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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 12 5064-5068
Copyright © 2006 by The Endocrine Society

Impaired Caspase-3 Expression by Peripheral T Cells in Chronic Autoimmune Thyroiditis and in Autoimmune Polyendocrine Syndrome-2

Francesco Vendrame, Maria Segni, Daniele Grassetti, Valeria Tellone, Giovanni Augello, Vincenzo Trischitta, Massimo Torlontano and Francesco Dotta

Diabetes Unit, Department of Internal Medicine, Endocrine and Metabolic Sciences and Biochemistry (F.V., V.Te., F.D.), University of Siena, 53100 Siena, Italy; Departments of Pediatrics (M.S.) and Clinical Sciences-Endocrinology (D.G., V.Tr.), University La Sapienza, 00161 Rome, Italy; and Unit of Endocrinology (G.A., V.Tr., M.T.), Scientific Institute Casa Sollievo della Sofferenza, 71013 San Giovanni Rotondo, Italy

Address all correspondence and requests for reprints to: Francesco Dotta, M.D., Unità Operativa di Diabetologia, 3 Lotto 5S, Policlinico Le Scotte, Viale Bracci 18, 53100 Siena, Italy. E-mail: fradotta{at}tin.it.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Activation-induced cell death (AICD) is a major mechanism in the regulation of peripheral tolerance, and caspase-3 represents its major executioner. AICD impairment contributes to the persistence of autoreactive T cells, and defective AICD has been reported in autoimmune thyroiditis as well as in type 1 diabetes mellitus.

Objective: The objective of this study was to evaluate the involvement of caspase-3 in the regulation of AICD resistance in thyroid and polyendocrine autoimmunity.

Design/Settings/Patients/Intervention: Caspase-3 expression was analyzed in peripheral blood lymphocytes from 26 adults (A-AT) and 25 children (Y-AT) affected by autoimmune thyroiditis and 13 individuals affected by chronic autoimmune thyroiditis plus Addison’s disease [autoimmune polyendocrine syndrome-2 (APS-2)] in comparison with 32 age-matched normal control subjects (NC).

Outcome Measures: Caspase-3 mRNA expression in peripheral T cells was evaluated by quantitative real-time PCR; protein expression of both procaspase-3 and activated caspase-3 by Western blot analysis was followed by scanning densitometry.

Results: Caspase-3 mRNA expression was significantly reduced in resting lymphocytes from both A-AT (P = 0.001) and Y-AT (P = 0.016) compared with NC. After lymphocyte activation, protein levels of caspase-3 active form were significantly reduced in A-AT (P = 0.023) and Y-AT (P = 0.001) compared with NC. The APS-2 group displayed characteristics similar to the A-AT group because both caspase-3 mRNA and protein active form levels were significantly reduced compared with NC (P = 0.004 and 0.002, respectively).

Conclusion: Our data show that peripheral lymphocytes of subjects affected by thyroid autoimmunity or APS-2 show defective expression of the major executioner of AICD, thus potentially contributing to AICD resistance and to the development of autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
IN THE IMMUNE system, activation-induced cell death (AICD) plays a crucial role in limiting the immune responses and in deleting autoreactive T cells (1, 2, 3). Freshly isolated T cells are resistant to AICD because activation is required to up-regulate death receptors (3). Then, activated T cells may die by fratricide behavior, after making contact with their activated neighbors, or by suicide when the cell, upon activation, kills itself (4, 5). The process is mainly dependent on the cross-linking of cell surface death receptors; for example, Fas with its natural ligand FasL, although the cytokine withdrawal represents another important pathway (6, 7). Once AICD is triggered, different molecules, called caspases, are involved in signal transduction (8, 9). Caspases are a family of aspartate-specific cysteine proteases required for the execution of apoptosis (3). They are synthesized as proenzymes, activated by proteolytic cleavage, and the active form is a heterotetrameric complex. Among caspases, caspase-3 represents the main executioner of the apoptotic machinery (10, 11). The molecule has the highest expression in cells of lymphocytic origin. It is synthesized as proenzyme of 32 kDa, which, once activated, produces two active subunits that account for the selective cleavage of vital substrates and the apoptotic death (12). Furthermore, caspase-3 plays an important role in T cell homeostasis because it is required for T cell proliferation (13), and in mice deficient for such protein, peripheral T lymphocytes are less susceptible to anti-CD3- or anti-Fas-mediated apoptosis (14).

