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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0093
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 7 4138-4144
Copyright © 2005 by The Endocrine Society

Innate and Acquired Immune System in Patients Developing Interferon-{alpha}-Related Autoimmune Thyroiditis: A Prospective Study

G. Mazziotti, F. Sorvillo, M. Piscopo, F. Morisco, M. Cioffi, G. Stornaiuolo, G. B. Gaeta, A. M. Molinari, J. H. Lazarus, G. Amato and C. Carella

Departments of Clinical and Experimental Medicine "F. Magrassi & A. Lanzara" (G.M., F.S., M.P., G.A., C.C.), Clinical Pathology (M.C., A.M.M.), and Infectious Disease (G.S., G.B.G.), Second University of Naples, 80121 Naples, Italy; Department of Food Science (F.M.), University "Federico II" of Naples, 80138 Naples, Italy; and Department of Medicine (J.H.L.), University of Wales College of Medicine, Cardiff CF10 3XP, United Kingdom

Address all correspondence and requests for reprints to: Carlo Carella, M.D., Department of Clinical and Experimental Medicine, "F. Magrassi and A. Lanzara," Second University of Naples, Via Crispi 44, 80121 Naples, Italy. E-mail: carlo.carella{at}unina2.it.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Objective: In this prospective study, we investigated whether the development of interferon (IFN)-{alpha}-related autoimmune thyroiditis (IFN-AT) was correlated with the sequential changes of cytokine pattern induced by IFN{alpha} in the peripheral lymphocytes.

Patients and Methods: We enrolled 18 hepatitis C virus (HCV)-positive patients who developed IFN-AT, eight patients with euthyroidism [IFN-AT(Eu)] and 10 with thyroid dysfunction [IFN-AT(Dy)]. Twenty HCV-positive patients without IFN-AT acted as control group (Co-HCV+). Intracellular expression of IFN{gamma} and IL-4 was evaluated by multicolor flow-cytometry analysis in peripheral lymphocytes in vitro stimulated by phorbol-12-myristate-13-acetate (PMA) (25 ng/ml) and ionomycin (1 µg/ml) in presence of monensin (5 µM).

Results: At the appearance of thyroid disease, both IFN-AT(Eu) and IFN-AT(Dy) patients showed a significant increase of IFN{gamma} expression in CD3+CD56+ and CD3–CD56+ cells but not in CD4+ and CD8+ cells. At this time point, IFN-AT(Eu) but not IFN-AT(Dy) patients also showed an increase of IL-4 expression in CD3+CD56+ cells and CD4+ cells. Six months later, IFN-AT(Eu) patients maintained high expression of IL-4 in CD4+ and CD3+CD56+ cells without any further increase in IFN{gamma} expression. By contrast, IFN-AT(Dy) patients showed an increase of IFN{gamma} expression in CD4+ and CD8+ cells, with a concomitant decrease of IL-4 expression in CD4+ cells.

Conclusions: Type 2 immune response is activated early and specifically in patients with IFN-AT who remain euthyroid throughout the follow-up. Predominant in patients developing thyroid dysfunction, by contrast, is the type 1 immune response that seems to occur earlier in innate than acquired immune system.


   Introduction
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 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THYROID DISORDERS ARE a frequent side effect of interferon (IFN)-{alpha} therapy (reviewed in Refs. 1 , 2). Positive thyroid antibodies with normal thyroid function tests is the commonest finding in patients treated with IFN{alpha}, whereas thyroid dysfunction is usually described in no more than 15% of all treated patients (3). Thyroid dysfunction may present as destructive thyrotoxicosis, Graves’ thyrotoxicosis, and hypothyroidism. Indeed, destructive thyrotoxicosis and hypothyroidism are more frequent than Graves’ hyperthyroidism because only 20–25% of all patients with IFN-related thyrotoxicosis are due to Graves’ disease induced by circulating thyroid receptor antibodies (TRAb) (3, 4). Destructive thyrotoxicosis and hypothyroidism have been considered as different expressions of the same disease at different time of development (5). It has been pointed out that IFN-related thyroid autoimmune disease (IFN-AT) may reproduce in its short-time and long-term outcome the natural history of Hashimoto’s thyroiditis (HT) (6). Indeed, IFN-AT has been proposed as a model for thyroid autoimmunity (7), although the immunological aspects of this relationship remain to be clarified.

