To better understand the genesis of autoimmunity in Graves’ disease (GD), it is essential to study the mechanism of apoptosis and cell proliferation in thyroid cells and intrathyroidal lymphocytic infiltrate of GD patients.
Graves’ disease (GD) is an autoimmune thyroid disease that involves [
BCL-2 (B-cell lymphoma/leukemia 2) is an antiapoptotic molecule from the BCL-2 protein family. Other molecules of the family participate in the control of apoptosis: antiapoptotic MCL-1 (myeloid cell leukemia 1) and proapoptotic BID (
Autoimmune thyroid diseases and their clinical characteristics are distinguished by an imbalance between the degree of apoptosis and cell proliferation. Ki-67 and p27Kip1 proteins are markers related to cell proliferation in thyroid cells. Ki-67 is a nuclear protein produced in dividing cells that is used as a marker to identify active dividing cells. Doganay et al. [
The study was carried out at the University of Campinas Teaching Hospital. Paraffin blocks of thyroidectomy products with anatomopathological and clinical diagnoses of GD and normal thyroid were selected. New sections were prepared for the immunohistochemical analysis of markers of apoptosis (Fas, Fas ligand, and BID), antiapoptosis (BCL-2, MCL-1), cell proliferation (Ki-67), and antiproliferation (p27Kip1) in thyroid cells and intrathyroidal lymphocytic infiltrate, as well as an evaluation of the lymphocytic infiltrate pattern in haematoxylin-eosin (HE) stained cells by an experienced pathologist. Clinical and laboratory parameters such as age, sex, serum thyroid-stimulating hormone (TSH) and free levothyroxine (FT4) concentrations, and antithyroperoxidase (anti-TPO) and antithyroglobulin (anti-Tg) antibodies were collected, retrospectively, by reviewing the chart.
The institutional review board approved the study under the opinion number 783.083, and no competing financial interests exist.
We selected 67 individuals from an iodine area who underwent thyroidectomy between 1996 and 2015 and whose specimens were maintained by the tissue bank of the hospital; 53 were GD patients, and 14 had normal thyroid samples in the control group. Out of 67 individuals studied, 52 were female. The clinical diagnosis of GD was confirmed based on the presence of goiter, hyperthyroidism, or euthyroidism using thionamide, with at least one of the antithyroid serum antibodies in high concentrations. The indications of thyroidectomy were difficult disease control even in optimized therapy or serious adverse reactions due to the use of antithyroid drugs such as hepatitis drug, cutaneous reaction, or agranulocytosis. We included in the GD sample, eight patients who were not using ATD at the time of surgery. Among patients with GD, 54.72% were smokers, 88.68% were using thionamide at the time of surgery, 84.91% had exophthalmos, and only 11.33% underwent previously radioiodine therapy. In the control group, the indications of thyroidectomy were thyroid infiltrative malignant neoplasm of larynx or suspicion of thyroid neoplasia. The inclusion criteria were the absence of antithyroid antibodies, euthyroidism, and normal thyroid tissue by anatomopathological analysis. If there were thyroid neoplasia in the sample, that case was excluded.
Serum TSH concentrations were measured by the electrochemiluminescence (EIA) method—a sandwich technique used in the Elecsys TSH immunoassay analyser (Roche), and a range of 0.4 to 4.5
We selected 10% formalin-fixed paraffin-embedded blocks of 67 thyroidectomies. We performed new histological sections for lymphocytic infiltrate evaluation and immunohistochemical analysis with proliferation, antiproliferation, cell apoptosis, and antiapoptotic markers.
The selected paraffin blocks were submitted to 4
Characteristic of primary antibodies.
