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Limits Experimental Autoimmune Thyroiditis by Suppressing Lymphocyte Activation in Cervical Lymph Nodes1
* Department of Pathology, School of Medicine, and
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205
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Abstract |
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in the pathogenesis of autoimmune disease is controversial, being described as immunostimulatory in some studies and immunosuppressive in others. To determine the contribution of local expression of IFN-, we derived NOD.H-2h4 transgenic mice overexpressing IFN- in a thyroid-restricted manner. Transgenic mice, which had serum IFN- levels similar to wild-type littermates, showed up-regulation of MHC class II on thyrocytes, but did not develop spontaneous thyroiditis. Upon immunization with murine thyroglobulin, transgenic mice developed milder disease and reduced IgG1 responses compared with wild type. The milder disease was associated with decreased frequency of activated CD44+ lymphocytes in the cervical lymph nodes. This suppressive effect was confirmed by showing that blockade of systemic IFN- with mAb enhanced disease and increased IgG1 responses. The study supports a disease-limiting role of IFN- in autoimmune thyroiditis. Furthermore, it provides the first evidence that local IFN- activity in the thyroid is sufficient for disease suppression. |
Introduction |
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, the prototypic Th1 cytokine (1, 2), promotes Th1 development, activates macrophages, induces MHC class II expression on hemopoietic and epithelial cells, and up-regulates vascular adhesion molecules, leading to leukocyte recruitment to the site of inflammation (3). Although these functions would predict that IFN- promotes cell-mediated autoimmune diseases, the role of IFN- in these diseases is controversial. In some autoimmune diseases, such as spontaneous murine models of type 1 diabetes (4, 5) and thyroiditis (6), IFN- enhances disease. In others, such as in murine models of experimentally induced encephalomyelitis (7, 8, 9), uveitis (10, 11), arthritis (12), and myocarditis (13, 14), IFN- limits disease.
IFN- has also been considered a disease-promoting factor in human chronic lymphocytic (Hashimoto’s) thyroiditis, one of the most frequent autoimmune diseases (15), which is characterized by mononuclear cell infiltration of the thyroid gland, production of autoantibodies against thyroid-specific Ags, and hypothyroidism. In fact, patients with Hashimoto’s thyroiditis exhibit increased serum levels of IFN- (16) and increased expression of intrathyroidal IFN-, as demonstrated by immunohistologic studies (17) and high levels of IFN- production by clones derived from infiltrating lymphocytes (18, 19).
This report addresses the role of IFN- in the pathogenesis of murine experimental autoimmune thyroiditis (EAT),3 a model of thyroiditis that can be induced in mice by immunization with thyroglobulin and adjuvant (20, 21). Susceptibility to murine EAT induction is predominantly controlled by the MHC class II locus; I-Ak-, I-Aq-, or I-As-bearing strains are susceptible, while I-Ab- or I-Ad-bearing strains are resistant to thyroiditis (22, 23). Previous studies have provided conflicting results on the role of IFN- in murine EAT. IFN- has been shown to promote (24, 25), suppress (26), or have no influence on EAT induction (27, 28, 29). All these studies have relied on manipulations influencing systemic effects of IFN-, either by the injection of Abs that block IFN- or by the use of knockout mice that lack IFN- or its receptor.
In this report, we focused on the local action of IFN-. We induced EAT in transgenic mice that overexpress IFN- specifically in the thyroid gland (30), using the NOD.H-2h4 background, a susceptible I-Ak-bearing strain (31). The results presented here show that thyroid-specific overexpression of IFN- in NOD.H-2h4 mice limits autoimmune thyroiditis, indicating that local IFN- activity is effective for disease control.
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Materials and Methods |
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NOD.H-2h4 mice, originally the gift of Dr. L. Wicker (Merck Laboratories, Rahway, NJ), were bred in specific pathogen-free facilities at Johns Hopkins University School of Medicine (Baltimore, MD). NOD.H-2h4 mice are I-E-negative and express the thyroiditis-susceptible I-Ak allele on the nonobese diabetic (NOD) background (31). Although the original NOD.H-2h4 strain was shown to develop spontaneous thyroiditis at low incidence (31) and iodine-induced thyroiditis at higher incidence (32), the present line has lost this phenotype, as we and others have observed (33). NOD.H-2h4 mice were used in two series of experiments.