So far, almost all studies have focused on the clinical aspects of IFN-AT, by the evaluation of thyroid autoantibody patterns and clinical pictures of thyroid dysfunction (3). However, there are limited data about the cellular immunological events accompanying the IFN-AT development. The main effect of IFN{alpha} on the immune system is the enhancement of cell cytotoxicity, very likely sustained by suppression of T helper (Th) 2 and an increase in Th1 immune response (8, 9, 10, 11, 12, 13). Although these effects seem to be transient (14), it has been suggested that the generalized Th1 activation induced by IFN{alpha} may be important for the occurrence of thyroid autoimmunity (15). However, IFN{alpha} was also shown to induce the production of type 2 cytokines (16, 17). In thyroid autoimmunity both Th1 and Th2 responses are found, although with different reciprocal intensity in relationship with the clinical expression of the disease process (18, 19, 20). The type 1 response, in particular, seems to be dominant in hypothyroid patients with HT, whereas type 2 immune response has been found in patients with thyroid autoimmunity and normal thyroid function (21). These differences were demonstrated at thyroid levels and in the peripheral lymphocytes (19, 21).

In this study we analyzed the dynamics of peripheral lymphocyte responses during IFN{alpha} plus ribavirin treatment in relation to the development of autoimmune thyroiditis. The evaluation of sequential modifications of Th1 and Th2 immune response in patients developing IFN-AT would provide information about the evolution of cellular immune response in the early phase of development of thyroid autoimmune process.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The study group included 18 patients with hepatitis C virus (HCV)-related chronic hepatitis (10 females, eight males, median age 42 yr, range 21–56) who developed thyroid autoimmune disease during IFN{alpha} plus ribavirin treatment. All of them were without thyroid disorders before the antiviral treatment. Twenty HCV-positive patients who did not develop any thyroid disorder during IFN{alpha} treatment were enrolled as control group (Co-HCV+ group). The Co-HCV+ subjects had comparable sex (13 females, seven males) and age (44.5 yr, range 20–65) with the patients developing IFN-AT. The patients and control subjects were retrospectively selected from a population of 90 patients affected by HCV-related chronic hepatitis who performed a full course of treatment with pegylated-IFN{alpha} (1.5 µg/kg·wk sc) and ribavirin (1.0–1.2 g/d orally) in the period between 2002 and 2004. These patients were enrolled, taking into account the following exclusion criteria: 1) thyroid autoimmunity and/or dysfunction before starting IFN{alpha} treatment; 2) pregnancy in the latter 12 months prior the enrollment; and 3) duration of IFN{alpha} treatment shorter than 12 months. At the study entry, both the IFN-AT patients and Co-HCV+ subjects were asked for the presence of thyroid autoimmunity in their relatives. Thyroid function [serum TSH, free T4 (FT4) and free T3 (FT3)] and autoimmunity [serum thyroglobulin (TgAb), thyroperoxidase (TPOAb), and TSH receptor antibodies (TRAb)] were evaluated at baseline and every 4–6 wk for the first 6 months of treatment and then every 12–15 wk until the end of the treatment. The patients developing thyroid autoimmunity (23 cases) were selected on the basis of appearance of thyroid autoantibodies with or without thyroid dysfunction [thyrotoxicosis alone (suppressed serum TSH values, normal or high serum FT3 and FT4 values, a low radioiodine uptake, and negative TRAb), hypothyroidism alone (high serum TSH values, low or normal serum FT4 and FT3 values), thyrotoxicosis followed by hypothyroidism, hyperthyroidism (suppressed serum TSH values, normal or high serum FT3 and FT4 values, a high radioiodine uptake and positive TRAb)]. For the purpose of this study, the patients with hyperthyroidism (two cases) were excluded from the analysis. Moreover, three patients with IFN-AT were excluded because they stopped the IFN{alpha} treatment early. Therefore, 18 patients IFN-AT were investigated in the present study.