Marker | Antibody | Dilution | Positive control | Producer |
---|---|---|---|---|
BCL-2 | Mouse monoclonal, clone 124 | 1 : 150 | Colon carcinoma | Dako, USA |
MCL-1 | Rabbit monoclonal, clone Y37 | 1 : 50 | Tonsil | Abcam, Cambridge, USA |
FasL | Rabbit polyclonal | 1 : 70 | Tonsil | Abcam, Cambridge, USA |
Fas | Rabbit polyclonal | 1 : 25 | Tonsil | Abcam, Cambridge, USA |
BID | Rabbit monoclonal, clone Y8 | 1 : 150 | Prostate carcinoma | Abcam, Cambridge, USA |
Ki-67 | Mouse monoclonal, clone MIB-1 | 1 : 500 | Lymph nodes | Dako, CA, USA |
p27Kip1 | Mouse monoclonal, clone SX53G8 | 1 : 50 | Tonsil | Dako, USA |
Anti-MCL-1 monoclonal antibody detects the long chain of MCL-1 protein (37 kDa), called isoform 1, and is defined as an antiapoptotic protein [
All analyses were performed under light microscopy (Eclipse E200, Nikon Instruments Inc., NY, USA). Two blinded observers, one pathology expert in thyroid (IBS) and the first author (JCV), reviewed all thyroid slides at different times individually (Cohen’s kappa = 0.628 − substantial agreement), and cases with inconsistent scores were reevaluated to achieve a consensus score. An evaluation of semiquantitative and visual immunohistochemistry (IHC) was done in follicular epithelium and lymphoid follicles, in each case, in at least three representative tissue areas, and different cut-off points were used for each marker, based on the extent of expression at 40x magnification (calculating the percentage of immunoreactive cells of at least 500 cells). For cell proliferation evaluation, we used Ki-67 and p27kip1, with nuclear positivity on thyrocytes and lymphocytes. For apoptosis, we used BID, Fas, and FasL with cytoplasm positivity in thyroid cells and cytoplasm or nuclear membrane positivity in lymphocytes. For antiapoptosis, we used BCL-2 and MCL-1, with cytoplasm positivity in thyroid cells and lymphocytes. It was considered strong expression of Fas/FasL when a minimum of 50% of cells were clearly positive. The staining intensity of Ki-67 was scored as expression less than 1% and greater than 1%. The marker BID was evaluated and classified as expression lower and greater than 50%. For BCL-2, the staining was divided into no or weak reactivity (0–25%) and positive (>25%). For p27Kip1, the expression was classified into three groups: no expression (0%), weak positivity (1 to 25%), and strong (>25%) positivity. MCL-1 protein expression was classified as strong positivity (>20%) and weak positivity (<20%).
Aiming to describe the sample profile according to the variables studied, we used frequency tables of categorical variables (sex, presence of lymphocytic infiltrate, and antithyroid antibodies in the two groups, as well as exophthalmos and radioiodine treatment only in cases of GD) with absolute frequency (
The younger patients were those with GD (mean 39.51 years,
We could observe that as lymphocytic infiltrate (Figure
Lymphocytic infiltrate degrees: (a) without infiltrate or infiltrate 0 (×10); (b) light grade or focal infiltrate (×10); (c) moderate degree infiltrate or 2 (×10); (d) intense degree infiltrate or 3 (no magnification).
Expression of markers in thyroid specimens. (a) Fas (×40); (b) FasL (×40); (c) BID (×40); (d) BCL2 (×10); (e) BCL2 in intrathyroidal secondary lymphoid follicle (×40); (f) MCL1 (×10); (g) Ki67 in intrathyroidal secondary lymphoid follicle (×40); (h) p27Kip1 (×10).
