was also produced by a transformed human thyroid cell line (HT-ori3), thus indicating that thyroid follicular cells themselves (not only the infiltrating hematopoietic cells) are capable of expressing IL-12 (19). These findings support the report of Phenekos et al. (20), who showed that patients with Hashimoto’s thyroiditis have higher serum IL-12 levels than patients with Graves’ disease, toxic nodular goiter, or healthy controls. In mouse models of autoimmune (lymphocytic) thyroiditis, Cooke’s laboratory has shown a dual role for IL-12. When administered at the time of immunization with thyroglobulin (one or two ip doses of 300 ng), IL-12 increased disease severity (21). Similarly, lymph node cells from thyroglobulin-immunized mice were able to transfer a more severe form of thyroiditis if cultured with IL-12 for 3 d before transfer. On the other hand, thyroiditis was milder when induced in mice that do not have functional IL-12 p40 gene, or in mice treated with an antibody that blocks the endogenous IL-12 (21). In contrast, when administered for longer periods (300 ng ip, five times/wk for 2 or 3 wk, starting the day before immunization), IL-12 inhibited the induction of thyroiditis (21). These findings have been confirmed in other disease models. Tarrant et al. (22) found that early administration of IL-12 (injections on d 0–4) was more effective than late injections (on d 7–11) in suppressing uveitis. Gran et al. (23) recently reported that early administration of IL-12 (10–200 ng/mouse·d on d 0–5 post immunization) suppressed encephalomyelitis severity.
During autoimmune (lymphocytic) thyroiditis, IL-12 is produced in the thyroid gland early and throughout the course of the disease (24). To assess the effects of prolonged and localized production of IL-12, we have developed transgenic mice in which the expression of heterodimeric IL-12 p70 was targeted to the thyroid follicular cell under transcriptional control of the thyroglobulin promoter.
Materials and Methods
Construction and screening of the thyr-IL-12 transgenic mice
The thyr-IL-12 transgene was made by joining the rat thyroglobulin promoter, the mouse IL-12 cDNA, and part of the human GH gene (as a source of introns and polyadenylation signal). Both IL-12 subunits, p35 and p40, were present in the same construct to allow synthesis of the mature IL-12 p70 heterodimer by the same cell (Fig. 1A). The 4.4-kb transgene was excised by BamHI and KpnI digestion and injected into fertilized eggs from (CBA/J x C57BL/6J) F1 females. Founders were identified by Southern blot, performed as described (25), using a 753-bp PCR-amplified probe that hybridizes to the p35 subunit and part of the thyroglobulin promoter (Fig. 1A, arrows). Transgene-positive mice were maintained as hemizygous by backcrossing for at least 12 generations to wild-type CBA/J or SJL/J mice (from The Jackson Laboratory, Bar Harbor, ME), inbred strains that have an MHC class II haplotype conducive to the induction of experimental autoimmune thyroiditis (26).
FIG. 1. The thyr-IL-12 transgene. A, Construction of the transgene. The transgene was made by joining rat thyroglobulin promoter, mouse IL-12 p35, and part of the human GH for p35 expression, followed by rat thyroglobulin promoter, mouse IL-12 p40, and part of the human GH for p40 expression. The two IL-12 subunits are present in the same construct to allow the expression of IL-12 p70 by the thyrocytes. B and C, Transfection of FRTL-5 cells to assess the in vitro expression of the IL-12 transgene. The plasmid vector containing the transgene induced the synthesis of IL-12 p70, as assessed by indirect immunofluorescence with a specific IL-12 p70 antibody (from Biosource International) (B); transfection of the empty vector, not containing the IL-12 transgene, did not induce IL-12 p70 synthesis (C). D, Southern blot screening from tail genomic DNA. When genomic DNA from the three transgenic lines (LW1, LW2, and LW3) and wild-type control was probed with a fragment comprising a portion of the thyroglobulin promoter and p35 subunit (probe location indicated by the arrows in A), it showed integration of the IL-12 transgene; E, Representative PCR screening from tail genomic DNA. Lane 1, 100-bp DNA ladder; lane 2, water control; lane 3, plasmid-positive control; lanes 4 and 6, IL-12 transgenics; lanes 5 and 7, wild-type littermates.
After establishment of the transgenic lines, mice were screened at each filial generation by PCR from tail genomic DNA, using an upstream primer (5'-TgccTTggTAgcATcTATgAg-3') that binds to exon 6 of the murine IL-12 p35 gene, and a downstream primer (5'-TTcAggcggAgcTcAgATAg-3') on exon 7. This primer pair amplifies a 2609-bp band from the wild-type IL-12 allele and a 294-bp band from the transgene.
Analysis of thyr-IL-12 transgene expression
The ability of the transgene to synthesize IL-12 p70 was first assessed in vitro by transfecting rat FRTL-5 cells with the IL-12 construct cloned into pBluescript II KS (+/–) plasmid (Stratagene, La Jolla, CA). FRTL-5 cells (CRL-1468; ATCC, Manassas, VA) were grown in Coon’s modified F-12 medium containing 5% heat-inactivated, mycoplasma-free calf serum, and 1 mM nonessential amino acids supplemented with a mixture of six hormones, including bovine TSH (1 x 10–10 M), insulin (10 μg/ml), cortisol (0.4 ng/ml), transferrin (5 μg/ml), glycil-L-histydil-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml). One microgram of plasmid was mixed with 4 μl lipofectamine transfection reagent (Invitrogen, Carlsbad, CA) and incubated for 3 h with FRTL-5 cells cultured in 6-well plates. After addition of fresh growth medium, cells were incubated for 24 h and then treated for 5 h with 2 μM monensin, a well-characterized inhibitor of protein secretion that allows accumulation of cytokines within the cells. Cells were finally washed, fixed, and stained with a fluorescein isothiocyanate-conjugated IL-12 p70 antibody (Biosource International, Camarillo, CA) to evaluate, by direct immunofluorescence, the expression of mature IL-12.
In vivo, IL-12 expression was evaluated at the RNA level by RT-PCR performed on various tissues (thyroid, salivary and lachrymal glands, heart, lung, kidney, and liver), and at the protein level by measuring IL-12 p70 in sera. For RNA analysis, tissues were first digested by collagenase and dispase as previously described (27), to prepare a single cell suspension that was then incubated with CD45 magnetic beads (Miltenyi Biotec, Auburn, CA). The CD45-postive fraction, a potential source of IL-12, was removed, and the remaining cells were used to extract mRNA (Dynal Biotech, Brown Deer, WI). mRNA was treated with DNase I (Invitrogen) and reverse transcribed using Superscript II (Invitrogen) and oligo(d)T primers. PCR was performed with primers for murine IL-12 p35 (forward: 5'-TgccTTggTAgcATcTATgAg-3' and reverse 5'-TTcAggcggAgcTcAgATAg-3'), and with primers for G3PDH as control (forward: 5'-gcATcTTgggcTAcAcTgAg-3' and reverse: 5'-TcTcTTgcTcAgTgTccTTg-3').
Serum IL-12 p70 was measured by ELISA and cytokine array, using blood immediately chilled after drawing and separated by a microtainer tube (Becton Dickinson and Co., Franklin Lakes, NJ). ELISA was performed with a commercially available kit (R&D Systems Inc, Minneapolis, MN), following the manufacturer’s recommendations. Cytokine arrays, chosen to detect the greatest number of cytokines with the smallest volume of serum, were purchased from RayBiotech (Norcross, GA). The array allows for the simultaneous detection of 31 molecules distributed as follows: eleven cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-17, IFN, and TNF-); eleven chemokines [CCL2 (MCP-1/JE), CCL3 (MIP-1 ), CCL5 (Rantes), CCL11 (Eotaxin), CCL12 (MCP-5), CCL17 (TARC), CCL19 (MIP-3 ?), CCL21 (6Ckine), CCL27 (CTAK), CXCL1 (KC), and CXCL2 (MIP-2)]; five hematopoietic promoting molecules (IL-3, G-CSF, GM-CSF, stem cell factor, and thrombopoietin); and four other molecules (leptin, tissue inhibitor of metalloproteinases 1, soluble TNF receptor 1, and vascular endothelial growth factor). Sera were diluted 1:10 in the provided blocking buffer, and incubated at 4 C overnight with the arrayed antibody membrane. After addition of biotinylated anticytokine antibody cocktail and horseradish peroxidase-conjugated streptavidin, a colororimetric signal was induced by addition of the provided detection buffer. The membrane was finally exposed to radiographic film [x-omat AR (Kodak, Rochester, NY)], and the signal was analyzed for gray scale intensity using the free software Image J (http://rsb.info.nih.gov/ij).
Growth curves, thyroid histopathology and immunohistochemistry, thyroglobulin immunization, and thyroglobulin antibodies
Mice were weighed at several time points throughout their lifetime to compare growth between transgenics and wild-type littermates.
For histopathology, thyroids were removed after euthanasia and fixed for 48 h in the zinc-based Beckstead’s solution. After processing and embedding in paraffin, six to eight nonsequential sections (5-μm thick) were cut from tissue blocks and stained with hematoxylin and eosin. Immunohistochemistry was performed to determine the nature of the cells infiltrating the thyroid, as described (28). Briefly, sections were deparaffinated, rehydrated, and blocked with 2% normal goat serum. Sections were incubated overnight at 4 C with rat antimouse CD45 (BD PharMingen, San Diego, CA), hamster antimouse CD3 (BD PharMingen), rat antimouse B220 (BD PharMingen), or rat antimouse F4/80 (Serotec, Raleigh, NC). After washing, addition of the biotinylated anti-IgG secondary antibody, and incubation with peroxidase-conjugated streptavidin, the brown positive color was revealed by the addition of diaminobenzidine substrate (Sigma, St. Louis, MO).