In the first one (transgenic experiment), transgenic mice expressing IFN- specifically in the thyroid gland (originally on the C57BL/6 background) (30) were backcrossed to the NOD.H-2h4 background for seven successive generations (identified as thyr-IFN- transgenic). At each generation, tail genomic DNA was screened by PCR for the presence of the transgene as previously described (30). Briefly, upstream (5'-cAcggcAcAgTcAATgAAAg-3') and downstream (5'-ccTTgcTgTTgcTgAAgAAg-3') primers were designed against exons 1 and 4 of the murine IFN- gene to produce a 258-bp cDNA amplicon, distinct from the 1531-bp genomic amplicon. In the second series (blockade experiment), NOD.H-2h4 mice were injected with an Ab that blocks IFN-, as indicated below. All protocols conformed to Johns Hopkins Animal Care and Use Committee guidelines.
In the second series of experiments (blockade experiment), NOD.H-2h4 mice were injected with a rat IgG1 anti-mouse IFN- mAb (clone R4-6A2; American Type Culture Collection, Manassas, VA), or a rat IgG1 anti-Escherichia coli 
-galactosidase mAb as control (clone GL113; gift of F. Finkelman, University of Cincinnati, Cincinnati, OH). The hybridomas secreting these two Abs were grown by the Cell Core Facility (Johns Hopkins University), purified by affinity chromatography with HiTrap Protein G columns (Amersham Biosciences, Piscataway, NJ), and administered i.p. at a dose of 1 mg in 0.1 ml of sterile PBS on days -1, 6, and 12 after immunization.
Immunization with mouse thyroglobulin and assessment of thyroid histopathology
On days 0 and 7, 8- to 10-wk-old mice were injected s.c. with 50 µg of purified mouse thyroglobulin emulsified in CFA (Sigma-Aldrich, St. Louis, MO). Mice were sacrificed on either day 21 (blockade experiments) or day 24 (transgenic experiments) after the first immunization. Severity of thyroid infiltration was determined using H&E-stained sections, read by two masked examiners and scored from 0 to 5 based on the percentage of thyroidal inflammatory involvement: grade 0, no disease; grade 1, 
20% involvement; grade 2, 21–30%; grade 3, 31–50%; grade 4, >50%; grade 5, >90% with involvement of surrounding striated muscles.
Determination thyroglobulin Abs and IFN- in serum
Ether anesthetized mice were bled from the retroorbital venous plexus on days 0, 9, and 21 in the blockade experiments, and on days 0, 7, and 24 in the transgenic experiments. Sera were diluted in PBS (1/400, 1/1600, 1/6400) and incubated on microtiter plates coated with 2 µg/ml mouse thyroglobulin. Mouse thyroglobulin-specific IgG subclasses, IgG1 and IgG2b, were detected using secondary Abs against IgG1 or IgG2b conjugated to alkaline phosphatase (ICN Pharmaceuticals, Irvine, CA). IgG2a was not assayed, as the NOD.H-2h4, similar to the parental NOD strain, is deficient in switching to IgG2a (our unpublished observation). The color change of para-nitrophenyl phosphate substrate (Sigma-Aldrich) was measured at 405 nm by a microplate reader (Thermo Lab Systems, Chantilly, VA). Serum IFN- was measured in 50 µl of serum diluted 1/10, using the mouse IFN- Quantikine kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.
Flow cytometry analysis of draining cervical lymph nodes
Cervical lymph nodes (four to six per mouse) were isolated and mechanically disrupted to prepare a single cell suspension. After a 10 min Fc block at 4°C, cells were stained for 30 min at 4°C using the following mAbs (all from BD PharMingen, San Diego, CA): FITC-conjugated anti-B220 and anti-CD8; PE-conjugated anti-CD19 and anti-CD4; CyChrome-conjugated anti-CD44 and anti-CD3. Data were collected on a FACSCalibur cytometer (BD Biosciences, San Diego, CA), gated, compensated, and displayed using CellQuest software (BD Biosciences). The gate was drawn based on the forward vs side scatter display, to exclude events with low forward light scatter, indicative of cell debris, isolated nuclei, and cells in advanced apoptosis.
Cytokine measurement in splenocyte supernatants
At sacrifice, spleens were removed, single cell suspension prepared, and splenocytes cultured in RPMI 1640, supplemented with 10% FCS, at a concentration of 2.5 x 106 cells per ml/well. Culture supernatants were collected 48 h later, and assayed undiluted by sandwich ELISA for the following cytokines: IFN-, IL-2, TNF-
, IL-13, IL-5, TGF-, and IL-10 (all kits from R&D Systems). Results were expressed as units per milliliter over medium alone.