Expression of proliferation (Ki-67) and antiproliferation (p27) cell markers in thyrocytes and lymphocytes according to the diagnosis: Graves’ disease versus normal control (chi-square test/
Markers | % | Graves | Control | |
---|---|---|---|---|
Ki-67 in thyrocytes | <1% | 56.82% | 85.71% | 0.1254 |
>1% | 43.18% | 14.29% | ||
Ki-67 in lymphocytes | <1% | 38.64% | 71.43% | 0.0001 |
>1% | 61.36% | 28.57% | ||
p27 in thyrocytes | <25% | 18.18% | 21.43% | 0.6892 |
>25% | 81.82% | 78.57% | ||
p27 in lymphocytes | <25% | 63.64 | 92.86% | <0.0001 |
>25% | 36.36% | 7.14% |
Expression of proliferation markers in thyrocytes and lymphocytes in patients with Graves’ disease according to the type of treatment (chi-square test/
Markers | Cells | ATD (%) | Without ATD (%) | RIT (%) | Beta-blocker (%) | |
---|---|---|---|---|---|---|
Ki-67 | Thyrocytes | <1% | 56.82 | 25 | 83.33 | |
>1% | 43.18 | 75 | 16.67 | |||
0.1337 | 0.1919 |
|||||
Lymphocytes | <1% | 38.64 | 37.5 | 66.67 | 45.95 | |
>1% | 61.36 | 62.5 | 33.33 | 54.05 | ||
1.0 | 0.1710 |
0.0608 | ||||
p27 | Thyrocytes | <25% | 18.18 | 50 | 50 | 29.73 |
>25% | 81.82 | 50 | 50 | 70.27 | ||
0.0715 | 0.1480 |
0.1433 | ||||
Lymphocytes | <25% | 63.64 | 37.5 | 64.86 | ||
>25% | 36.36 | 62.5 | 35.14 | |||
0.4115 | 0.4798 |
Expression of apoptotic markers in thyrocytes and lymphocytes in patients with Graves’ disease according to the type of treatment (chi-square test/
Markers | Cells | ATD (%) | Without ATD (%) | RIT (%) | Beta-blocker (%) | |
---|---|---|---|---|---|---|
BID | Thyrocytes | <50% | 27.27 | 37.5 | 16.67 | |
>50% | 72.73 | 72.5 | 83.33 | |||
0.6756 | 0.6627 |
|||||
Lymphocytes | <50% | 100 | 97.30 | |||
>50% | 0 | 2.70 | ||||
1.0 |
0.0770 | |||||
Fas | Thyrocytes | <50% | 2.27 | 0 | 0 | 2.70 |
>50% | 97.73 | 100 | 100 | 97.30 | ||
— | — | — | ||||
Lymphocytes | <50% | 65.91 | 32.5 | 83.33 | ||
>50% | 34.09 | 62.5 | 16.67 | |||
0.2346 | 0.3794 |
|||||
Fas-L | Thyrocytes | <50% | 2.27 | 12.5 | 0 | 0 |
>50% | 97.73 | 87.5 | 100 | 100 | ||
0.2866 | — | — | ||||
Lymphocytes | <50% | 72.73 | 75 | 100 | 72.97 | |
>50% | 27.27 | 25 | 0 | 27.03 | ||
1.0 | 0.1639 |
1.0 |
If we divided these patients according to the use of ATD, those who were not using ATD had higher expression of the proapoptotic marker BID (greater than 50%) in intrathyroid lymphocytes (
Expression of antiapoptotic markers in thyrocytes and lymphocytes in patients with GD according to the type of treatment (chi-square test/
Markers | Cells | ATD (%) | Without ATD (%) | RIT (%) | Beta-blocker (%) | |
---|---|---|---|---|---|---|
BCL-2 | Thyrocytes | <30% | 6.82 | 25 | 16.67 | 13.51 |
>30% | 93.18 | 75 | 83.33 | 86.49 | ||
0.1643 | 0.4952 |
0.3049 | ||||
Lymphocytes | <30% | 84.09 | 87.5 | 100 | ||
>30% | 15.91 | 12.5 | 0 | |||
1.0 | 0.5742 |
|||||
MCL-1 | Thyrocytes | <20% | 4.55 | 25 | 16.67 | 8.11 |
>20% | 95.45 | 75 | 83.33 | 91.89 | ||
0.1072 | 0.4175 |
1.0 | ||||
Lymphocytes | <20% | 75 | 37.5 | 100 | ||
>20% | 25 | 62.5 | 0 | |||
0.0889 | 0.1588 |
Comparison of markers of cell proliferation and apoptosis among patients with Graves’ disease according to radioiodine, thionamide, and beta-blocker therapy.