Thyroglobulin immunization was done to assess whether the presence of transgenic IL-12 within the thyroid would enhance the incidence and/or severity of thyroiditis. Given the severity of disease that we achieve by our standard immunization protocol [75 μg thyroglobulin in complete Freund’s adjuvant on d 0 and 7 (25)], we considered unlikely the detection of any further enhancement of disease by IL-12 in this system. We therefore used, for this experiment, a suboptimal immunization protocol (25 μg thyroglobulin rather than 75 μg) and the transgenic line with the weakest expression of IL-12 (the LW2 line). We also used the SJL, rather than the CBA strain, because, while responding equally well to thyroglobulin immunization (26), it has a superior breeding performance (http://aretha.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home). All experimental protocols conformed to Johns Hopkins Animal Care and Use Committee guidelines.
Thyroglobulin antibodies were measured as described (25). Briefly, sera were added in triplicates to Immulon2 ELISA plates (DYNEX Technologies, Inc., Chantilly, VA) coated with 100 ng/well murine thyroglobulin. After overnight incubation, plates were washed and incubated with secondary antibodies against IgG1, IgG2c, and IgG2b conjugated to alkaline phosphatase. Each plate included a standard curve derived from serial dilutions of a pool serum with known mouse thyroglobulin antibodies. The IgG2c (IgG2ab) isotype was chosen because in the SJL strain, as in the C57BL6 and NOD strains, the IgG2a (IgG2aa) gene is deleted (29).
Assessment of thyroid function by total T4 and TSH levels
Total T4 levels were determined using a commercial competitive RIA (GammaCoat 125I-T4; DiaSorin, Inc., Stillwater, MN). Mouse TSH was measured with a highly sensitive, double-antibody RIA developed by A.F.P (30). Briefly, the assay employs a highly purified rat TSH (AFP11542B) as the iodinated ligand, a selected guinea pig antimouse TSH (AFP98991 as the primary antibody, and a partially purified extract of mouse pituitary containing TSH (AFP5171.8MP) as the reference preparation.
Changes in thyroid gene expression induced by IL-12
The effect of IL-12 on thyroid gene expression was studied in vivo and in vitro. For in vivo studies, thyr-IL-12 transgenic mice and wild-type littermates were killed and thyroidectomized. After dissection and mechanical disruption of the thyroid lobes, mRNA was extracted (mRNA direct kit from Dynal Biotech), treated with DNase I (Invitrogen), and reverse transcribed. cDNA was then amplified to assess the expression of sodium-iodide symporter (5'-gcTcTcATcAgcTAccTAAcTgg-3' and 5'-cTcAgAggTTggTcTcAAcATc-3'), thyroglobulin (5'-cgTgTTTgTcccTgAgAAccTg-3' and 5'-TccgTTgAgAAgTAgcccTggTAg-3'), thyroid peroxidase (5'-TgccAAcAgAAgcATggTcAAc-3' and 5'-gcAcAAAgTTcccATTgTccAc-3'), and TSH receptor (5'-cggTTccTcATgTgcAAcTTg-3' and 5'-ccTcTTggcAATcTTggTgTc-3'). To compare gene expression between transgenics and wild-type littermates, semiquantitative RT-PCR was performed using G3PDH as the gene against which to compare the expression of the other messages.
For in vitro studies, rat FRTL-5 cells, maintained in the complete six-hormones medium as described (31), were incubated with 10 ng/ml recombinant mouse IL-12 (R&D Systems, Minneapolis, MN) and harvested 12, 24, or 48 h thereafter. Total RNA was then extracted (RNeasy mini kit from QIAGEN, Valencia, CA), separated by agarose gel electrophoresis, and analyzed by Northern blot for the expression of sodium-iodide symporter, thyroglobulin, thyroperoxidase, and TSH receptor, as described (31). The signal from G3PDH was also obtained to adjust for the amount of RNA loaded in each lane. Probe signals were quantified using the BAS-1500 Bioimaging Analyzer (Fuji Photo Film Co., Ltd., Japan).
Statistical analysis
The population of mice used for the analyses presented in this study included a total of 244 mice: 221 on the CBA/J background and 23 on the SJL/J background. The CBA/J mice comprised 60 wild-type (14 males and 46 females) and 161 thyr-IL-12 transgenics (62 males and 99 females) and were used to analyze statistically the following outcomes: body weight; thyroid histopathology in baseline conditions; and serum levels of IL-12 p70, total T4, and TSH. The SJL/J mice comprised 10 thyr-IL-12 transgenics of the LW2 line and 13 wild-type littermates and were used to analyze thyroid histopathology, thyroglobulin antibodies, and total T4 after mouse thyroglobulin immunization.
Body weight was measured longitudinally in 91 thyr-IL-12 transgenics (25 LW1: 13 males and 12 females; 40 LW2: 22 males and 18 females; and 26 LW3: 15 males and 11 females) and 21 wild-type controls (11 males and 10 females) on d 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 90, 120, 180, 300, and 365 after birth. Differences in weight between transgenics and controls were assessed by multiple linear regression with generalized estimating equations, as described (32).
Thyroid histopathology in basal conditions was analyzed in 99 IL-12 CBA/J transgenics (21 LW1, 47 LW2, and 31 LW3) and 39 wild-type CBA/J controls, 38–466 d old. Thyroid sections were scored by digital microscopy (27) and assessed in a multiple linear regression model including as covariates: sex, age, and the four genotypes (wild type, LW1, LW2, and LW3). Thyroid histopathology, after thyroglobulin immunization, was analyzed in 10 LW2 SJL/J transgenics and 13 wild-type SJL/J littermates. Differences in the score were evaluated by the Wilcoxon rank-sum test.
Serum IL-12 p70 was measured in 39 transgenics (13 LW1, 17 LW2, and 9 LW3) and eight wild-type controls, total T4 in 83 transgenics (24 LW1, 32 LW2, and 27 LW3) and 20 controls, and TSH in 42 transgenics (15 LW1, 13 LW2, and 14 LW3) and 19 controls. Differences in the mean serum IL-12, T4, or TSH levels among the four groups were assessed by the Kruskal-Wallis test, followed by pairwise comparisons using the Wilcoxon rank-sum test. Serum TSH was also assessed in a multiple linear regression model including as covariates: T4, sex, age, and the four genotypes.
All analyses were performed using Stata statistical software, release 8 (from Stata Corp., College Station, TX).
Results
Thyr-IL-12 transgene expression and basal phenotype
The ability of the transgene to support synthesis of IL-12 was first assessed in vitro by transfection of FRTL-5 cells. The mature IL-12 p70 heterodimer was produced upon transfection of the IL-12 transgene (Fig. 1B) but not of the control plasmid (Fig. 1C). In vivo, the transgene stably integrated into the genome of six of the 114 injected mice. Three of these six founders, named thyr-IL-12 transgenic LW1, LW2, and LW3 line (Fig. 1D), were maintained as hemizygous by mating to normal inbred strains (CBA/J for most of the experiments and SJL/J for thyroglobulin immunization) and screened by PCR (Fig. 1E) to form the cohort described in the present study.
Transgenic IL-12 was expressed in the thyroid of the three transgenic lines but not in the other organs examined or in the thyroid of wild-type controls (Fig. 2A). The thyroidal production of IL-12 p70 was also capable of reaching the systemic circulation, as demonstrated by the significantly higher serum IL-12 levels in the three transgenic lines compared with wild-type controls (P = 0.0002 by Kruskal-Wallis test) (Fig. 2B). Mice of the LW2 line tended to have lower serum IL-12 levels than those of the LW1 or LW3 lines, although the difference was not statistically significant. The increased serum levels were restricted to IL-12 and not seen for IFN (Fig. 2, C and D). In addition, the other cytokines and chemokines assayed by the array were not detectable or were no different between transgenics and controls (data not shown).
FIG. 2. Expression of IL-12 in thyr-IL-12 transgenic mice. A, IL-12 mRNA measured by RT-PCR. RT+ and RT– indicate that the PCR amplification of the cDNA was performed with or without previous reverse transcription. The organs used were thyroids, lungs, lachrymal glands (lac.), kidneys (kid.), liver (liv.), heart and salivary glands (sal.), all depleted of CD45-positive hematopoietic cells following the procedure described in (27 ). N is the water control, P is the plasmid-positive control. Note that only transgenic thyroids express IL-12 mRNA. B, Serum IL-12 p70 measured by ELISA. Note the increased expression in the three transgenic lines, compared with wild-type controls. C and D, Serum IL-12 p70 and IFN measured by cytokine arrays. IL-12 was increased in transgenic sera, whereas IFN was undetectable in both IL-12 transgenic and controls. The signal of the positive control provided by the manufacturer is also shown to adjust for differences between wild-type and transgenic array membranes. To create the graph shown in D, signals from the radiographic film were scanned and analyzed on a gray scale comprised between 0 and 255.
IL-12 transgenic mice showed no gross physical and behavioral abnormalities and delivered a number of offspring similar to that of normal CBA/J (4.2 average pups per litter) or SJL/J (6.2 pups per litter) strains. There was, however, a distortion in the frequency by which the transgenic allele was transmitted to the offspring. According to Mendel’s first law (principle of segregation), the cross between a hemizygous transgenic and a wild-type animal should yield an equal number of transgenics and wild-types in the progeny. In contrast, the transgenic father-to-normal female mating scheme yielded a transgenic-to-wild-type ratio of 0.31 for the LW1 line (90:294 mice), of 1.01 for the LW2 line (167:165), and 0.25 for LW3 line (78:316). This ratio departed even further from one in the transgenic mother-to-normal male mating scheme, being 0.25 for LW1 (4:16), 0.67 for LW2 (16:24), and 0.06 for LW3 (3:52). The reasons for this reduced transmission of the IL-12 transgene to the progeny are unknown at the moment.
Growth defect and spontaneous, although minimal, lymphocytic thyroiditis
Thyr-IL-12 transgenic mice were smaller than wild-type littermates (Fig. 3A). In particular, regression analysis showed that, holding sex and age constant, transgenics were 3.26 g lighter than controls (95% confidence interval, 2.87–3.65, P < 0.0001). The growth defect became clearly evident at the time of weaning (d 21) and persisted throughout life. Similarly to wild-type littermates, females were 2.98 g smaller than males (95% confidence interval, 2.61–3.56; P < 0.0001) (data not shown). There was no significant difference in weight among the three transgenic lines, which therefore were combined in the analysis shown in Fig. 3A.