Assessment of thyroid-specific IFN- and MHC class II expression
Mouse thyroids were dissected and digested for 20 min at 37°C with collagenase-II (Sigma-Aldrich) and dispase-II (Roche, Indianapolis, IN) in Eagle’s MEM, as previously described (34). Following digestion, cells were incubated for 15 min at 4°C with anti-CD45 paramagnetic beads (Miltenyi Biotec, Auburn, CA) and applied to a magnetic separator column (Miltenyi Biotec). The eluate was used for mRNA extraction and flow cytometry. mRNA was isolated with oligo-d(T) paramagnetic beads (Dynal, Lake Success, NY) and reverse-transcribed by Superscript II (Invitrogen, Carlsbad, CA) for 1 h at 42°C. After RNaseH digestion, the cDNA was amplified with the same IFN--specific primers used for the genomic DNA screening. For flow cytometry, freshly isolated thyrocytes were stained for 30 min at 4°C with PE-conjugated anti-I-Ak mAb (Caltag Laboratories, Burlingame, CA) and acquired on a FACSCalibur cytometer (BD PharMingen).
Statistics
Differences between the medians of two or three groups were assessed by the Mann-Whitney U test or the Kruskal-Wallis test, respectively. Differences between the means of two or three groups were assessed by the unpaired Student t test or one-way analysis of variance, respectively. All statistical analyses were performed with Stata 7 (Stata Corporation, College Station, TX).
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is functional in the thyroid gland
To determine the impact of local, thyroid-specific overexpression of IFN- on the pathogenesis of EAT, a transgene expressing murine IFN- under the control of the rat thyroglobulin promoter (30) was backcrossed onto the NOD.H-2h4 background. Whole thyroids from both thyr-IFN- transgenic and wild-type mice expressed IFN- mRNA (data not shown), most likely due to the presence of hemopoietic cells within the thyroid. To exclude this contamination, thyroids were depleted of the CD45+ population by paramagnetic negative selection. CD45-depleted thyroids from transgenic mice expressed IFN- mRNA, whereas thyroids from wild-type littermates did not (Fig. 1A). This difference in thyroidal IFN- expression was not due to differences in mRNA input quantities, as expression levels of thyroglobulin and G3PDH mRNAs were comparable in both groups.
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is a potent inducer of MHC class II expression, we examined expression of I-Ak on the CD45- thyroid cell population to verify that transgenic IFN- was not only expressed in the thyroid, but also exerted a functional effect. CD45- thyrocytes from transgenic mice showed a 22-fold increase in I-Ak mean fluorescence intensity compared with wild-type controls (Fig. 1B).
To assess whether IFN- expression was confined to the thyroid or also reached the systemic circulation, serum was assayed by sandwich ELISA for IFN-. As shown in Fig. 1C, serum IFN- levels were not different between transgenic and wild-type controls at days 0 or 24 postimmunization. These results indicate that elevated IFN- levels are present in the thyroid environment, but not systemically.
Thyroid-specific transgenic expression of IFN- protects NOD.H2h4 mice from EAT
Young (
20 wk), unimmunized thyr-IFN- transgenic mice and wild-type littermates did not develop spontaneous thyroiditis (data not shown). Similarly, they did not develop thyroiditis after injection of CFA without thyroid Ag (data not shown). These findings speak against a primary role of IFN- in the spontaneous initiation of autoimmune thyroiditis. To assess the effect of IFN- following immunization, thyr-IFN- transgenic (n = 26) and wild-type littermates (n = 28) were immunized with mouse thyroglobulin in CFA, and sacrificed on day 24 postimmunization. thyr-IFN- transgenic mice exhibited marked reduction in both incidence and severity of thyroiditis (Fig. 2A), as compared with wild-type littermates (Fig. 2B). The median lesion grade in thyr-IFN- transgenic mice was 1, significantly lower (p = 0.0002) than the median score of 3 observed in wild-type littermates (Fig. 2C). Histologically, thyr-IFN- transgenic mice on the NOD.H-2h4 background showed only focal mononuclear cell infiltration and minimal changes in the thyroid follicular cells (Fig. 2A).
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on the humoral response to thyroglobulin in immunized mice, sera were collected on days 0, 7, and 24 postimmunization for the determination of thyroglobulin-specific IgG subclass Abs. thyr-IFN- transgenic mice produced less thyroglobulin-specific IgG1 at day 24 (p = 0.005) than wild-type littermates (Fig. 2D). This decrease in IgG1 responses to thyroglobulin in the transgenic animals was also seen at other serum dilutions (data not shown). Thyroglobulin-specific IgG1 Abs were not detectable on days 0 and 7 (Fig. 2D). No differences between the two groups were seen in thyroglobulin-specific IgG2b responses at any time point tested (data not shown).
thyr-IFN- transgenic mice demonstrate reduced activation of lymphocytes isolated from cervical lymph nodes
To gain insight into the mechanisms by which IFN- limits EAT, we studied the hemopoietic population of the spleen and of the draining cervical lymph nodes. Splenocytes isolated from thyr-IFN- transgenic and wild-type control were not different in terms of absolute lymphoid number, proportion of T, B, and NK lymphoid subsets, and spontaneous production of IFN-, IL-2, TNF-, IL-5, IL-13, TGF-, and IL-10 (data not shown). These results suggest that a systemic mode of action of IFN- is unlikely.
Cervical lymph nodes in transgenic and controls showed similar cellular composition, and similar proportions of the B220+, CD19+, CD4+, and CD8+ lymphoid subsets (Table I). Interestingly, T lymphocytes in the transgenic group showed a significant reduction in the expression of CD44 (Table I and Fig. 3), suggesting that IFN- locally suppresses lymphocyte activation.
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via systemic injection of a mAb exacerbates EAT
Because we found that increased IFN- expression in the thyroid limits disease, we tested next whether blocking IFN- in NOD.H-2h4 mice by administration of a mAb would increase EAT. NOD.H-2h4 mice were immunized with mouse thyroglobulin in CFA as before, and sacrificed 21 days postimmunization. One group (n = 8) was treated with a blocking mAb to IFN- during the induction of disease; a second group (n = 12) was given isotype control at an identical regimen, and a third group (n = 11) received PBS alone. The isotype control group developed thyroid infiltration, with a median grade of 3.5, not significantly different from the infiltration in the thyroids of mice treated with PBS alone (Fig. 4A). This infiltration was composed mainly of mononuclear cells that surrounded and, in some cases, destroyed thyroid follicles. In contrast, all mice receiving the anti-IFN- treatment, but none of the controls, developed grade 5 thyroid lesions, the highest disease score possible (Fig. 4B). In these mice, the inflammatory infiltrate almost completely destroyed the normal thyroidal architecture and extended into surrounding striated muscle (Fig. 4B). Although primarily mononuclear, this infiltration was also characterized by an abundance of eosinophils (Fig. 4B, inset). The increased severity of disease in mice receiving anti-IFN- was highly statistically different (p < 0.001) from both the isotype control and PBS groups (Fig. 4C).
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blockade on the humoral response to thyroglobulin, sera were collected at days 0, 9, and 21 after immunization and assayed for the presence of thyroglobulin-specific IgG subclass Abs. At day 9 postimmunization, mice treated with anti-IFN- had significantly increased thyroglobulin-specific IgG1 responses compared with both isotype (p = 0.013) and PBS controls (p = 0.030) (Fig. 4D). These differences at day 9, confirmed at other serum dilutions (data not shown), became less pronounced by day 21 (Fig. 4D). The absence of statistically significant differences in thyroglobulin-specific IgG1 responses at day 21 postimmunization is most likely explained by a robust IgG1 response in the control group by day 21 due to the potency of the adjuvant, leaving little room for enhancement of humoral responses. Thyroglobulin-specific IgG1 Abs were undetectable in all treatment groups at day 0 (data not shown). Levels of IgG2b Abs were not different between the groups at any time point or serum dilution (data not shown).
The production of IFN-, IL-2, TNF-, IL-5, IL-13, TGF-, and IL-10 in supernatants of cultured spleen cells was determined by ELISA. Spleen cells from mice injected with the mAb blocking IFN- or with control vehicles (isotype or PBS alone) produced similar levels of TNF-, IL-5, and IL-10 (data not shown). In contrast, splenocytes from the anti-IFN- group more rapidly produced IL-13 (Fig. 5A), IFN- (Fig. 5B), and IL-2 (Fig. 5C), likely indicating a more activated state and a rebound response to the administration of the Ab. Interestingly, splenocytes from the anti-IFN- group produced significantly lower levels of TGF-, a cytokine known for its immunosuppressive effects (35).
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Discussion |
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and IL-2) stimulate cell-mediated immune responses, whereas Th2 lymphocytes (mainly producers of IL-4, IL-5, and IL-13) drive allergic reactions and Ab production. Indeed, early studies showed that Th1 cytokines promote cell-mediated autoimmune diseases, such as type 1 diabetes (4), whereas Th2 cytokines down-regulate the same autoimmune disease (37). However, more recent studies have shown that the model based on a simple Th1/Th2 dichotomy oversimplifies reality, and that the same cytokine originally described as immunostimulatory may also have immunosuppressive activities (3, 38). These findings suggest that cytokines act in a dynamic interactive network so that changes in one cytokine inevitably perturb others.
In the context of this realization of the great complexity of cytokine interactions, we have conducted the present work to further examine the role of IFN- in EAT. Previous publications have provided seemingly conflicting information regarding the contribution of IFN- in EAT, where it has been shown to exacerbate, ameliorate, or be dispensable for disease. Specifically, initial studies characterized IFN- as a disease promoter, considering that systemic injection of IFN- enhanced EAT (24), and systemic injection of a mAb neutralizing IFN- abrogated EAT (25). Then IFN- was shown to suppress EAT because when blocked systemically by the injection of a neutralizing Ab, a more severe form of thyroiditis ensued (26). Finally, mice lacking the IFN- receptor developed EAT equally well (28), and adoptive transfer of splenocytes from donors immunized with thyroglobulin and restimulated in vitro with thyroglobulin and anti-IL-2 receptor Ab induced EAT independently of whether the splenocytes came from IFN- knockout or wild-type donors (27). Although some of these differences may be explained by the varying experimental conditions (such as different sources of thyroglobulin used for immunization, different dosages, and timing), a common theme of these experiments is that they have all relied on systemic manipulation of the IFN- pathway, such as by injection of mAbs or by using mice with germline deletion of IFN- or its receptor. Because the local and systemic actions of cytokines may differ, we reasoned that it would be more instructive to study the effect of locally produced IFN-, simulating the scenario in EAT. We describe for the first time that the local intrathyroidal expression of IFN- in EAT, achieved via transgenesis, limits EAT, as indicated by reduced thyroid histopathology and thyroglobulin-reactive IgG1.
IFN- can attenuate EAT and other autoimmune diseases by at least three mechanisms, all operative on hemopoietic cells. One mechanism is the induction of regulatory proteins that dampen or terminate immunostimulatory signals. Of these proteins, the best characterized are the members of the suppressor of cytokine signaling (SOCS) family, and SOCS-1 in particular. SOCS-1, expressed only in hemopoietic cells, especially in the thymus, binds and blocks the Janus kinases, which are in turn noncovalently bound to the cytokine receptor, thus interfering with downstream cytokine signaling (39). SOCS-1 is not specific for IFN-, but can be induced by a bewildering diversity of cytokines, such as IL-2, IL-4, IL-6, IFN-, G-CSF, and also hormones, such as prolactin, thyrotropin, and growth hormone. Considering the lack of specificity for IFN-, it is unlikely that SOCS-1 induction alone explains our findings. However, formal experiments addressing the role of SOCS-1, such as crossing our thyr-IFN- transgenic to the SOCS-1 knockout mice (40, 41), need to be performed. A second potential immunosuppressive mechanism of IFN- relates to the inclusion of CFA in the immunization protocol. It has been proposed that IFN- has protective effects by inhibiting CFA-induced proliferation of Mac-1+ (myeloid) splenocytes (42). This mechanism seems unlikely in our transgenic model, where the expression of IFN- is restricted to the thyroid gland, with no detectable changes of its systemic levels. Finally, IFN- could suppress the lymphocytic infiltration the target organ during the autoimmune attack by inducing apoptosis or reducing activation. For example, IFN- has been shown to enhance apoptosis in experimental models of encephalomyelitis (9) and uveitis (11). Consistent with these observations, we found that draining cervical nodes isolated from thyr-IFN- transgenic mice contained a significantly lower number of lymphocytes expressing CD44, a marker of activation.
CD44 is a transmembrane glycoprotein, belonging to a class of cell adhesion molecules. Although encoded by a single highly conserved gene, CD44 displays great structural heterogeneity, as a result of extensive alternative splicing and posttranslational modifications (43). The smallest CD44 isoform, which lacks all the 10 variant exons, is widely expressed on hemopoietic cells, and its expression increases upon activation (44). CD44 has been shown to possess many functions which can be classified under three types of molecular properties (43): it can act as a receptor, binding ligands such as hyaluronan, in the extracellular matrix; it can act as a coreceptor, cooperating with receptor tyrosine kinases, such as Met and ERBB; it can bind proteins that bind to the actin cytoskeleton, thus functioning as a linker between the plasma membrane and the cytoskeleton. All of these functions influence cell signaling and activity, which is reduced when CD44 expression is reduced. We found that increased levels of IFN- are associated with decreased expression of CD44 in draining lymph nodes and suggest that IFN- may suppress the activation of naive, potentially thyroiditogenic, T cells. Data from two transgenic mouse models of type 1 diabetes have shown that the local draining lymph nodes are the sites where naive T cells first encounter their Ags and become activated (45, 46). Our findings support this observation and represent the first description of this type of mechanism in thyroiditis.
In addition to acting upon hemopoietic cells, cytokines may directly influence the target organ. Increased expression of IFN- could, for example, decrease the amount of thyroid Ags available for uptake by thyroid-resident APCs. IFN- could also induce thyrocytes to express molecules that form a "protective shield." Paradoxically, these molecules could be the MHC class II products themselves. The thyr-IFN- transgenic mice showed a marked up-regulation of MHC class II, yet they did not develop spontaneous thyroiditis. In addition, unpublished findings in our laboratory have shown that transgenic mice expressing the transcription factor class II transactivator under control of the thyroglobulin promoter do not develop spontaneous thyroiditis. These concepts contradict the hypothesis of Bottazzo et al. (47), which states that aberrant expression of MHC class II on epithelial cells confers Ag-presenting functions, favoring the initiation of autoimmune disease.
The transgenic expression of IFN- in the thyroid of NOD-H2h4 mice did not cause the same disruption of thyroid architecture and hypothyroidism we described in the C57BL/6 mice (30). Strain differences are known to influence the outcome of genetic changes. For example, mice with a homozygous loss of function mutation of the gene encoding Fas (Faslpr) develop a lupus-like disease only on the MRL/Mp background, but not on the C57BL/6 or C3H background (48). Similarly, the homozygous deletion of PD-1, a transmembrane receptor expressed on the T cell that acts as negative regulator of immune responses, causes a late onset and progressive arthritis and lupus-like glomerulonephritis in the C56BL/6 strain (49), but an early onset dilated cardiomyopathy in the BALB/c strain (50). Studies are presently ongoing in our laboratory aimed to assess whether IFN- causes the same protective effect in the CBA mouse, another thyroiditis-prone strain.
The protective effect of IFN- on EAT seen in the transgenic experiment was confirmed by the blockade experiment. We showed that administration of a mAb that neutralizes IFN- exacerbates disease. These results agree with our previous findings (29) and those by Stull et al. (26), who showed that adoptive transfer of splenocytes cultured with thyroglobulin and a IFN--neutralizing Ab causes a more severe granulomatous form of thyroiditis. The findings of these three studies contrast to those by Tang et al. (25), who reported that blockade of IFN- with a different neutralizing Ab abrogates EAT. The discrepancy could be explained by the fact that the authors used a different IFN- Ab and porcine, rather than murine, thyroglobulin to induce EAT. It is known, in fact, that susceptible mice develop more severe and long-lasting thyroiditis when immunized with murine compared with heterologous thyroglobulin (51, 52, 53).
In conclusion, this study demonstrates a disease-limiting role of IFN- in autoimmune thyroiditis. Furthermore, we provide the first evidence that local IFN- activity in the thyroid is sufficient for disease suppression. The same cytokine appears to have both immunostimulatory and immunosuppressive activities. The decision on which effect will predominate, likely dependent on the timing and location of its secretion and duration of exposure, needs to be better understood, because it may have therapeutic implications.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Patrizio Caturegli, Department of Pathology, School of Medicine, Johns Hopkins University, Ross 656, 720 Rutland Avenue, Baltimore, MD 21205. E-mail address: pcat{at}jhmi.edu
3 Abbreviations used in this paper: EAT, experimental autoimmune thyroiditis; NOD, nonobese diabetic; SOCS, suppressor of cytokine signaling.
Received for publication February 3, 2003. Accepted for publication March 20, 2003.
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) and WT littermate control (
) mice as determined by sandwich ELISA at days 0 and 21 postimmunization. Data represent mean ± SD.
) 9 and 21 days after immunization; values are expressed as OD adjusted to a normal mouse serum control. Values of p of the difference between medians (histopathology) and means (Ab levels) calculated by the Mann-Whitney U test and the unpaired Student t test, respectively.