In IFN-AT patients the immunological evaluations were performed on frozen samples drawn before starting antiviral treatment (T0), at the time of appearance of thyroid disease (T1), and 6 months later (T2). In Co-HCV+ patients the immunological evaluations were performed before commencing IFN treatment (T0) and at 3–5 (T1) and 9–11 month (T2) of the treatment.

Informed consent was obtained in each IFN-AT and Co-HCV+ patient, and the study was approved by local ethical committee.

Methods

Serum FT4 and FT3 concentrations were measured by double-antibody RIA (Technogenetics, Milan, Italy); serum TSH was assayed by an immunoradiometric method (DIA-Sorin, Saluggia, Italy). Samples were assayed in duplicate for each hormone. Normal ranges are as follows: TSH, 0.3–3.5 µU/ml (SI: 0.3–3.5 mU/liter), FT4, 7.0–17.9 pg/ml (SI: 9.0–23.1 pmol/liter), FT3, 2.5–5.0 pg/ml (SI: 3.8–7.7 pmol/liter). The sensitivity and the intraassay coefficients of variation for TSH measurements were 0.05 µU/ml (SI: 0.05 mU/liter) and 3.1%, respectively.

TgAbs (negative < 100 U/ml) were measured using the immunoradiometric assay (BioChem ImmunoSystem, Bologna, Italy) with intraassay and limit of 3.9% and 5.0 U/ml, respectively. TPOAbs (negative < 10 U/ml) were tested by RIA set (DIA-Sorin) with intraassay and detection limit of 2.5% and 0.7 U/ml, respectively. Serum TRAbs were measured with the DYNOtest TRAK human (BRAHMS AG, Berlin, Germany). This radioreceptor assay uses human TSH receptor on coated tubes and human antibodies for standard material and expresses the results in international units based on a World Health Organization standard (22). For TRAb values between 0.5 and 5.0 IU/liter, intraassay and interassay coefficients of variation were 9.6 and 13.0%, respectively. TRAb positivity was defined for values above 1.5 IU/liter.

The immunological analyses were performed according to the procedure already described (21). Peripheral blood mononuclear cells (PBMCs) were obtained from peripheral blood collected in EDTA (15 ml) by density gradient centrifugation over Ficoll Histopaque-1077. The PBMCs were resuspended in aliquots of 2 x 106 cells in 1 ml of freezing mixture (50% fetal calf serum, 40% RPMI 1640, 10% dimethyl sulfoxide) and frozen at a rate of 1 C/min until the temperature reached –85 C when they were transferred for long-term storage to liquid nitrogen.

The membrane antigen expression was investigated using a panel of monoclonal antibodies directly coupled to allophycocyanin or peridin chlorophyll protein, including anti-CD3, -CD4, -CD8, and -CD56 (Becton Dickinson, San Jose, CA). Stained cells were analyzed using a FACSscan flow cytometer (Becton Dickinson). A minimum of 10,000 events was acquired and all analyses were carried out in duplicate. Data were processed using CellQuest software (Becton Dickinson). IgG1 isotype controls (Becton Dickinson) were used to establish background fluorescence.

The functional analysis consisted of evaluation of cytokine production from the above cell populations before and after pharmacological stimulation. Aliquots of PBMCs in RPMI 1640 medium (500 µl for each well: 1 x 106 cells) were incubated for 4 h at 37 C in a humidified atmosphere containing 5% CO2, in the presence of activation reagent [25 ng/ml phorbol-12-myristate-13-acetate (PMA) plus 1 µg/ml ionomycin (PMA+I; Sigma, St. Louis, MO)] and in the presence of 5 µM monensin (Sigma), which inhibited cytokine secretion leading to their intracellular accumulation (23). After 4 h incubation, cell viability was assessed by Trypan-Blue exclusion. Subsequently, the cells were incubated with allophycocyanin-labeled anti-CD4 or anti-CD8 monoclonal antibodies for 20 min at 25 C. After washing [phosphate-buffered saline containing 0.5% BSA and 0.1% NaN3], the PBMCs were incubated [30 min, room in the dark] with FACS permeabilizing solution (Becton Dickinson) and conjugated anticytokine monoclonal antibodies [isothiocyanate anti-IFN{gamma} and phycoerythrin anti-IL-4 antibodies] (24). IFN{gamma} was investigated as marker of type 1 immune response, whereas IL-4 was detected as markers of type 2 response. CD8+ and CD3–CD56+ cells did not show detectable IL-4, even after stimulation; therefore, this cytokine was studied only in CD4+ and CD3+CD56+. The cells were washed once more before resuspension in 1% paraformaldehyde before analysis on a FACS scan flow cytometer. The CD4+ and CD8+ cells were gated from CD3+ cells, and the intracellular cytokine expression was evaluated in each population separately. CD56+ cells were subdivided in two subpopulations (CD3+ and CD3–).

Data were presented as mean ± SEM. Paired and unpaired data were compared using Wilcoxon’s and Mann-Whitney’s U tests, respectively. Multiple comparisons were made by Friedman’s and Kruskal-Wallis tests, with post hoc Bonferroni’s correction. Frequencies were compared using {chi}2 test, with Fisher’s correction, when appropriate. Statistical significance was assumed when the P ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
In all IFN-AT patients, thyroid disease appeared during the first 6 months of IFN{alpha} treatment. In 10 of them, thyroid autoimmunity was accompanied by different degree of thyroid dysfunction [IFN-AT(Dy)] (Table 1Go), whereas eight patients remained biochemically and clinically euthyroid [IFN-AT(Eu)] throughout the treatment, even if serum TSH concentrations decreased slightly in some of them (Table 2Go).


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  		TABLE 1. Thyroid function (serum TSH: µU/ml = mU/liter) and FT4 (pg/ml = pmol/1.287) and autoimmunity (TgAbs and TPOAbs) in 10 patients [four males (M) and six females (F)] who developed IFN-AT with thyroid dysfunction

 

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  		TABLE 2. Thyroid function [serum TSH (µU/ml = mU/liter) and FT4 (pg/ml = pmol/1.287)] and autoimmunity (TgAbs and TPOAbs) in eight patients [four males (M) and four females (F)] who developed IFN-AT without thyroid dysfunction

 
At the study entry, two Co-HCV+ subjects (10.0%) and seven IFN-AT patients (38.9%) were aware that their relatives had autoimmune thyroid disease ({chi}2 4.37; P = 0.06). Before starting IFN{alpha} treatment, patients developing IFN-AT with or without thyroid dysfunction showed no significant differences in cytokine pattern with respect to Co-HCV+ subjects who did not develop any thyroid abnormalities during IFN{alpha} treatment (Fig. 1GoA, B).



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  		FIG. 1. Intracellular IFN{gamma} expression (A) in peripheral CD4+, CD8+, CD3+CD56+, and CD3-CD56+ lymphocytes and IL-4 expression (B) in CD4+ and CD3+CD56+ cells in IFN-AT analyzed separately for IFN-AT(Eu) (eight cases) and IFN-AT(Dy) (10 cases) patients in comparison with Co-HCV+ (20 cases). We report the data at the start of IFN{alpha} treatment. The data are expressed as mean ± SEM.

 
IFN{alpha} treatment induced a significant increase in IFN{gamma}+ and IL-4 expression in all of the lymphocyte populations investigated, at higher extent in IFN-AT than in Co-HCV+ patients. IFN-AT(Dy) (10 cases) and IFN-AT(Eu) (eight cases) patients showed different sequential modifications of cytokine expression in the peripheral lymphocytes during the development of thyroid disease. IFN-AT(Eu) patients showed a significant increase of IFN{gamma} in CD8+ (Fig. 2BGo), CD3+CD56+ (Fig. 2CGo), and CD3-CD56+ (Fig. 2DGo) cells but not in CD4+ cells (Fig. 2AGo). However, the patients with IFN-AT(Dy) showed a significant increase of IFN{gamma}+ expression in all of lymphocyte populations (Fig. 2Go, A–D). In CD3+CD56+ and CD3-CD56+ cells, we found no significant difference in IFN{gamma}+ expression between IFN-AT(Dy) and IFN-AT(Eu) patients. By contrast, IFN-AT(Dy) patients showed higher IFN{gamma}+ expression in CD4+ and CD8+ cells than IFN-AT(Eu) patients. In CD3+CD56+ (Fig. 2CGo) and CD3-CD56+ (Fig. 2DGo) cells, the increase of IFN{gamma} expression occurred earlier than that found in CD4+ cells in which the IFN{gamma} expression increased during the 6 months after the diagnosis of IFN-AT (Fig. 2AGo). In CD8+ cells, IFN{gamma} expression increased progressively throughout the period of the study (Fig. 2BGo).