FIG. 3. Growth curves and thyroid pathology. A, Growth: thyr-IL-12 transgenic mice showed significant growth retardation compared with wild-type littermates. B–D, Thyroid histopathology: thyroid glands showed enlarged and flattened follicular cells (B) and a clear, albeit minimal, mononuclear cell infiltration (C), frequently found around vessels (D), and connective tissue capsule (C and D). E and F, Nature of the infiltrate: immunohistochemistry indicated that the infiltrate was mainly composed of B220+ B lymphocytes (E) and also CD3+ T lymphocytes (F).
Thyroid follicles were often enlarged and lined by a flattened epithelium (Fig. 3B and Table 1), an appearance seen in patients with long-standing hypofunctioning goiter (such as the colloid involution phase of the diffuse nontoxic goiter). Transgenic thyroids also showed, at baseline (i.e. before immunization), a mononuclear cell infiltration (Fig. 3C) that was frequently localized around vessels (Fig. 3D), and underneath the connective tissue capsule (Fig. 3, C and D). The infiltrating cells were lymphocytes, mainly comprised of B220-positive B cells (Fig. 3E), and CD3-positive T cells (Fig. 3F). There were only few F4/80-positive macrophages, with no difference between transgenics and controls (data not shown). Prevalence and severity of this infiltration were greater in transgenic lines expressing the higher levels of IL-12 (LW1 and LW3) than in the LW2 line (Fig. 4A and Table 1). Overall, the infiltration was clear (>2% of the total thyroid area), but of moderate intensity; none of the transgenic mice, in fact, developed spontaneously an overt infiltration comprising more than 15% of the total thyroid area (Fig. 4A). Thyroglobulin antibodies were absent when measured at several time points throughout the life of the mice (data not shown), suggesting perhaps that, in patients with Hashimoto’s thyroiditis, these antibodies are not related to the initial immunopathology.
TABLE 1. Morphologic changes seen in the thyroids of the three thyr-IL-12 transgenic lines (LW1, LW2, and LW3)
FIG. 4. Effect of thyroglobulin immunization. A, Baseline infiltration score. Before immunization, thyroids of IL-12 transgenic mice showed mononuclear cell infiltration, which was significantly greater in the lines expressing the higher IL-12 levels (LW1 and LW3) than in the low-expressor line (LW2). P values are based on pairwise comparisons using the Wilcoxon rank-sum test, performed after the Kruskal-Wallis test (P = 0.0001). The infiltration was clear (>2% of the total thyroid area) but overall of low severity (<15% of the total thyroid area). B, Infiltration after challenging LW2 transgenic mice with low doses (25 μg on d 0 and 7) of mouse thyroglobulin. This suboptimal immunization regimen induced a more frequent and severe thyroiditis in transgenics than controls (P = 0.0003 by Wilcoxon rank-sum test). C, Thyroid function after immunization. Total T4, which at baseline was slightly lower in LW2 transgenics than controls (see also Fig. 5A), decreased significantly 21 d after immunization (P = 0.023 vs. d 0 by Wilcoxon matched-pairs signed-ranks test), reaching similar levels in transgenics and controls. WT, Wild type.
Considering that transgenic IL-12 was capable of inducing minimal lymphocytic thyroiditis, we tested whether these infiltrates could lead to an increased incidence and severity of disease upon induction of experimental autoimmune thyroiditis. We therefore immunized 10 thyr-IL-12 SJL/J transgenics of the LW2 line and 13 SJL/J littermates with a suboptimal dose of mouse thyroglobulin (25 μg), assessing thyroid morphology, thyroglobulin antibodies, and total T4 3 wk thereafter. As shown in Fig. 4B, the presence of IL-12 in the thyroid gland resulted in a more incident and severe thyroiditis (P = 0.0003), indicating a disease-promoting role for this cytokine. Total T4, which began at lower levels in the LW2 transgenic line, decreased significantly 21 d after immunization (Fig. 4C; P = 0.023, comparing d 0 vs. d 21 with the Wilcoxon matched-pairs signed-rank test). This decrease in T4 after immunization, which confirms what we previously published (25), reached similar levels in both genotypes, suggesting that the lower T4 levels observed at baseline in LW2 transgenic mice (see also Fig. 5A) represent the direct effect of IL-12 on thyrocytes rather than the consequence of the lymphocytic infiltration. Thyroglobulin antibodies at d 21 were present at similar levels in transgenics and controls and overall at low titer (data not shown), likely a consequence of the mild immunization protocol.
FIG. 5. Thyroid function. A, Serum total T4. T4 was decreased in thyr-IL-12 transgenic mice compared with wild-type controls. The decrease reached statistical significance for the LW1 (P = 0.0007 vs. control) and LW3 line (P = 0.0001) but not for the LW2 line (P = 0.065). P values are based on pairwise comparisons using the Wilcoxon rank-sum test, performed after the Kruskal-Wallis test (P = 0.0002). B, Serum TSH. TSH was increased in thyr-IL-12 transgenic mice compared with wild-type controls. The increase reached statistical significance for the LW1 (P = 0.016 vs. control) and LW3 line (P = 0.0001) but not for the LW2 line (P = 0.108). C, Relationship between serum T4 and TSH. Regression analysis showed that T4 significantly predicted TSH values (P = 0.007), although the proportion of variation in TSH that could be predicted by T4 was small (r2 = 0.39). CI, Confidence interval.
Primary hypothyroidism
Serum total T4, examined at multiple times throughout life, was lower in all three transgenic lines than in wild-type controls (Fig. 5A). The decrease reached statistical significance for the LW1 line (P = 0.0007 vs. wild-type) and LW3 line (P = 0.0001) but not for the LW2 line (P = 0.065). There was no statistical difference in the mean T4 levels among the three transgenic lines, although T4 was less decreased and closer to the normal range in the LW2 line (Fig. 5A).
Serum TSH levels followed a similar pattern but in the opposite direction. They were significantly increased in the LW1 (P = 0.016) and LW3 (P = 0.0001) transgenic lines compared with wild-type controls (Fig. 5B). In the LW2 line, TSH levels were higher but not statistically different from those observed in normal controls (P = 0.108). Multiple linear regression modeling showed that T4 significantly predicted the serum TSH levels (for every 1 μg/dl increase in T4 there is a 16.9 U/ml decrease in TSH, holding age, sex, and genotype constant; P = 0.007; Fig. 5C). The proportion of the variation in TSH that could be predicted by T4 in this model was, however, small (0.39; Fig. 5C), confirming the observation that, in mice, serum T4 levels do not correlate strongly with TSH levels (33).
Taken together, the histological and biochemical data indicate that the presence of IL-12 within the thyroid gland induces a moderate primary hypothyroidism. The hypothyroidism was more severe in the two transgenic lines that expressed higher IL-12 levels (LW1 and LW3) than in the low expressor line (LW2), suggesting that it is a direct consequence of the action of IL-12 on the thyroid follicular epithelium rather than secondary to the minimal lymphocytic infiltrate.
Mechanisms of hypothyroidism
To understand how IL-12 induced primary hypothyroidism, we extracted thyroid mRNA and performed semiquantitative RT-PCR to evaluate the expression of some thyroid-restricted genes. Sodium iodide symporter (NIS) was strongly up-regulated in transgenics, showing a 4-fold increase over wild-type controls (Fig. 6, A and B). Thyroid peroxidase and thyroglobulin were also increased in transgenics, although less markedly (Fig. 6, A and B). In contrast, the expression of the TSH receptor gene was reduced (Fig. 6, A and B). This gene expression profile could, overall, be reconciled with the increased serum TSH levels present in the thyr-IL-12 transgenic mice. It is, in fact, well established that increased TSH levels up-regulate NIS (34, 35), thyroperoxidase (36, 37), and thyroglobulin (38) and, at the same time, down-regulate the TSH receptor (39, 40).
FIG. 6. Analysis of thyroid gene expression. A and B, In vivo analysis. mRNA was extracted from the thyroids of 2- to 3-month-old LW3 transgenic mice and age-matched controls, reverse transcribed, and PCR amplified to assess the expression of four thyroid-specific genes. PCR products were fractionated by agarose gel electrophoresis and stained with ethidium bromide (A). Images were then acquired with a digital camera to express the intensity of the bands on a continuous numeric scale. Results were finally expressed as a ratio of intensities between transgenics and wild types, adjusting for the intensity of a housekeeping gene (G3PDH). A change in intensity greater than 50% was considered significant. Thyroids from LW3 thyr-IL-12 transgenics showed increased expression of NIS and also thyroglobulin (TG) and thyroperoxidase (TPO). On the contrary, the expression of TSH receptor (TSH-R) was reduced. C and D, Time course expression of four thyroid-specific genes upon addition of recombinant IL-12 (10 ng/ml) to FRTL-5 cultured cells. FRTL-5 cells were collected 12, 24, and 48 h after addition of IL-12 to the culture medium to extract total RNA. RNA was then fractionated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized to specific radioactive probes (C). Probe signals were quantified with a phosphoroimager and expressed as a ratio of intensity between thyroid genes and housekeeping gene (G3PDH), adjusting for the intensity at time zero. A change in intensity greater than 50% was considered significant. Addition of IL-12 induced a 2-fold increase in NIS expression.
To distinguish whether the striking increase in NIS expression was simply the consequence of increased TSH levels or, rather, also a direct (TSH-independent) effect of IL-12 on thyrocytes, we added IL-12 (10 ng/ml) to FRTL-5 cells grown in complete (six hormones) medium and measured gene expression 12, 24, or 48 h thereafter. In keeping with the in vivo results, IL-12 increased NIS expression (Fig. 6, C and D), which peaked at 12 h and remained elevated for 24 h. NIS then declined to baseline at 48 h, possibly suggesting a diminished IL-12 activity in a culture medium that allows for continuous thyrocyte growth. In contrast, IL-12 did not increase, or change significantly, the expression of the other thyroid-specific genes (Fig. 6, C and D), suggesting that their changes observed in vivo were mainly the consequence of increased TSH stimulation. These novel findings indicate that IL-12 is capable of increasing NIS expression and, at the same time, inducing hypothyroidism.