Treatment | Ki-67 | p27 | BCL-2 | MCL-1 | BID | Fas | |
---|---|---|---|---|---|---|---|
RIT | Thyrocytes | ||||||
Lymphocytes | ↓ | ||||||
ATD use | Thyrocytes | ||||||
Lymphocytes | ↓ | ||||||
Beta-blocker use | Thyrocytes | ↓ | ↑ | ||||
Lymphocytes | ↓ | ↓ | ↓ |
GD pathophysiology involves dysregulation of apoptosis and lymphocytic infiltration, in addition to the result of the balance between proapoptotic and antiapoptotic factors, as well as proliferative and antiproliferative factors in thyroid cells and intrathyroidal lymphocytes [
When analysing the expression of markers between GD patients and the control group, we found that Ki-67 (proliferation) and p27Kip1 (antiproliferation) expression in intrathyroidal lymphocytes was lower in the control group than that in GD patients using antithyroid drugs (ATD). Studies have shown a tendency toward apoptosis of intrathyroidal lymphocytes in patients with GD who take ATD and exhibit less cell proliferation, with an elevation of the p27Kip1 marker and its regulator Ki-67 [
According to the literature [
We divided GD patients into two groups: with and without the use of ATD at the time of surgery. The expression of BID in intrathyroidal lymphocytes was lower in patients using ATD, suggesting that they had less lymphocytic apoptosis, which stimulates less activity of thyrocytes and may be another disease control mechanism of ATD.
In relation to MCL-1 in GD, patients with the lowest expression of this marker in lymphocytes are those with the largest goiters; those using beta-blockers also had lower expression of MCL-1 and BCL-2 in lymphocytes. If MCL-1 or BCL-2 is decreased in lymphocytes, they survive less and have less stimulating thyrocyte apoptosis, leading to a greater goiter volume. There are already studies evaluating melanoma and thyroid carcinoma [
The higher proportion of positive detectable antithyroid antibodies found in patients with GD with more prominent lymphocytic infiltrate suggests that a larger lymphocytic infiltrate and activation of T cells led to a higher recruitment of B cells and the development of immune response via antithyroid antibodies [
The study limitations were as follows: the impossibility to show a predominance of one marker over another. We evaluated the expression of markers separately, and there was a lower number of individuals in the control group due to the lower indication of thyroidectomy in these cases. The strengths were as follows: the study was carried out with anatomopathological findings, thyrocytes, and intrathyroidal lymphocytes; the study contained many patients and immunohistochemical markers, as well as an evaluation of drugs in use and their effects on apoptotic mechanisms and cell proliferation.
In conclusion, the recognition of thionamides as immunomodulatory drugs, with alteration of the lymphocytic infiltrate in patients who use them, and RIT as a lymphocyte sparer represents significant findings obtained from this study. We believe that these findings in the immunohistochemical expression of cell antiproliferation (p27Kip1 on intrathyroidal lymphocytes), apoptotic (BID on thyroid cells and intrathyroidal lymphocytes), and antiapoptotic (MCL-1 on thyroid cells and intrathyroidal lymphocytes) markers may be relevant in the development of new drugs and a better understanding of the pathophysiology of these diseases. Additionally, understanding the role of beta-blockers, more specifically propranolol, in GD has been poorly explored in thyroid studies, although it has been described as an important aid in melanoma cells and cardiac cells. Therefore, the greater BID (apoptotic) expression in the thyrocytes and lower expression of MCL-1 (antiapoptotic) in lymphocytes may lead to the routine use of these drugs in therapy for Graves’ disease, changing its indication. Further studies are needed to better understand the pathophysiology of these diseases and to better target the therapeutic regimen for less need for surgeries and complications.
The authors declare that they have no conflict of interest.
Jessica Castro de Vasconcelos conducted the histopathological and immunochemical review and participated in the conception, database collection, statistical analysis, interpretation, and review of the literature. Icléia Siqueira Barreto conducted and coordinated the immunochemical review and study design. Patrícia Sabino Matos conducted the histopathological review and interpretation. Frederico Fernandes Ribeiro Maia, Marcos Antônio Tambascia, and Maria Cândida Ribeiro Parisi conceived the study and participated in the design. Denise Engelbrecht Zantut-Wittmann conceived the study and participated in the design, interpretation of the results, and coordination. All authors read and approved the final version of the manuscript.
This study was funded by FAPESP (State of São Paulo Research Foundation).