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  		FIG. 2. Outcome of intracellular IFN{gamma} expression in peripheral CD4+ (A), CD8+ (B), CD3+CD56+ (C), and CD3-CD56+ (D) lymphocytes in IFN-AT analyzed separately for IFN-AT(Eu) (eight cases) and IFN-AT(Dy) (10 cases) patients in comparison with Co-HCV+ (20 cases) during IFN{alpha} treatment. We report the data at the start of IFN{alpha} treatment (T0), at the appearance of thyroid disease (T1), and 6 months later (T2). In Co-HCV+ subjects, T1 and T2 correspond to 3–5 months and 9–11 months of treatment, respectively. The data are expressed as mean ± SEM [a, P < 0.05 vs. T0; b, P < 0.05 vs. T0 and T1; c, P < 0.05 vs. Co-HCV+; d, P < 0.05 vs. Co-HCV+ and IFN-AT(Eu)]. Multiple comparisons were made by Friedman’s (for repeated measures) and Kruskal-Wallis (for unpaired data) tests, with post hoc Bonferroni’s correction.

 
At diagnosis of thyroid disease (T1), both IFN-AT(Eu) and IFN-AT(Dy) patients showed higher expression of IFN{gamma}+ in CD3+CD56+ (Fig. 2CGo) and CD3-CD56+ (Fig. 2DGo) cells as compared with the Co-HCV+ subjects, whereas the IFN{gamma}+ expression in CD4+ (Fig. 2AGo) and CD8+ (Fig. 2BGo) cells of both IFN-AT(Eu) and IFN-AT(Dy) remained comparable with the control subjects. At this time point, IFN-AT(Eu) and IFN-AT(Dy) patients showed no significant differences in CD4+IFN{gamma}+, CD8+IFN{gamma}+, CD3+CD56+IFN{gamma}+, and CD3-CD56+IFN{gamma}+ cells (Fig. 2Go, A–D).

Six months after the diagnosis of thyroid disease (T2), both IFN-AT(Eu) and IFN-AT(Dy) patients showed higher IFN{gamma}+ expression than the Co-HCV+ subjects in CD8+ (Fig. 2BGo), CD3+CD56+ (Fig. 2CGo), and CD3-CD56+ (Fig. 2DGo) cells, whereas in CD4+ cells, the IFN{gamma}+ expression increased only in IFN-AT(Dy) but not in IFN-AT(Eu) patients (Fig. 2AGo). At this time point, IFN-AT(Dy) showed higher numbers of CD4+IFN{gamma}+ (Fig. 2AGo) and CD8+IFN{gamma}+ (Fig. 2BGo) cells as compared with IFN-AT(Eu), without any significant difference in CD3+CD56+IFN{gamma}+ (Fig. 2CGo) and CD3-CD56+IFN{gamma}+ (Fig. 2DGo) cells.

The IFN-AT(Eu) patients showed a significant increase of IL-4 expression in both CD4+ (Fig. 3AGo) and CD3+CD56+ (Fig. 3BGo) cells at diagnosis of IFN-related thyroid disease. At this time point, the patients with thyroid dysfunction had lower expression of IL-4 in the both cell populations as compared with the euthyroid patients (Fig. 3Go, A and B). This difference was still present 6 months after the diagnosis of thyroid disease, especially in CD4+ cells (Fig. 3AGo).