Discussion
This study reports that transgenic mice expressing IL-12 specifically in the thyroid gland develop moderate lymphocytic thyroiditis and primary hypothyroidism. Studies in other murine models of autoimmunity (encephalomyelitis, type 1 diabetes, and myocarditis) had indicated a similar disease-promoting role for this cytokine. It was unclear, however, whether IL-12 was sufficient to initiate disease or, rather, was only one of the many cofactors associated with disease. Even when considering solely murine studies of lymphocytic thyroiditis, the role played by IL-12 remains uncertain (21, 41). To gain insights on the function of IL-12 in Hashimoto’s thyroiditis, we created transgenic mice that secrete relatively low levels of IL-12 inside the thyroid in a chronic fashion, with the intent of mimicking one aspect of disease pathogenesis.
Our results show that IL-12 induces a moderate lymphocytic thyroiditis that, despite never reaching the severity seen in patients with frank Hashimoto’s thyroiditis, clearly and significantly differed from the normal thyroid morphology. This disease-promoting role of IL-12 showed dose-dependence because it was stronger in the transgenic lines expressing the higher IL-12 levels (LW1 and LW3) than in the low expressor line (LW2). It was also confirmed by immunization experiments that used a suboptimal regimen and the low expressor line. Here IL-12 was capable of inducing a more incident and severe thyroiditis than that seen in wild-type littermates.
Uncertainty remains on the mechanism through which IL-12 exerts its disease-promoting effect. IL-12 may enhance the antigen presenting capacity of resident dendritic cells (42) or their ability to release IFN (43). In our thry-IL-12 transgenic mice, however, the effect of IL-12 appears independent of IFN. IFN, in fact, was undetectable when assayed in properly collected sera. Although a formal proof of the IFN independence would likely require a cross between thyr-IL-12 transgenic and IFN knock-out mice, previous work has shown that systemic administration of IL-12 is not associated with increased serum IFN levels (44, 45). Our findings are in keeping with recent studies that have revealed contrasting roles of the two prototypic Th1 cytokines: IL-12 and IFN. Using the murine model of autoimmune myocarditis, Afanasyeva et al. (17) have shown that IL-12 is capable of inducing disease without using the IFN pathway. We have also previously shown that IFN has a protective, rather than disease-promoting, effect in experimental autoimmune thyroiditis (46). These findings are consistent with the results of the present study, and they highlight a novel, dichotomous role for IL-12 and IFN, two classic Th1 cytokines commonly considered to act in synchrony.
The different outcomes induced by IL-12 may be explained by the redundancy and complexity of the IL-12 system, which also includes IL-23 and IL-27. IL-23 is a heterodimer composed of the same p40 subunit as IL-12 and a novel p19 subunit. Using the murine model of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein, Cua et al. (47) showed that disease susceptibility was retained in p35 (IL-12)-deficient mice but was abolished in p19 (IL-23)-deficient mice or p40 (IL-12 and IL-23)-deficient mice, indicating that disease induction mainly depends upon IL-23 signaling. Similar results were obtained with IL-27, a heterodimer composed of EBI3 (a p40-related protein) and p28 (a p35-related protein). Blockade of p28 with a specific antibody significantly decreased disease severity in experimental autoimmune encephalomyelitis (48). These findings suggest that IL-12, originally considered necessary for encephalomyelitis induction (49), may behave more as a disease modulator rather than a promoter of encephalomyelitis (23) and that the coordinated expression of the two subunits forming each cytokine is crucial for appropriate immune responses in timing, location, and magnitude (50).
Our study reveals a novel effect of IL-12 on thyroid function: primary hypothyroidism associated with increased NIS mRNA expression. The hypothyroidism is likely a direct effect of IL-12, rather than the consequence of the lymphocytic infiltration, because it was present even in transgenic mice that did not have apparent infiltration, and the extent of the infiltration was too small to cause loss of function of the entire thyroid gland observed in our transgenic mice. It is interesting to speculate on the mechanism by which IL-12 causes hypothyroidism, in the context of thyroid hormonogenesis. Thyroid hormone synthesis begins with the NIS-mediated influx of iodine from the blood into the thyroid cell. Iodine then moves through the cytosol by still-undefined mechanisms, reaches the apical membrane, and is transported into the follicular lumen by specific proteins, such as pendrin and apical iodide transporter. Within the follicle, on the luminal side of the apical membrane, iodine is then oxidized by thyroperoxidase in the presence of hydrogen peroxide and subsequently placed on selected tyrosyl residues of thyroglobulin, a process referred to as organification or iodination. Thyroperoxidase also catalyzes the next step, which is the coupling of two iodinated tyrosyl residues. When needed, the iodinated thyroglobulin, stored as colloid in the follicular lumen, reenters the thyrocyte via micropinocytosis. Here, thyroglobulin-containing vesicles fuse with lysosomes that degrade thyroglobulin, releasing thyroid hormones that enter the bloodstream at the basolateral membrane, likely by way of specific thyroidal channels that still await identification. The gene expression profile of the IL-12 transgenic thyroids (increased expression of NIS, thyroperoxidase, and thyroglobulin) suggests that IL-12 inhibits thyroid hormone synthesis downstream of the organification reaction, although the precise location of the block is unknown at the moment. This inhibitory effect is strong enough to override the thyroid stimulation induced by the increased serum TSH levels.
The biological significance of the increased NIS transcription induced by IL-12 remains unknown, considering that increased NIS mRNA levels do not always correlate with greater capacity of the thyrocyte to uptake iodine (35, 51). The stimulatory effect of IL-12 on NIS mRNA expression, however, is worth of attention. The best-known NIS regulators are TSH, which increases NIS transcription, biosynthesis, and presence in the plasma membrane, and iodine itself (52). Cytokines, however, also have an effect on NIS expression, usually a decrease. TGF-? (53), IL-1, IL-6, and TNF- (54), and IFN (55) have all been shown to suppress NIS mRNA expression when added in vitro to FRTL-5 cell cultures. We now report the first cytokine, IL-12, that is capable of increasing NIS expression in vivo. The effect of IL-12 is direct because it can be reproduced by addition of IL-12 to cultured thyroid cells. The effect also highlights the complexity and the contrasting roles of the two prototypic Th1 cytokines, considering that we have previously reported that transgenic mice expressing IFN in the thyroid develop primary hypothyroidism associated with suppression of NIS gene transcription, NIS protein expression, and iodine uptake (56).
This observation has potential clinical implications. IL-12 has, in fact, shown promising results in treating differentiated thyroid carcinoma. When murine IL-12 p70 was expressed and delivered via an adenovirus vector, it induced effective antitumor activity and long-term immunity against medullary (57, 58, 59) and follicular (60) rat thyroid carcinomas. The use of IL-12 to treat thyroid cancers could be of value not only for the above described antitumor activity but also for its ability to increase NIS expression and, consequently, the uptake of therapeutic radioactive iodine into thyroid follicular cells.
The effect of IL-12 on thyroid function could be more global than the effect shown by this paper on the thyrocytes. For example, Boelen et al. (62) have reported that the lipopolysaccharide-induced decrease in type I deiodinase [the selenoprotein that converts T4 to T3 (61)] within the pituitary is less pronounced in IL-12-deficient mice than in wild-type controls. Thus, IL-12-deficient mice are predicted to have a greater T4 to T3 conversion, with resulting pituitary-specific thyrotoxicosis and inhibition of TSH secretion. The opposite scenario could occur in the presence of increased serum IL-12 levels that should lead to reduced T4 to T3 conversion within the pituitary and increased TSH secretion.
In conclusion, we have shown, for the first time, that local production of IL-12 in the thyroid enhances the expression of sodium-iodide symporter and inhibits thyroid hormonogenesis downstream of the organification, thus inducing primary hypothyroidism. The effect on NIS is independent of, and opposite to, the action of the other prototypic proinflammatory cytokine, IFN. The results provide new insights into the complex checks and balances of the inflammatory response.
Acknowledgments
The authors acknowledge Mehrdad Hejazi and Liwen He for their contribution in the initial stages of the project.
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Address all correspondence and requests for reprints to: Patrizio Caturegli, Johns Hopkins Pathology, Ross Building, Room 656, 720 Rutland Avenue, Baltimore, Maryland 21205. E-mail: pcat@jhmi.edu.
Abstract
IL-12, a prototypic T helper 1 cytokine, has been implicated in the pathogenesis of organ-specific autoimmune diseases, such as Hashimoto’s thyroiditis, but reported to give conflicting results in murine models of lymphocytic thyroiditis. To determine the effects of chronic, local production of IL-12 within the thyroid gland, we created transgenic mice that express IL-12 p70 under the transcriptional control of the thyroglobulin promoter. Transgenics developed growth retardation, moderate primary hypothyroidism, and mild lymphocytic infiltration of the thyroid gland. The hypothyroidism was associated with increased mRNA levels of the sodium-iodide symporter, an increase partly due to a direct effect of IL-12 on the thyrocyte. Upon immunization with a suboptimal dose of mouse thyroglobulin, IL-12 transgenic mice developed a lymphocytic thyroiditis that was more frequent and severe than that observed in wild-type littermates. The disease-promoting effect of IL-12 was independent of interferon-, as shown by the similar interferon- levels in transgenics and controls. These findings highlight the contrasting roles of two T helper 1 cytokines and report a novel role of IL-12 on thyroid hormonogenesis.
Introduction
IL-12 IS A PROINFLAMMATORY cytokine mainly produced by activated macrophages, dendritic cells, and granulocytes (1, 2, 3). It acts upon T, B, and NK lymphocytes, although it is best known for inducing the differentiation of CD4+ T lymphocytes from a Th0 to a Th1 phenotype (4). IL-12 is a heterodimer (p70) composed of a p40 subunit that is expressed predominantly on antigen-presenting cells and a p35 subunit that is present constitutively in numerous cells. Both subunits, which are encoded by separate chromosomes, have to be secreted by the same cell for production of a bioactive molecule. IL-12 binds to a specific plasma membrane receptor, composed of a ?1- and a ?2-subunit; the p40 subunit binds to ?1, whose intracellular domain associates with Tyk2; and p35 binds to ?2, associated with Jak2. Ligation of the IL-12 receptor activates Tyk2 and Jak2 kinases, which, in turn, induce phosphorylation, dimerization, and nuclear translocation of STAT4 (5), ultimately resulting in transcription of the genes involved in prototypic Th1 responses, such as interferon (IFN)-.