Different studies have reported the existence of impaired apoptosis in peripheral T cells of subjects affected by autoimmune diseases (15, 16). Mutations of the Fas gene result in defective Fas-mediated apoptosis, lymphoproliferation, and development of autoimmune diseases such as hemolytic anemia and thrombocytopenia (17). In subjects affected by multiple sclerosis, AICD resistance in peripheral T cells is associated with an overexpression of the apoptosis inhibitor FLICE-like inhibitory protein (18), as well as with an increased expression of the inhibitor of apoptosis proteins (19). Finally, in subjects affected by Hashimoto thyroiditis, a resistance to both Fas- and ceramide-induced apoptosis can occur in peripheral T cells, partially as a consequence of an impaired activity of caspase-8 and/or caspase-9 (20).

We recently reported that peripheral blood lymphocytes of patients affected by type 1 diabetes mellitus present a reduced expression of caspase-3 and that such defects contribute to the development of AICD resistance in peripheral T cells (21). In the present study, we investigated the mRNA and protein caspase-3 expression in peripheral T cells of adults and children affected by chronic autoimmune thyroiditis, including individuals affected by chronic autoimmune thyroiditis plus Addison disease [namely, autoimmune polyendocrine syndrome-2 (APS-2)] in comparison with normal individuals. Our results show that an impaired expression of caspase-3 active form is a frequent trait in peripheral T cells of subjects affected by single and multiple autoimmune organ-specific endocrinopathies.


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

Peripheral blood mononuclear cells (PBMCs) were obtained from 26 adult subjects affected by chronic autoimmune thyroiditis (A-AT) (47.0 ± 13.0 yr old; 22 females, four males; disease duration, 1.4 ± 0.7 yr), 13 individuals affected by APS-2 (46.0 ± 10.6 yr old; 11 females, two males; disease duration, 1.3 ± 0.9 yr), and 10 healthy adult normal controls (A-NC) (40.0 ± 3.4 yr old; seven females, three males) recruited among students, residents, and staff members of the University La Sapienza. In addition, 25 young individuals with chronic autoimmune thyroiditis (Y-AT) (12.0 ± 4.1 yr old; 21 females, four males; disease duration, 3.5 ± 2.9 yr) and 22 age-matched young normal control subjects (Y-NC) (11.2 ± 4.0; 13 females, nine males) followed at the outpatient Pediatric Clinic of the University of Rome La Sapienza and tested negative for thyroid autoantibodies were included in the study. At the time of the study, all patients were in the euthyroid state (nine A-AT and 10 Y-AT, respectively, were under L-thyroxine treatment). The diagnosis of chronic autoimmune thyroiditis was based on clinical presentation, thyroid hormone levels, positivity for thyroid autoantibodies (antithyroperoxidase and antithyroglobulin) measured by RIA, and evidence of decreased or dishomogenous echogenicity at thyroid ultrasonography. Documented hypocortisolemia, positivity for 21-hydroxilase autoantibodies evaluated by RIA, and a positive ACTH stimulation test substantiated the diagnosis of Addison’s disease. Normal control subjects had no family history of autoimmune endocrinopathies, no biochemical sign of chronic autoimmune thyroiditis or Addison’s disease, and were tested negative for thyroid autoantibodies. The study was conducted in accordance with the indications of The Declaration of Helsinki and approved by the ethical committee of the University of Rome La Sapienza.

Cell separation and cultures

PBMCs were isolated from heparinized blood by Ficoll-Hystopaque density gradient centrifugation, washed twice with RPMI 1640 medium (Life Technologies, Inc.-BRL, Grand Island, NY), and either frozen and stored in liquid nitrogen until used for caspase-3 expression studies or cultured (1 x 106/ml) in 25-cm2 flasks (Falcon Labware, Becton Dickinson, Lincoln Park, NJ) with RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics. PBMCs contained about 90% CD3+ T lymphocytes by cytofluorimetric analysis (EPICS XL-MCL cytometer, Coulter Electronics, Hialeah, FL) employing an anti-CD3 monoclonal antibody (Becton Dickinson, San Jose, CA). PBMCs were then activated with phytohemagglutinin (PHA) (5 µg/ml) (Sigma, St. Louis, MO) for 72 h. Cell activation was determined by cytofluorimetric analysis employing an anti-CD25 monoclonal antibody (Dako Corporation, Santa Barbara, CA). At d 3 of culture, flow cytometry analysis showed that the proportion of T lymphocytes was more than 95%.