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  		FIG. 3. Outcome of intracellular IL-4 expression in peripheral CD4+ (A) and CD3+CD56+ (B) lymphocytes in IFN-AT analyzed separately for IFN-AT(Eu) (eight cases) and IFN-AT(Dy) (10 cases) patients in comparison with Co-HCV+ (20 cases) during IFN{alpha} treatment. We report the data at the start of IFN{alpha} treatment (T0), at the appearance of thyroid disease (T1), and 6 months later (T2). In Co-HCV+ subjects, T1 and T2 correspond to 3–5 months and 9–11 months of treatment, respectively. The data are expressed as mean ± SEM [a, P < 0.05 vs. T0; b, P < 0.05 vs. T0 and T1; c, P < 0.05 vs. Co-HCV+; d, P < 0.05 vs. Co-HCV+ and IFN-AT(Eu); e, P < 0.05 vs. Co-HCV+ and IFN-AT(Dy)]. Multiple comparisons were made by Friedman’s (for repeated measures) and Kruskal-Wallis (for unpaired data) tests, with post hoc Bonferroni’s correction.

 
The Th1 to Th2 ratio in CD4+ and CD3+CD56+ cells increased significantly during IFN{alpha} treatment in IFN-AT(Dy) but not IFN-AT(Eu) patients (Fig. 4Go, A and B). In IFN-AT(Eu) patients, the Th1 to Th2 ratio remained significantly lower than the control HCV+ patients who did not develop any thyroid disease during IFN{alpha} treatment (Fig. 4Go, A and B).



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  		FIG. 4. Outcome of Th1 to Th2 ratio in peripheral CD4+ (A) and CD3+CD56+ (B) lymphocytes in IFN-AT analyzed separately for IFN-AT(Eu) (eight cases) and IFN-AT(Dy) (10 cases) patients in comparison with Co-HCV+ (20 cases) during IFN{alpha} treatment. We report the data at the start of IFN{alpha} treatment (T0), at the appearance of thyroid disease (T1), and 6 months later (T2). In Co-HCV+ subjects, T1 and T2 correspond to 3–5 months and 9–11 months of treatment, respectively. The data are expressed as mean ± SEM [a, P < 0.05 vs. T0; b, P < 0.05 vs. T0 and T1; c, P < 0.05 vs. Co-HCV+; d, P < 0.05 vs. Co-HCV+ and IFN-AT(Eu); e, P < 0.05 vs. Co-HCV+ and IFN-AT(Dy)]. Multiple comparisons were made by Friedman’s (for repeated measures) and Kruskal-Wallis (for unpaired data) tests, with post hoc Bonferroni’s correction.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This prospective study shows that the sequential modifications of type 1 and type 2 immune responses induced by IFN{alpha} treatment in peripheral lymphocyte of HCV+ patients are in relation to the development of thyroid autoimmune disease in its different clinical expressions.

Following the original description of IFN-related hypothyroidism (25), several studies have demonstrated that thyroid disease is a frequent side effect of IFN{alpha} treatment (1, 2, 3). In the previous studies, the immunological evaluations of IFN-AT were limited to the analysis of serum thyroid autoantibody patterns (2). To our knowledge this is the first study analyzing the lymphocyte immune response in patients with IFN-AT.

Various methods are currently employed to detect cytokine production and secretion in humans (24, 26, 27). In the present study, we used the multiparameter flow cytometry analysis to investigate the immune response separately for CD4+ and CD8+ lymphocytes among the total PBMCs. Because unstimulated T cells produce a low amount of cytokines, in vitro stimulation is required (28). Moreover, the stimulation of lymphocytes could allow to reproduce in vitro the in vivo activation of these cells by specific and unspecific stimuli. There are various means of artificial stimulation, and advantages and disadvantages have been described for each of them (29, 30). The pharmacological stimulus, as used in the present study, is not physiological but permits a rapid and reproducible stimulation of T cells by the activation of the transduction mechanisms used by T-cell receptor (24). Furthermore, the pharmacological stimulus permits to evidence the expression of low-degree expressed cytokine, as IL-4 (29). Finally, the pharmacological stimulus gave us the opportunity to evaluate the immune response in natural killer (NK) cells, which are not provided antigen-specific receptors.

Cellular immune responses have been studied in circulating (21, 31, 32, 33) and infiltrating lymphocytes (18, 19, 20, 34, 35, 36) of the patients with thyroid autoimmune disease. Because in the organ-specific diseases, the reliable reflection of the autoimmune state lies in the target organ, the analysis of the intrathyroid lymphocytes is probably the best manner to investigate the immunological status in the patients with thyroid autoimmunity (37). However, a prospective study such as ours would force us to perform repeated cytological evaluations that would have been not justifiable and not easily feasible. Therefore, the analysis of peripheral lymphocytes results in the easiest manner to study the immunological status of the patients with thyroid autoimmunity. Indeed, previous studies found an activated phenotype in peripheral blood from HT patients (21, 31, 32, 38), suggesting a generalized immune dysregulation in such disease (39).

According to the Th1/Th2 paradigm, the clinical expressions of autoimmune thyroid disease are sustained by different polarization of immune response (19, 20). Here we studied the patients with IFN-related autoimmune thyroiditis, which resembles Hashimoto’s thyroiditis (6, 7). Our study confirms previous observations that both Th1 and Th2 immune responses are activated in autoimmune thyroiditis, the former being more evident in presence of thyroid dysfunction and the latter more pronounced in euthyroidism (21). The dynamic model of IFN-related thyroid autoimmune disease allowed us to observe that Th2 activation in euthyroid patients was an early event during the development of the disease. Throughout the 6 months after the IFN-AT diagnosis, the patients with euthyroidism maintained high Th2 activation, whereas the patients with thyroid dysfunction showed a decrease in Th2 immune response with a concomitant increase in Th1 immune response. A similar outcome was already found in women developing postpartum thyroiditis with thyroid dysfunction in whom the physiological Th2/Th1 shift occurred early during the postpartum period (32). Future prospective studies will clarify whether the different time-dependent activation of Th1 and Th2 immune responses also occurs in IFN-related hyperthyroidism, as already suggested in patients with Graves’ disease (40).

Innate and acquired immune system have synergic effects in the control of immunological tolerance in adult subjects (41). It is well known that innate cells (NK and NKT cells) provide an early defense, serving to prevent replication and dissemination of infection before efficient adaptive responses are activated. However, new insights into innate immunity propose major roles for innate responses in driving, shaping, and even regulating adaptive immune responses (42). Recent studies using animal models suggest that the innate response is instrumental in determining whether an autoimmune reaction will occur (43, 44). A type 1 bias in NK cells was demonstrated in experimental models of autoimmune diabetes and encephalomyelitis (44, 45, 46). Here we found a type 1 and type 2 activation of peripheral NK and NKT CD56+ cells. The type 1 activation of innate immune system occurred at the same extent in patients with euthyroidism and those with thyroid dysfunction, whereas the type 2 activation occurred specifically in patients who remained euthyroid for the whole period of follow-up as that observed in CD4+ cells. It is intriguing that the type 1 activation in CD56+ cells was earlier than that observed in CD4+ cells, according to the experimental evidence of an early involvement of innate immune system in the development of autoimmunity (43). One could argue that the type 1 polarization in NK and NKT cells is an important factor for the development of thyroid autoimmunity but it not capable alone to drive the damage process of thyroid gland. By contrast, the type 1 activation of CD4+ and CD8+ T cells may be critical for inducing the destruction of thyroid parenchyma, as previously suggested in other experiences (47, 48, 49).

According to this model, innate and acquired immune system may play different roles in the thyroid autoimmune process. Future studies will be needed to demonstrate whether the specific modulation of IFN{gamma} (Th1) and IL-4 (Th2) polarization of NK, NKT, CD4+, and CD8+ cells may modify the evolution of autoimmune process in patients with thyroid autoimmunity.


   Footnotes
 
First Published Online April 26, 2005

Abbreviations: Co-HCV+, HCV-positive patients without IFN-AT acting as control; FT3, free T3; FT4, free T4; HCV, hepatitis C virus; HT, Hashimoto’s thyroiditis; IFN, interferon; IFN-AT, IFN-related thyroid autoimmune disease; IFN-AT(Dy), IFN-AT accompanied by different degrees of thyroid dysfunction; IFN-AT(Eu), IFN-AT patients who developed euthyroidism; NK, natural killer; PBMC, peripheral blood monuclear cell; TgAb, thyroglobulin antibody; Th, T helper; TPOAb, thyroperoxidase antibody; TRAb, TSH receptor antibody.

Received January 18, 2005.

Accepted April 20, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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