In light of its ability to stimulate Th1 responses, IL-12 has been invoked as a key cytokine in the pathogenesis of organ-specific autoimmune diseases, which are often mediated by cellular immunity (6). In a murine model of autoimmune diabetes in the NOD mouse, IL-12 is up-regulated in the initial phases of the disease and accelerates disease (7) via the generation of pathogenic autoreactive Th1 cells (8). In addition, in the NOD mouse, a susceptibility locus for type 1 diabetes (Idd4) is located near the IL-12 p40 gene (9). Similarly, IL-12 is up-regulated in patients suffering from multiple sclerosis attacks (10) and in experimental autoimmune encephalomyelitis (11, 12). Pagenstecher et al. (13) expressed IL-12 via transgenesis specifically in the cerebellum. With age, transgenic mice developed a neurological disorder characterized by modest mononuclear cell infiltration, up-regulation of proinflammatory cytokines, hypomyelination, and calcification at the site of transgene expression. Immunization with a neural protein induced an earlier and more frequent disease, which could also be induced by complete Freund’s adjuvant lacking neural proteins (14) or by infecting newborn mice with the Borna disease virus (15). By crossing the IL-12 transgenic to RAG2-deficient mice, the authors subsequently showed that mature T and B lymphocytes and IFN are required for disease induction (16). A similar disease-promoting role for IL-12 has been shown in experimental autoimmune myocarditis, where this cytokine acted independently of IFN (17) and through induction of pathogenic CD8+ effector T cells (18).
Relatively few studies are available on the role of IL-12 in autoimmune (lymphocytic) thyroiditis, either in patients with Hashimoto’s thyroiditis or in mice. In patients, Weetman’s laboratory reported that IL-12 p40 mRNA was detected in three of four thyroid specimens removed at surgery from Hashimoto’s patients; it was also produced by a transformed human thyroid cell line (HT-ori3), thus indicating that thyroid follicular cells themselves (not only the infiltrating hematopoietic cells) are capable of expressing IL-12 (19). These findings support the report of Phenekos et al. (20), who showed that patients with Hashimoto’s thyroiditis have higher serum IL-12 levels than patients with Graves’ disease, toxic nodular goiter, or healthy controls. In mouse models of autoimmune (lymphocytic) thyroiditis, Cooke’s laboratory has shown a dual role for IL-12. When administered at the time of immunization with thyroglobulin (one or two ip doses of 300 ng), IL-12 increased disease severity (21). Similarly, lymph node cells from thyroglobulin-immunized mice were able to transfer a more severe form of thyroiditis if cultured with IL-12 for 3 d before transfer. On the other hand, thyroiditis was milder when induced in mice that do not have functional IL-12 p40 gene, or in mice treated with an antibody that blocks the endogenous IL-12 (21). In contrast, when administered for longer periods (300 ng ip, five times/wk for 2 or 3 wk, starting the day before immunization), IL-12 inhibited the induction of thyroiditis (21). These findings have been confirmed in other disease models. Tarrant et al. (22) found that early administration of IL-12 (injections on d 0–4) was more effective than late injections (on d 7–11) in suppressing uveitis. Gran et al. (23) recently reported that early administration of IL-12 (10–200 ng/mouse·d on d 0–5 post immunization) suppressed encephalomyelitis severity.
During autoimmune (lymphocytic) thyroiditis, IL-12 is produced in the thyroid gland early and throughout the course of the disease (24). To assess the effects of prolonged and localized production of IL-12, we have developed transgenic mice in which the expression of heterodimeric IL-12 p70 was targeted to the thyroid follicular cell under transcriptional control of the thyroglobulin promoter.
Materials and Methods
Construction and screening of the thyr-IL-12 transgenic mice
The thyr-IL-12 transgene was made by joining the rat thyroglobulin promoter, the mouse IL-12 cDNA, and part of the human GH gene (as a source of introns and polyadenylation signal). Both IL-12 subunits, p35 and p40, were present in the same construct to allow synthesis of the mature IL-12 p70 heterodimer by the same cell (Fig. 1A). The 4.4-kb transgene was excised by BamHI and KpnI digestion and injected into fertilized eggs from (CBA/J x C57BL/6J) F1 females. Founders were identified by Southern blot, performed as described (25), using a 753-bp PCR-amplified probe that hybridizes to the p35 subunit and part of the thyroglobulin promoter (Fig. 1A, arrows). Transgene-positive mice were maintained as hemizygous by backcrossing for at least 12 generations to wild-type CBA/J or SJL/J mice (from The Jackson Laboratory, Bar Harbor, ME), inbred strains that have an MHC class II haplotype conducive to the induction of experimental autoimmune thyroiditis (26).
FIG. 1. The thyr-IL-12 transgene. A, Construction of the transgene. The transgene was made by joining rat thyroglobulin promoter, mouse IL-12 p35, and part of the human GH for p35 expression, followed by rat thyroglobulin promoter, mouse IL-12 p40, and part of the human GH for p40 expression. The two IL-12 subunits are present in the same construct to allow the expression of IL-12 p70 by the thyrocytes. B and C, Transfection of FRTL-5 cells to assess the in vitro expression of the IL-12 transgene. The plasmid vector containing the transgene induced the synthesis of IL-12 p70, as assessed by indirect immunofluorescence with a specific IL-12 p70 antibody (from Biosource International) (B); transfection of the empty vector, not containing the IL-12 transgene, did not induce IL-12 p70 synthesis (C). D, Southern blot screening from tail genomic DNA. When genomic DNA from the three transgenic lines (LW1, LW2, and LW3) and wild-type control was probed with a fragment comprising a portion of the thyroglobulin promoter and p35 subunit (probe location indicated by the arrows in A), it showed integration of the IL-12 transgene; E, Representative PCR screening from tail genomic DNA. Lane 1, 100-bp DNA ladder; lane 2, water control; lane 3, plasmid-positive control; lanes 4 and 6, IL-12 transgenics; lanes 5 and 7, wild-type littermates.
After establishment of the transgenic lines, mice were screened at each filial generation by PCR from tail genomic DNA, using an upstream primer (5'-TgccTTggTAgcATcTATgAg-3') that binds to exon 6 of the murine IL-12 p35 gene, and a downstream primer (5'-TTcAggcggAgcTcAgATAg-3') on exon 7. This primer pair amplifies a 2609-bp band from the wild-type IL-12 allele and a 294-bp band from the transgene.
Analysis of thyr-IL-12 transgene expression
The ability of the transgene to synthesize IL-12 p70 was first assessed in vitro by transfecting rat FRTL-5 cells with the IL-12 construct cloned into pBluescript II KS (+/–) plasmid (Stratagene, La Jolla, CA). FRTL-5 cells (CRL-1468; ATCC, Manassas, VA) were grown in Coon’s modified F-12 medium containing 5% heat-inactivated, mycoplasma-free calf serum, and 1 mM nonessential amino acids supplemented with a mixture of six hormones, including bovine TSH (1 x 10–10 M), insulin (10 μg/ml), cortisol (0.4 ng/ml), transferrin (5 μg/ml), glycil-L-histydil-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml). One microgram of plasmid was mixed with 4 μl lipofectamine transfection reagent (Invitrogen, Carlsbad, CA) and incubated for 3 h with FRTL-5 cells cultured in 6-well plates. After addition of fresh growth medium, cells were incubated for 24 h and then treated for 5 h with 2 μM monensin, a well-characterized inhibitor of protein secretion that allows accumulation of cytokines within the cells. Cells were finally washed, fixed, and stained with a fluorescein isothiocyanate-conjugated IL-12 p70 antibody (Biosource International, Camarillo, CA) to evaluate, by direct immunofluorescence, the expression of mature IL-12.
In vivo, IL-12 expression was evaluated at the RNA level by RT-PCR performed on various tissues (thyroid, salivary and lachrymal glands, heart, lung, kidney, and liver), and at the protein level by measuring IL-12 p70 in sera. For RNA analysis, tissues were first digested by collagenase and dispase as previously described (27), to prepare a single cell suspension that was then incubated with CD45 magnetic beads (Miltenyi Biotec, Auburn, CA). The CD45-postive fraction, a potential source of IL-12, was removed, and the remaining cells were used to extract mRNA (Dynal Biotech, Brown Deer, WI). mRNA was treated with DNase I (Invitrogen) and reverse transcribed using Superscript II (Invitrogen) and oligo(d)T primers. PCR was performed with primers for murine IL-12 p35 (forward: 5'-TgccTTggTAgcATcTATgAg-3' and reverse 5'-TTcAggcggAgcTcAgATAg-3'), and with primers for G3PDH as control (forward: 5'-gcATcTTgggcTAcAcTgAg-3' and reverse: 5'-TcTcTTgcTcAgTgTccTTg-3').
Serum IL-12 p70 was measured by ELISA and cytokine array, using blood immediately chilled after drawing and separated by a microtainer tube (Becton Dickinson and Co., Franklin Lakes, NJ). ELISA was performed with a commercially available kit (R&D Systems Inc, Minneapolis, MN), following the manufacturer’s recommendations. Cytokine arrays, chosen to detect the greatest number of cytokines with the smallest volume of serum, were purchased from RayBiotech (Norcross, GA). The array allows for the simultaneous detection of 31 molecules distributed as follows: eleven cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-17, IFN, and TNF-); eleven chemokines [CCL2 (MCP-1/JE), CCL3 (MIP-1 ), CCL5 (Rantes), CCL11 (Eotaxin), CCL12 (MCP-5), CCL17 (TARC), CCL19 (MIP-3 ?), CCL21 (6Ckine), CCL27 (CTAK), CXCL1 (KC), and CXCL2 (MIP-2)]; five hematopoietic promoting molecules (IL-3, G-CSF, GM-CSF, stem cell factor, and thrombopoietin); and four other molecules (leptin, tissue inhibitor of metalloproteinases 1, soluble TNF receptor 1, and vascular endothelial growth factor). Sera were diluted 1:10 in the provided blocking buffer, and incubated at 4 C overnight with the arrayed antibody membrane. After addition of biotinylated anticytokine antibody cocktail and horseradish peroxidase-conjugated streptavidin, a colororimetric signal was induced by addition of the provided detection buffer. The membrane was finally exposed to radiographic film [x-omat AR (Kodak, Rochester, NY)], and the signal was analyzed for gray scale intensity using the free software Image J (http://rsb.info.nih.gov/ij).