Real-time PCR

Total RNA was extracted from 107 purified PBMCs with an RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. First strand cDNA was prepared using Superscript II (Invitrogen, Paisley, UK). Message levels for caspase-3 and ß-actin were quantified using Assay on Demand real-time PCR kits (Applied Biosystems, Foster City, CA) with TaqMan Master Mix (Applied Biosystems) and ABI 7700 Sequence Detection System (Applied Biosystems). Amplification was carried out in a total volume of 25 µl for 40 cycles of 15 sec at 95 C and 1 min at 60 C. Samples were run in triplicate, and their relative expression was determined by normalizing expression of each target to ß-actin and then comparing this normalized value with the normalized expression in a reference sample to calculate a fold change value.

Western blot

Activated lymphocytes were washed twice with Ca2+- and Mg2+-free PBS buffer. Cell extracts were prepared by lysing 107 cells in 80 µl lysis buffer containing 50 mM Tris buffer (pH 7.6), 150 mM sodium chloride, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 0.5% sodium deoxycholate, 100 µg/ml phenylmethylsulfonyl fluoride, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin A at 4 C. Insoluble materials were removed by centrifugation for 20 min at 14,000 rpm at 4 C. Protein content was determined using the BCA protein assay kit (Pierce, Rockford, IL). Protein samples (20 µg protein) were boiled for 5 min in a SDS buffer and separated in 12% SDS-PAGE according to Laemmli (22). Proteins were electroblotted to nitrocellulose membranes (Schleicher & Schuell GmbH, Dassel, Germany). The loading and transfer of equal amounts of protein were confirmed by staining the membrane with Ponceau S. The blots were blocked by incubation in 25 mM Tris-Hcl (pH 8.0), 125 mm NaCl (Tris-buffered saline) with 0.05% Tween 20 [Tris-buffered saline/Tween 20 (TBST)] and 5% nonfat dry milk at room temperature for 1 h. After a brief rinse, blots were incubated overnight at 4 C in TBST-5% milk with rabbit anticaspase-3 polyclonal antibody (Abcam Limited, Cambridge, UK) (1:1000). Mouse anti-ß-actin monoclonal antibody (Sigma) (1:5000) was used as an internal control protein. Blots were washed four times with TBST, incubated with peroxidase conjugated with antirabbit or antimouse IgG (Santa Cruz, Palo Alto, CA) (1:2000) in TBST for 1 h at room temperature, washed again, and developed with a enhanced chemiluminescence reagent (Pierce). For each sample, expression levels of caspase-3 active form were determined by the ratio between the cleaved form and whole protein by densitometry scanning of bands using the Image J software (National Institutes of Health, Bethesda, MD).

Statistical analysis

The Mann-Whitney U test was employed to compare patients and normal controls in terms of caspase-3 expression levels. Fisher’s exact test was used to compare the frequency of subjects with pathologically reduced caspase-3 expression levels in patients and normal controls. Both caspase-3 mRNA and protein active form levels less than mean – 3 SD of control subjects were considered as pathologically reduced. The Spearman correlation test was used to evaluate the relationship between the expression levels of caspase-3 mRNA and caspase-3 protein in the active form. P < 0.05 was considered significant. Data were analyzed using the GraphPad statistical software (GraphPad, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Impaired lymphocyte caspase-3 mRNA expression in chronic autoimmune thyroiditis and in APS-2

Caspase-3 mRNA expression was analyzed by real-time PCR in resting lymphocytes of 26 A-AT, 13 APS-2, and 25 Y-AT subjects and compared with their age-matched control individuals. As for adult control individuals, caspase-3 expression levels were virtually superimposable to those previously reported by us in a study (21) performed on a larger group of subjects. In A-AT and in APS-2 subjects, caspase-3 mRNA expression levels were significantly reduced compared with A-NC (P = 0.001 and 0.004 respectively, by Mann-Whitney U test) (Fig. 1AGo). In particular, caspase-3 mRNA levels less than mean – 3 SD of control subjects were found in 15 of 26 A-AT subjects (P = 0.002 by Fisher’s test vs. A-NC) and in seven of 13 APS-2 individuals (P = 0.007 by Fisher’s test vs. A-NC) (Fig. 1AGo). In addition, because age is correlated with changes in immunological profiles (23), we investigated whether the defects detected in the A-AT and APS-2 subjects were also detectable in children affected by chronic autoimmune thyroiditis in comparison with age-matched healthy individuals. Indeed, Y-AT subjects showed significantly reduced levels of caspase-3 mRNA expression compared with Y-NC (P = 0.016 by Mann-Whitney U test) (Fig. 1BGo), confirming the data found in the adult groups.