Growth curves, thyroid histopathology and immunohistochemistry, thyroglobulin immunization, and thyroglobulin antibodies
Mice were weighed at several time points throughout their lifetime to compare growth between transgenics and wild-type littermates.
For histopathology, thyroids were removed after euthanasia and fixed for 48 h in the zinc-based Beckstead’s solution. After processing and embedding in paraffin, six to eight nonsequential sections (5-μm thick) were cut from tissue blocks and stained with hematoxylin and eosin. Immunohistochemistry was performed to determine the nature of the cells infiltrating the thyroid, as described (28). Briefly, sections were deparaffinated, rehydrated, and blocked with 2% normal goat serum. Sections were incubated overnight at 4 C with rat antimouse CD45 (BD PharMingen, San Diego, CA), hamster antimouse CD3 (BD PharMingen), rat antimouse B220 (BD PharMingen), or rat antimouse F4/80 (Serotec, Raleigh, NC). After washing, addition of the biotinylated anti-IgG secondary antibody, and incubation with peroxidase-conjugated streptavidin, the brown positive color was revealed by the addition of diaminobenzidine substrate (Sigma, St. Louis, MO).
Thyroglobulin immunization was done to assess whether the presence of transgenic IL-12 within the thyroid would enhance the incidence and/or severity of thyroiditis. Given the severity of disease that we achieve by our standard immunization protocol [75 μg thyroglobulin in complete Freund’s adjuvant on d 0 and 7 (25)], we considered unlikely the detection of any further enhancement of disease by IL-12 in this system. We therefore used, for this experiment, a suboptimal immunization protocol (25 μg thyroglobulin rather than 75 μg) and the transgenic line with the weakest expression of IL-12 (the LW2 line). We also used the SJL, rather than the CBA strain, because, while responding equally well to thyroglobulin immunization (26), it has a superior breeding performance (http://aretha.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home). All experimental protocols conformed to Johns Hopkins Animal Care and Use Committee guidelines.
Thyroglobulin antibodies were measured as described (25). Briefly, sera were added in triplicates to Immulon2 ELISA plates (DYNEX Technologies, Inc., Chantilly, VA) coated with 100 ng/well murine thyroglobulin. After overnight incubation, plates were washed and incubated with secondary antibodies against IgG1, IgG2c, and IgG2b conjugated to alkaline phosphatase. Each plate included a standard curve derived from serial dilutions of a pool serum with known mouse thyroglobulin antibodies. The IgG2c (IgG2ab) isotype was chosen because in the SJL strain, as in the C57BL6 and NOD strains, the IgG2a (IgG2aa) gene is deleted (29).
Assessment of thyroid function by total T4 and TSH levels
Total T4 levels were determined using a commercial competitive RIA (GammaCoat 125I-T4; DiaSorin, Inc., Stillwater, MN). Mouse TSH was measured with a highly sensitive, double-antibody RIA developed by A.F.P (30). Briefly, the assay employs a highly purified rat TSH (AFP11542B) as the iodinated ligand, a selected guinea pig antimouse TSH (AFP98991 as the primary antibody, and a partially purified extract of mouse pituitary containing TSH (AFP5171.8MP) as the reference preparation.
Changes in thyroid gene expression induced by IL-12
The effect of IL-12 on thyroid gene expression was studied in vivo and in vitro. For in vivo studies, thyr-IL-12 transgenic mice and wild-type littermates were killed and thyroidectomized. After dissection and mechanical disruption of the thyroid lobes, mRNA was extracted (mRNA direct kit from Dynal Biotech), treated with DNase I (Invitrogen), and reverse transcribed. cDNA was then amplified to assess the expression of sodium-iodide symporter (5'-gcTcTcATcAgcTAccTAAcTgg-3' and 5'-cTcAgAggTTggTcTcAAcATc-3'), thyroglobulin (5'-cgTgTTTgTcccTgAgAAccTg-3' and 5'-TccgTTgAgAAgTAgcccTggTAg-3'), thyroid peroxidase (5'-TgccAAcAgAAgcATggTcAAc-3' and 5'-gcAcAAAgTTcccATTgTccAc-3'), and TSH receptor (5'-cggTTccTcATgTgcAAcTTg-3' and 5'-ccTcTTggcAATcTTggTgTc-3'). To compare gene expression between transgenics and wild-type littermates, semiquantitative RT-PCR was performed using G3PDH as the gene against which to compare the expression of the other messages.
For in vitro studies, rat FRTL-5 cells, maintained in the complete six-hormones medium as described (31), were incubated with 10 ng/ml recombinant mouse IL-12 (R&D Systems, Minneapolis, MN) and harvested 12, 24, or 48 h thereafter. Total RNA was then extracted (RNeasy mini kit from QIAGEN, Valencia, CA), separated by agarose gel electrophoresis, and analyzed by Northern blot for the expression of sodium-iodide symporter, thyroglobulin, thyroperoxidase, and TSH receptor, as described (31). The signal from G3PDH was also obtained to adjust for the amount of RNA loaded in each lane. Probe signals were quantified using the BAS-1500 Bioimaging Analyzer (Fuji Photo Film Co., Ltd., Japan).
Statistical analysis
The population of mice used for the analyses presented in this study included a total of 244 mice: 221 on the CBA/J background and 23 on the SJL/J background. The CBA/J mice comprised 60 wild-type (14 males and 46 females) and 161 thyr-IL-12 transgenics (62 males and 99 females) and were used to analyze statistically the following outcomes: body weight; thyroid histopathology in baseline conditions; and serum levels of IL-12 p70, total T4, and TSH. The SJL/J mice comprised 10 thyr-IL-12 transgenics of the LW2 line and 13 wild-type littermates and were used to analyze thyroid histopathology, thyroglobulin antibodies, and total T4 after mouse thyroglobulin immunization.
Body weight was measured longitudinally in 91 thyr-IL-12 transgenics (25 LW1: 13 males and 12 females; 40 LW2: 22 males and 18 females; and 26 LW3: 15 males and 11 females) and 21 wild-type controls (11 males and 10 females) on d 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 90, 120, 180, 300, and 365 after birth. Differences in weight between transgenics and controls were assessed by multiple linear regression with generalized estimating equations, as described (32).
Thyroid histopathology in basal conditions was analyzed in 99 IL-12 CBA/J transgenics (21 LW1, 47 LW2, and 31 LW3) and 39 wild-type CBA/J controls, 38–466 d old. Thyroid sections were scored by digital microscopy (27) and assessed in a multiple linear regression model including as covariates: sex, age, and the four genotypes (wild type, LW1, LW2, and LW3). Thyroid histopathology, after thyroglobulin immunization, was analyzed in 10 LW2 SJL/J transgenics and 13 wild-type SJL/J littermates. Differences in the score were evaluated by the Wilcoxon rank-sum test.
Serum IL-12 p70 was measured in 39 transgenics (13 LW1, 17 LW2, and 9 LW3) and eight wild-type controls, total T4 in 83 transgenics (24 LW1, 32 LW2, and 27 LW3) and 20 controls, and TSH in 42 transgenics (15 LW1, 13 LW2, and 14 LW3) and 19 controls. Differences in the mean serum IL-12, T4, or TSH levels among the four groups were assessed by the Kruskal-Wallis test, followed by pairwise comparisons using the Wilcoxon rank-sum test. Serum TSH was also assessed in a multiple linear regression model including as covariates: T4, sex, age, and the four genotypes.
All analyses were performed using Stata statistical software, release 8 (from Stata Corp., College Station, TX).
Results
Thyr-IL-12 transgene expression and basal phenotype
The ability of the transgene to support synthesis of IL-12 was first assessed in vitro by transfection of FRTL-5 cells. The mature IL-12 p70 heterodimer was produced upon transfection of the IL-12 transgene (Fig. 1B) but not of the control plasmid (Fig. 1C). In vivo, the transgene stably integrated into the genome of six of the 114 injected mice. Three of these six founders, named thyr-IL-12 transgenic LW1, LW2, and LW3 line (Fig. 1D), were maintained as hemizygous by mating to normal inbred strains (CBA/J for most of the experiments and SJL/J for thyroglobulin immunization) and screened by PCR (Fig. 1E) to form the cohort described in the present study.
Transgenic IL-12 was expressed in the thyroid of the three transgenic lines but not in the other organs examined or in the thyroid of wild-type controls (Fig. 2A). The thyroidal production of IL-12 p70 was also capable of reaching the systemic circulation, as demonstrated by the significantly higher serum IL-12 levels in the three transgenic lines compared with wild-type controls (P = 0.0002 by Kruskal-Wallis test) (Fig. 2B). Mice of the LW2 line tended to have lower serum IL-12 levels than those of the LW1 or LW3 lines, although the difference was not statistically significant. The increased serum levels were restricted to IL-12 and not seen for IFN (Fig. 2, C and D). In addition, the other cytokines and chemokines assayed by the array were not detectable or were no different between transgenics and controls (data not shown).
FIG. 2. Expression of IL-12 in thyr-IL-12 transgenic mice. A, IL-12 mRNA measured by RT-PCR. RT+ and RT– indicate that the PCR amplification of the cDNA was performed with or without previous reverse transcription. The organs used were thyroids, lungs, lachrymal glands (lac.), kidneys (kid.), liver (liv.), heart and salivary glands (sal.), all depleted of CD45-positive hematopoietic cells following the procedure described in (27 ). N is the water control, P is the plasmid-positive control. Note that only transgenic thyroids express IL-12 mRNA. B, Serum IL-12 p70 measured by ELISA. Note the increased expression in the three transgenic lines, compared with wild-type controls. C and D, Serum IL-12 p70 and IFN measured by cytokine arrays. IL-12 was increased in transgenic sera, whereas IFN was undetectable in both IL-12 transgenic and controls. The signal of the positive control provided by the manufacturer is also shown to adjust for differences between wild-type and transgenic array membranes. To create the graph shown in D, signals from the radiographic film were scanned and analyzed on a gray scale comprised between 0 and 255.
IL-12 transgenic mice showed no gross physical and behavioral abnormalities and delivered a number of offspring similar to that of normal CBA/J (4.2 average pups per litter) or SJL/J (6.2 pups per litter) strains. There was, however, a distortion in the frequency by which the transgenic allele was transmitted to the offspring. According to Mendel’s first law (principle of segregation), the cross between a hemizygous transgenic and a wild-type animal should yield an equal number of transgenics and wild-types in the progeny. In contrast, the transgenic father-to-normal female mating scheme yielded a transgenic-to-wild-type ratio of 0.31 for the LW1 line (90:294 mice), of 1.01 for the LW2 line (167:165), and 0.25 for LW3 line (78:316). This ratio departed even further from one in the transgenic mother-to-normal male mating scheme, being 0.25 for LW1 (4:16), 0.67 for LW2 (16:24), and 0.06 for LW3 (3:52). The reasons for this reduced transmission of the IL-12 transgene to the progeny are unknown at the moment.
Growth defect and spontaneous, although minimal, lymphocytic thyroiditis
Thyr-IL-12 transgenic mice were smaller than wild-type littermates (Fig. 3A). In particular, regression analysis showed that, holding sex and age constant, transgenics were 3.26 g lighter than controls (95% confidence interval, 2.87–3.65, P < 0.0001). The growth defect became clearly evident at the time of weaning (d 21) and persisted throughout life. Similarly to wild-type littermates, females were 2.98 g smaller than males (95% confidence interval, 2.61–3.56; P < 0.0001) (data not shown). There was no significant difference in weight among the three transgenic lines, which therefore were combined in the analysis shown in Fig. 3A.
FIG. 3. Growth curves and thyroid pathology. A, Growth: thyr-IL-12 transgenic mice showed significant growth retardation compared with wild-type littermates. B–D, Thyroid histopathology: thyroid glands showed enlarged and flattened follicular cells (B) and a clear, albeit minimal, mononuclear cell infiltration (C), frequently found around vessels (D), and connective tissue capsule (C and D). E and F, Nature of the infiltrate: immunohistochemistry indicated that the infiltrate was mainly composed of B220+ B lymphocytes (E) and also CD3+ T lymphocytes (F).
Thyroid follicles were often enlarged and lined by a flattened epithelium (Fig. 3B and Table 1), an appearance seen in patients with long-standing hypofunctioning goiter (such as the colloid involution phase of the diffuse nontoxic goiter). Transgenic thyroids also showed, at baseline (i.e. before immunization), a mononuclear cell infiltration (Fig. 3C) that was frequently localized around vessels (Fig. 3D), and underneath the connective tissue capsule (Fig. 3, C and D). The infiltrating cells were lymphocytes, mainly comprised of B220-positive B cells (Fig. 3E), and CD3-positive T cells (Fig. 3F). There were only few F4/80-positive macrophages, with no difference between transgenics and controls (data not shown). Prevalence and severity of this infiltration were greater in transgenic lines expressing the higher levels of IL-12 (LW1 and LW3) than in the LW2 line (Fig. 4A and Table 1). Overall, the infiltration was clear (>2% of the total thyroid area), but of moderate intensity; none of the transgenic mice, in fact, developed spontaneously an overt infiltration comprising more than 15% of the total thyroid area (Fig. 4A). Thyroglobulin antibodies were absent when measured at several time points throughout the life of the mice (data not shown), suggesting perhaps that, in patients with Hashimoto’s thyroiditis, these antibodies are not related to the initial immunopathology.
TABLE 1. Morphologic changes seen in the thyroids of the three thyr-IL-12 transgenic lines (LW1, LW2, and LW3)
FIG. 4. Effect of thyroglobulin immunization. A, Baseline infiltration score. Before immunization, thyroids of IL-12 transgenic mice showed mononuclear cell infiltration, which was significantly greater in the lines expressing the higher IL-12 levels (LW1 and LW3) than in the low-expressor line (LW2). P values are based on pairwise comparisons using the Wilcoxon rank-sum test, performed after the Kruskal-Wallis test (P = 0.0001). The infiltration was clear (>2% of the total thyroid area) but overall of low severity (<15% of the total thyroid area). B, Infiltration after challenging LW2 transgenic mice with low doses (25 μg on d 0 and 7) of mouse thyroglobulin. This suboptimal immunization regimen induced a more frequent and severe thyroiditis in transgenics than controls (P = 0.0003 by Wilcoxon rank-sum test). C, Thyroid function after immunization. Total T4, which at baseline was slightly lower in LW2 transgenics than controls (see also Fig. 5A), decreased significantly 21 d after immunization (P = 0.023 vs. d 0 by Wilcoxon matched-pairs signed-ranks test), reaching similar levels in transgenics and controls. WT, Wild type.
Considering that transgenic IL-12 was capable of inducing minimal lymphocytic thyroiditis, we tested whether these infiltrates could lead to an increased incidence and severity of disease upon induction of experimental autoimmune thyroiditis. We therefore immunized 10 thyr-IL-12 SJL/J transgenics of the LW2 line and 13 SJL/J littermates with a suboptimal dose of mouse thyroglobulin (25 μg), assessing thyroid morphology, thyroglobulin antibodies, and total T4 3 wk thereafter. As shown in Fig. 4B, the presence of IL-12 in the thyroid gland resulted in a more incident and severe thyroiditis (P = 0.0003), indicating a disease-promoting role for this cytokine. Total T4, which began at lower levels in the LW2 transgenic line, decreased significantly 21 d after immunization (Fig. 4C; P = 0.023, comparing d 0 vs. d 21 with the Wilcoxon matched-pairs signed-rank test). This decrease in T4 after immunization, which confirms what we previously published (25), reached similar levels in both genotypes, suggesting that the lower T4 levels observed at baseline in LW2 transgenic mice (see also Fig. 5A) represent the direct effect of IL-12 on thyrocytes rather than the consequence of the lymphocytic infiltration. Thyroglobulin antibodies at d 21 were present at similar levels in transgenics and controls and overall at low titer (data not shown), likely a consequence of the mild immunization protocol.
FIG. 5. Thyroid function. A, Serum total T4. T4 was decreased in thyr-IL-12 transgenic mice compared with wild-type controls. The decrease reached statistical significance for the LW1 (P = 0.0007 vs. control) and LW3 line (P = 0.0001) but not for the LW2 line (P = 0.065). P values are based on pairwise comparisons using the Wilcoxon rank-sum test, performed after the Kruskal-Wallis test (P = 0.0002). B, Serum TSH. TSH was increased in thyr-IL-12 transgenic mice compared with wild-type controls. The increase reached statistical significance for the LW1 (P = 0.016 vs. control) and LW3 line (P = 0.0001) but not for the LW2 line (P = 0.108). C, Relationship between serum T4 and TSH. Regression analysis showed that T4 significantly predicted TSH values (P = 0.007), although the proportion of variation in TSH that could be predicted by T4 was small (r2 = 0.39). CI, Confidence interval.
Primary hypothyroidism
Serum total T4, examined at multiple times throughout life, was lower in all three transgenic lines than in wild-type controls (Fig. 5A). The decrease reached statistical significance for the LW1 line (P = 0.0007 vs. wild-type) and LW3 line (P = 0.0001) but not for the LW2 line (P = 0.065). There was no statistical difference in the mean T4 levels among the three transgenic lines, although T4 was less decreased and closer to the normal range in the LW2 line (Fig. 5A).
Serum TSH levels followed a similar pattern but in the opposite direction. They were significantly increased in the LW1 (P = 0.016) and LW3 (P = 0.0001) transgenic lines compared with wild-type controls (Fig. 5B). In the LW2 line, TSH levels were higher but not statistically different from those observed in normal controls (P = 0.108). Multiple linear regression modeling showed that T4 significantly predicted the serum TSH levels (for every 1 μg/dl increase in T4 there is a 16.9 U/ml decrease in TSH, holding age, sex, and genotype constant; P = 0.007; Fig. 5C). The proportion of the variation in TSH that could be predicted by T4 in this model was, however, small (0.39; Fig. 5C), confirming the observation that, in mice, serum T4 levels do not correlate strongly with TSH levels (33).
Taken together, the histological and biochemical data indicate that the presence of IL-12 within the thyroid gland induces a moderate primary hypothyroidism. The hypothyroidism was more severe in the two transgenic lines that expressed higher IL-12 levels (LW1 and LW3) than in the low expressor line (LW2), suggesting that it is a direct consequence of the action of IL-12 on the thyroid follicular epithelium rather than secondary to the minimal lymphocytic infiltrate.
Mechanisms of hypothyroidism
To understand how IL-12 induced primary hypothyroidism, we extracted thyroid mRNA and performed semiquantitative RT-PCR to evaluate the expression of some thyroid-restricted genes. Sodium iodide symporter (NIS) was strongly up-regulated in transgenics, showing a 4-fold increase over wild-type controls (Fig. 6, A and B). Thyroid peroxidase and thyroglobulin were also increased in transgenics, although less markedly (Fig. 6, A and B). In contrast, the expression of the TSH receptor gene was reduced (Fig. 6, A and B). This gene expression profile could, overall, be reconciled with the increased serum TSH levels present in the thyr-IL-12 transgenic mice. It is, in fact, well established that increased TSH levels up-regulate NIS (34, 35), thyroperoxidase (36, 37), and thyroglobulin (38) and, at the same time, down-regulate the TSH receptor (39, 40).
FIG. 6. Analysis of thyroid gene expression. A and B, In vivo analysis. mRNA was extracted from the thyroids of 2- to 3-month-old LW3 transgenic mice and age-matched controls, reverse transcribed, and PCR amplified to assess the expression of four thyroid-specific genes. PCR products were fractionated by agarose gel electrophoresis and stained with ethidium bromide (A). Images were then acquired with a digital camera to express the intensity of the bands on a continuous numeric scale. Results were finally expressed as a ratio of intensities between transgenics and wild types, adjusting for the intensity of a housekeeping gene (G3PDH). A change in intensity greater than 50% was considered significant. Thyroids from LW3 thyr-IL-12 transgenics showed increased expression of NIS and also thyroglobulin (TG) and thyroperoxidase (TPO). On the contrary, the expression of TSH receptor (TSH-R) was reduced. C and D, Time course expression of four thyroid-specific genes upon addition of recombinant IL-12 (10 ng/ml) to FRTL-5 cultured cells. FRTL-5 cells were collected 12, 24, and 48 h after addition of IL-12 to the culture medium to extract total RNA. RNA was then fractionated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized to specific radioactive probes (C). Probe signals were quantified with a phosphoroimager and expressed as a ratio of intensity between thyroid genes and housekeeping gene (G3PDH), adjusting for the intensity at time zero. A change in intensity greater than 50% was considered significant. Addition of IL-12 induced a 2-fold increase in NIS expression.
To distinguish whether the striking increase in NIS expression was simply the consequence of increased TSH levels or, rather, also a direct (TSH-independent) effect of IL-12 on thyrocytes, we added IL-12 (10 ng/ml) to FRTL-5 cells grown in complete (six hormones) medium and measured gene expression 12, 24, or 48 h thereafter. In keeping with the in vivo results, IL-12 increased NIS expression (Fig. 6, C and D), which peaked at 12 h and remained elevated for 24 h. NIS then declined to baseline at 48 h, possibly suggesting a diminished IL-12 activity in a culture medium that allows for continuous thyrocyte growth. In contrast, IL-12 did not increase, or change significantly, the expression of the other thyroid-specific genes (Fig. 6, C and D), suggesting that their changes observed in vivo were mainly the consequence of increased TSH stimulation. These novel findings indicate that IL-12 is capable of increasing NIS expression and, at the same time, inducing hypothyroidism.
Discussion
This study reports that transgenic mice expressing IL-12 specifically in the thyroid gland develop moderate lymphocytic thyroiditis and primary hypothyroidism. Studies in other murine models of autoimmunity (encephalomyelitis, type 1 diabetes, and myocarditis) had indicated a similar disease-promoting role for this cytokine. It was unclear, however, whether IL-12 was sufficient to initiate disease or, rather, was only one of the many cofactors associated with disease. Even when considering solely murine studies of lymphocytic thyroiditis, the role played by IL-12 remains uncertain (21, 41). To gain insights on the function of IL-12 in Hashimoto’s thyroiditis, we created transgenic mice that secrete relatively low levels of IL-12 inside the thyroid in a chronic fashion, with the intent of mimicking one aspect of disease pathogenesis.
Our results show that IL-12 induces a moderate lymphocytic thyroiditis that, despite never reaching the severity seen in patients with frank Hashimoto’s thyroiditis, clearly and significantly differed from the normal thyroid morphology. This disease-promoting role of IL-12 showed dose-dependence because it was stronger in the transgenic lines expressing the higher IL-12 levels (LW1 and LW3) than in the low expressor line (LW2). It was also confirmed by immunization experiments that used a suboptimal regimen and the low expressor line. Here IL-12 was capable of inducing a more incident and severe thyroiditis than that seen in wild-type littermates.
Uncertainty remains on the mechanism through which IL-12 exerts its disease-promoting effect. IL-12 may enhance the antigen presenting capacity of resident dendritic cells (42) or their ability to release IFN (43). In our thry-IL-12 transgenic mice, however, the effect of IL-12 appears independent of IFN. IFN, in fact, was undetectable when assayed in properly collected sera. Although a formal proof of the IFN independence would likely require a cross between thyr-IL-12 transgenic and IFN knock-out mice, previous work has shown that systemic administration of IL-12 is not associated with increased serum IFN levels (44, 45). Our findings are in keeping with recent studies that have revealed contrasting roles of the two prototypic Th1 cytokines: IL-12 and IFN. Using the murine model of autoimmune myocarditis, Afanasyeva et al. (17) have shown that IL-12 is capable of inducing disease without using the IFN pathway. We have also previously shown that IFN has a protective, rather than disease-promoting, effect in experimental autoimmune thyroiditis (46). These findings are consistent with the results of the present study, and they highlight a novel, dichotomous role for IL-12 and IFN, two classic Th1 cytokines commonly considered to act in synchrony.
The different outcomes induced by IL-12 may be explained by the redundancy and complexity of the IL-12 system, which also includes IL-23 and IL-27. IL-23 is a heterodimer composed of the same p40 subunit as IL-12 and a novel p19 subunit. Using the murine model of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein, Cua et al. (47) showed that disease susceptibility was retained in p35 (IL-12)-deficient mice but was abolished in p19 (IL-23)-deficient mice or p40 (IL-12 and IL-23)-deficient mice, indicating that disease induction mainly depends upon IL-23 signaling. Similar results were obtained with IL-27, a heterodimer composed of EBI3 (a p40-related protein) and p28 (a p35-related protein). Blockade of p28 with a specific antibody significantly decreased disease severity in experimental autoimmune encephalomyelitis (48). These findings suggest that IL-12, originally considered necessary for encephalomyelitis induction (49), may behave more as a disease modulator rather than a promoter of encephalomyelitis (23) and that the coordinated expression of the two subunits forming each cytokine is crucial for appropriate immune responses in timing, location, and magnitude (50).
Our study reveals a novel effect of IL-12 on thyroid function: primary hypothyroidism associated with increased NIS mRNA expression. The hypothyroidism is likely a direct effect of IL-12, rather than the consequence of the lymphocytic infiltration, because it was present even in transgenic mice that did not have apparent infiltration, and the extent of the infiltration was too small to cause loss of function of the entire thyroid gland observed in our transgenic mice. It is interesting to speculate on the mechanism by which IL-12 causes hypothyroidism, in the context of thyroid hormonogenesis. Thyroid hormone synthesis begins with the NIS-mediated influx of iodine from the blood into the thyroid cell. Iodine then moves through the cytosol by still-undefined mechanisms, reaches the apical membrane, and is transported into the follicular lumen by specific proteins, such as pendrin and apical iodide transporter. Within the follicle, on the luminal side of the apical membrane, iodine is then oxidized by thyroperoxidase in the presence of hydrogen peroxide and subsequently placed on selected tyrosyl residues of thyroglobulin, a process referred to as organification or iodination. Thyroperoxidase also catalyzes the next step, which is the coupling of two iodinated tyrosyl residues. When needed, the iodinated thyroglobulin, stored as colloid in the follicular lumen, reenters the thyrocyte via micropinocytosis. Here, thyroglobulin-containing vesicles fuse with lysosomes that degrade thyroglobulin, releasing thyroid hormones that enter the bloodstream at the basolateral membrane, likely by way of specific thyroidal channels that still await identification. The gene expression profile of the IL-12 transgenic thyroids (increased expression of NIS, thyroperoxidase, and thyroglobulin) suggests that IL-12 inhibits thyroid hormone synthesis downstream of the organification reaction, although the precise location of the block is unknown at the moment. This inhibitory effect is strong enough to override the thyroid stimulation induced by the increased serum TSH levels.
The biological significance of the increased NIS transcription induced by IL-12 remains unknown, considering that increased NIS mRNA levels do not always correlate with greater capacity of the thyrocyte to uptake iodine (35, 51). The stimulatory effect of IL-12 on NIS mRNA expression, however, is worth of attention. The best-known NIS regulators are TSH, which increases NIS transcription, biosynthesis, and presence in the plasma membrane, and iodine itself (52). Cytokines, however, also have an effect on NIS expression, usually a decrease. TGF-? (53), IL-1, IL-6, and TNF- (54), and IFN (55) have all been shown to suppress NIS mRNA expression when added in vitro to FRTL-5 cell cultures. We now report the first cytokine, IL-12, that is capable of increasing NIS expression in vivo. The effect of IL-12 is direct because it can be reproduced by addition of IL-12 to cultured thyroid cells. The effect also highlights the complexity and the contrasting roles of the two prototypic Th1 cytokines, considering that we have previously reported that transgenic mice expressing IFN in the thyroid develop primary hypothyroidism associated with suppression of NIS gene transcription, NIS protein expression, and iodine uptake (56).
This observation has potential clinical implications. IL-12 has, in fact, shown promising results in treating differentiated thyroid carcinoma. When murine IL-12 p70 was expressed and delivered via an adenovirus vector, it induced effective antitumor activity and long-term immunity against medullary (57, 58, 59) and follicular (60) rat thyroid carcinomas. The use of IL-12 to treat thyroid cancers could be of value not only for the above described antitumor activity but also for its ability to increase NIS expression and, consequently, the uptake of therapeutic radioactive iodine into thyroid follicular cells.
The effect of IL-12 on thyroid function could be more global than the effect shown by this paper on the thyrocytes. For example, Boelen et al. (62) have reported that the lipopolysaccharide-induced decrease in type I deiodinase [the selenoprotein that converts T4 to T3 (61)] within the pituitary is less pronounced in IL-12-deficient mice than in wild-type controls. Thus, IL-12-deficient mice are predicted to have a greater T4 to T3 conversion, with resulting pituitary-specific thyrotoxicosis and inhibition of TSH secretion. The opposite scenario could occur in the presence of increased serum IL-12 levels that should lead to reduced T4 to T3 conversion within the pituitary and increased TSH secretion.
In conclusion, we have shown, for the first time, that local production of IL-12 in the thyroid enhances the expression of sodium-iodide symporter and inhibits thyroid hormonogenesis downstream of the organification, thus inducing primary hypothyroidism. The effect on NIS is independent of, and opposite to, the action of the other prototypic proinflammatory cytokine, IFN. The results provide new insights into the complex checks and balances of the inflammatory response.
Acknowledgments
The authors acknowledge Mehrdad Hejazi and Liwen He for their contribution in the initial stages of the project.
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