Figure 1
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  		FIG. 1. Impaired caspase-3 mRNA expression in resting peripheral T cells of patients affected by chronic autoimmune thyroiditis or by APS-2. Caspase-3 mRNA expression levels were evaluated by real-time PCR by normalizing expression of each target to ß-actin and then comparing this normalized value with the normalized expression in a reference sample to calculate a fold change value. The expression levels were significantly reduced in A-AT (n = 26) and APS-2 (n = 13) individuals compared with A-NC (n = 10) (A) and in Y-AT subjects (n = 25) compared with Y-NC (n = 22) (B). The statistical comparison among groups was performed by Mann-Whitney U test. Solid lines, Mean of caspase-3 mRNA expression levels. Horizontal dashed line, value corresponding to the mean – 3 SD of caspase-3 mRNA expression in normal control subjects; caspase-3 mRNA levels below this value were considered as pathologically reduced.

 
Impaired caspase-3 mRNA expression correlates with a defective expression of its protein active form in activated peripheral lymphocytes of subjects with chronic autoimmune thyroiditis and subjects with APS-2

In view of the fact that an impaired expression of caspase-3 mRNA in resting condition may impair the generation of the protein active form, we investigated the expression levels of caspase-3 active form after T cell activation in A-AT, APS-2, and Y-AT individuals in comparison with age-matched controls. Western blot analysis revealed that A-AT and APS-2 patients showed significantly lower expression levels of caspase-3 active form (P = 0.023 and 0.002, respectively, by Mann-Whitney U test) compared with A-NC (Fig. 2AGo). As shown in Fig. 2BGo, after lymphocyte activation in A-NC, caspase-3 resulted in its cleavage and generation of its active form; in contrast, in A-AT and APS-2 individuals, lymphocyte activation resulted in an impaired cleavage of caspase-3 with absence (lanes 4 for both A-AT and APS-2) or reduction (lanes 2 and 3 and 1–3, respectively, for A-AT and APS-2) of its active form. Pathologically reduced levels of caspase-3 protein active form defined as values less than mean – 3 SD of control group were present in nine of 17 A-AT patients, in four of five APS-2 patients, and in zero of 10 A-NC patients (P = 0.022 and 0.003, respectively, by Fisher’s test vs. A-NC). Next, we extended our investigation to Y-AT subjects. Similar to A-AT and APS-2 subjects, in Y-AT individuals, the expression levels of caspase-3 protein active form were significantly reduced compared with Y-NC (P = 0.001 by Mann-Whitney U test) (Fig. 3AGo). As shown in Fig. 3BGo, after T cell activation, caspase-3 resulted in almost complete lack of its active forms in the Y-AT group (Fig. 3BGo, lanes 2–4).


Figure 2
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  		FIG. 2. Defective expression of caspase-3 protein active form in activated peripheral T cells of adults affected by chronic autoimmune thyroiditis or by APS-2. The caspase-3 protein active form was evaluated by Western blot analysis in peripheral T cells after activation with PHA at 5 µg/ml for 3 d. A, Caspase-3 active form expression levels were significantly reduced in A-AT (n = 17) and APS-2 (n = 5) subjects compared with A-NC (n = 10). Solid lines, Mean values of the expression levels of caspase-3 protein active form. Horizontal dashed line, Lower limit of normal range, defined as the mean – 3 SD of caspase-3 active form expression in normal controls. B, Representative gel photographs show higher frequency of reduced caspase-3 protein active from expression in A-AT and APS-2 compared with normal controls. The band at 32 kDa represents procaspase-3 (inactive protein); bands at 19 and 17 kDa represent the active fragments after caspase-3 cleavage.

 

Figure 3
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  		FIG. 3. Defective expression of caspase-3 active form in activated peripheral T cells in children affected by chronic autoimmune thyroiditis. Caspase-3 protein active form was evaluated by Western blot analysis in peripheral T cells after activation with PHA at 5 µg/ml for 3 d. Caspase-3 active form expression levels were significantly reduced in Y-AT (n = 17) individuals compared with Y-NC (n = 10). Solid lines, Mean values of the expression levels of caspase-3 active form. B, Representative gel photographs show higher frequency of reduced caspase-3 protein active from expression in Y-AT subjects (B) compared with normal controls. The band at 32 kDa represents procaspase-3 (inactive protein); bands at 19 and 17 kDa represent the active fragments after caspase-3 cleavage.

 
With regard to the relationship between caspase-3 mRNA and caspase-3 active form protein expression, A-AT individuals studied for both of these parameters revealed that subjects with impaired expression of caspase-3 mRNA displayed a defective expression of caspase-3 protein active form as well (r = 0.92, P = 0.037 by Spearman correlation test). However, we observed 3 A-AT individuals with normal levels of caspase-3 mRNA expression but reduced expression of caspase-3 active form and two other A-AT individuals with the opposite pattern. A positive association between caspase-3 mRNA and protein levels was also found in the Y-AT group (r = 0.53, P = 0.039 by Spearman correlation test).

Relationship among age, disease duration, and hormone replacement therapy

In all the groups studied, caspase-3 expression was not affected by any of the following parameters: age, sex, autoantibody status, disease duration, and hormone replacement therapy.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
It is now well established that defects in the mechanisms underlying the regulation of peripheral tolerance in the immune system result in the development of autoimmune phenomena (1, 2, 3). To date, AICD represents one of the main players in this scenario. Defects in this process impair the turning-off system of the immune responses, may lead to the inappropriate exposure of self antigens, and favor the persistence of autoreactive T cells in the periphery (15, 16). We previously observed that in type 1 diabetes mellitus, peripheral T cells are resistant to Fas-mediated AICD and that this defect is associated with an impaired expression of caspase-3, the main executioner of AICD (21). In the present study, we extended our observation to adults and children affected by chronic autoimmune thyroiditis and to individuals with APS-2. Our results show an impaired expression of caspase-3 not only in subjects affected by chronic autoimmune thyroiditis alone but also in patients suffering from multiple endocrine organ-specific autoimmune diseases such as APS-2.

In Hashimoto thyroiditis, one of the most studied models of chronic autoimmune thyroiditis, thyrocytes appear to die by autocrine or paracrine interactions between Fas and its natural ligand, Fas ligand (24). In this context, infiltrating T cells play an important role by producing Th1-like cytokines able to up-regulate Fas and Fas ligand (25, 26), whereas peripheral T cells are resistant to Fas and ceramide-mediated AICD and present in some cases an impaired activity of caspase-8 and -9 (20). Our study reports the presence of an additional defect in the peripheral T cells of subjects affected by chronic autoimmune thyroiditis and may further explain the mechanisms underlying the Fas and ceramide resistance phenotype. Therefore, it is feasible that multiple defects in the same subject or in different individuals affect the AICD process and lead to an impairment of the immune homeostasis. This is suggested by our data, which show an impaired expression of caspase-3 mRNA in 58% of A-AT subjects that correlates with the defective expression of its protein active form. However, three A-AT subjects displayed normal expression levels of mRNA and reduced levels of caspase-3 active protein form, and two other individuals presented reduced caspase-3 mRNA levels despite the presence of normal protein expression. Thus, it is likely that compensatory and posttranslational events may be actively involved in caspase-3 processing and that multiple hits affect such a pathway.

Our study included a group of APS-2 subjects. The role of peripheral T regulatory cells has been recently investigated by Kriegel et al. (27), who found an impaired suppressive function of CD4+CD25+ regulatory T cells in APS-2 individuals. So far, however, no data are available on the role of AICD and its related molecules in the pathogenesis of such disease. Our findings suggest that the impaired expression of caspase-3 in APS-2 individuals represents another important defect that involves the AICD system. Interestingly, almost all APS-2 subjects (four of five) resulted in pathologically reduced levels of the caspase-3 protein active form, whereas in the A-AT group, the expression levels of this protein varied widely among the different subjects. In this regard, it is likely that in APS-2 subjects, different defects involve both the active (regulatory T cells) and passive (AICD) mechanisms of peripheral tolerance.

In conclusion, our study shows that in autoimmune endocrinopathies such as chronic autoimmune thyroiditis and APS-2, caspase-3 expression in peripheral T cells is defective. Such a feature represents a frequent trait in the studied populations and may favor the development of autoimmune phenomena. Rescuing T cells from this defect may represent a suitable strategy to restore the balance in the peripheral immune system and prevent the development of autoimmune phenomena.


   Footnotes
 
This work was supported by the Italian Ministry of Health, by the Italian Ministry of Research, and by the Fondazione Umberto Di Mario Organizzazione Non Lucrativa di Utilità Sociale (ONLUS).

First Published Online September 12, 2006

Abbreviations: A-AT, Adult affected by chronic autoimmune thyroiditis; AICD, activation-induced cell death; A-NC, adult normal control; APS-2, autoimmune polyendocrine syndrome-2; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; SDS, sodium dodecyl sulfate; TBST, Tris-buffered saline/Tween 20; Y-AT, young with chronic autoimmune thyroiditis; Y-NC, young normal control.

Received June 23, 2006.

Accepted September 1, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals