Hashimoto’s Thyroiditis

Anna Nandipati

Hashimoto Thyroiditis (HT) is one of the most common causes of hypo-thyroiditis and one of the main manifestations of autoimmune thyroid disease (4). HT is thought to be caused by a specific immune response to thyroid antigens which results in an intense lymphocytic infiltration of the thyroid gland. Lymphocytes become more sensitive to thyroidal antigens and auto-antibodies. The antibodies react with a variety of thyroid antigens, including thyroglobulin and thyroid peroxidases (3). As the disease evolves, it leads to heavy infiltration of lymphocytes leads to the destruction of thyrocytes through apoptosis, which is the major cause of HT. In general, the organs in the body maintain normal cell populations through a fixed rate of cellular proliferation and apoptosis (figure A). Apoptosis is a coordinated cycle of programmed events that result in cell death by changing the mitochondrial membrane permeability and proteolytic enzyme activity eventually the DNA is fragmented (11). Thyroid stimulating hormone (TSH) stimulates thyrocyte proliferation by blocking the apoptotic pathway. Tumor cells and CD8 cells often escape apoptosis by down-regulating Fas receptor or by inducing the expression of Fas ligand (FasL)(3). Fas receptors are needed for a cell to go through apoptosis. The presence of cytokines secreted by lymphocytes, such as IFN-γ, IL-1β, or TNF-α increases the sensitivity of thyrocytes to apoptosis by increasing Fas receptors(2). Most of the apoptotic cells are detected in areas of disrupted follicles, on the periphery of infiltrating lymphocytes. The number of apoptotic cells decreased in the thyroid follicles that are further away from lymphocyte germinal centers (3).

Approximately 4.6% of United States population is suffering from HT (1). Adult patients show mild symptoms such as, low tolerance to cold temperatures, low blood pressure, lethargy, muscle weakness, and unexplained weight gain. Infants and children show mental retardation and severe growth retardation (6). Some times these symptoms are irreversible in children. However, in adults the symptoms can be alleviated by taking hormone supplements. The actual mechanism by which the autoimmune process leads Hashimoto’s disease to death of thyrocytes is still not fully understood. There are various techniques that can be used to detect apoptosis of thyroid cells in Hashimoto’s thyroiditis. These techniques will be further examined.

Techniques for detection of thyrocyte apoptosis

Electron Microscopy

The apoptotic thyrocytes from patients with Hashimoto’s disease can be examined under electron microscopy. Fresh needle biopsies from HT patient are fixed and then stained with uranylacetate and lead citrate. The sections show shrunken and condensed nuclei under magnification, which is a characteristic of apoptosis. Electron Microscopy provides a direct visual evaluation of apoptosis and it shows subtle changes in nuclear density. Since the electron microscope is an expensive apparatus, it can be only used to confirm the results obtained with other methods (2).

DNA ladder

The fragmentation of genomic DNA is an irreversible event that commits the cells to death and this method is the gold standard for the detection of apoptosis. DNA is cleaved into fragments between nucleosomes that results in fragmented that are multiples 150bp. Nucleosomes are the basic packaging units of chromatin, which consists of about 150bp of DNA wrapped around a core of eight histone proteins (9). The DNA fragments can be separated on the gel electrophoresis.

This assay technique is can be used to identify apoptosis in thyrocytes. A sample of apoptotic cells should be incubated with an equal volume of binding/lysis buffer. The sample is then be poured into a filter tube containing glass fiber fleece. The DNA fragments are separated from the cellular components by centrifugation. After washing, the DNA is collected and run through gel electrophoresis. Apoptotic DNA produces a ladder of multiples of 180bp that can be observed clearly with ethidium bromide staining. Since apoptosis is a rapid phenomenon and it happens in less than 5% of the total cell population, DNA fragmentation may not be able to detect such a small percentage of apoptotic cells at one particular time. Therefore, complementary technique must always be used to confirm the diagnosis (2).

The terminal deoxynucleotidyl transferase mediated X-dUTP nick-end labelling (TUNEL) technique

Since endonucleases selectively cleave DNA at sites between nucleosomal units of the chromosomes, the fragments can be detected by enzymatic labeling of the free 3’OH of the fragment with modified nucleotides. As the DNA cleavage generally occurs at higher frequency in apoptotic cells than in non-apoptotic cells, the TUNEL method can be extensively used to detect apoptosis. This technique can be applied in association with flow cytometry to calculate the percentage of the apoptotic cells. To avoid the loss of DNA from the cells, frozen sections are fixed in formalin before permeabilization because the fragmented DNA might leak out through plasma membrane. Paraffin-embedded sections are then dewaxed, rehydrated and incubated with proteinase K. The 3’ ends of the DNA fragments generated in apoptotic cells are labeled with biotin-coupled uridine by using the enzyme terminal deoxynucleotidyl transferase (TdT). The biotin label is then detected with enzyme tagged streptavidin, which binds to biotin. Apoptosis is evaluated by calculating the percentage of positive nuclei in several fields. Fine needle biopsies produce higher positive nuclei counts than surgical specimens and similarly paraffin sections show higher positive counts than cryostat sections. However, the TUNEL technique cannot discriminate apoptosis from necrosis (2).

Fluorescence-activated cell sorting (FACS) analysis

Since activation of endonucleases induces DNA fragmentation, apoptosis can be measured by analyzing the remaining DNA in the cells using DNA binding dyes. In this method, thyrocytes are treated with hypotonic buffer containing a detergent at low concentration and the fragmented DNA is allowed to leak out during the rinse and staining procedure. Cultured thyroid cells are detached from the plates and re-suspended in a hypotonic solution to make the cell membrane leaky. After incubating the samples for about two hours a flow cytometry was used to calculate the percentage of apoptic cells. The propidium iodide fluorescence produced by individual nuclei is recorded (2).

Susceptibility of thyroid cells to apoptosis following incubation with Fas agonistic Ab and/or cytokines by FACS

Incubation of normal thyrocytes with IgM pro-apoptotic anti-FasnAb(CH11, 1µg/ml) alone for 18 hours was not able to induce apoptosis on thyroid cells. This indicates that these cells were resistant to apoptosis. However, a 24 hour pretreatment of these cells with IL-1β or IFNγ proved effective while thyroid stimulating hormone had an inhibitory effect on apoptosis. These results were also confirmed by morphological observations: the treated cells were seen to rounded up and detached from the plates (2).

Calorimetric Microtiter (MTT) assay

This method is based on the ability of normal cells to reduce 0.5 mg/ml tetrazolium salts to colored formazan compounds while dead cells do not. Tetrazolium should be incubated at 37°C with the thyroid cells for 4 hours. Acid isoproponolol is then be added and mixed to dissolve the dark blue product. Even though this method is easy to perform, it does not effectively detect apoptosis. It may underestimate cellular damage since it detects death at the later stage of apoptosis when the metabolic activity is clearly reduced. This test is more useful to observe apoptosis over a period of time (2).

Annexin V

This method is based on the changes in the plasma membrane of apoptotic cells. One of these changes includes the redistribution of neutral phospholipids such as phosphotidylserine from the inside to the outside of the cell. A molecule called annexin V will bind to the phosphatidylserine on the outer membrane of apoptotic cells. In this test a suspensions of 2-3x10⁶ thyroid cells from one HT patient’s thyroid gland were incubated with magnetic beads coated with antibody to the common leukocyte antigen, CD45, at 4°C for 45 minutes. The cells were then suspended in 100µl of binding buffer, 140mM NaCl and 10µl of fluorescein isothio cyanate (FITC) conjugated annexin V was added for 20 minutes. Double staining was performed by contra-staining the FITC-labeled annexin V stained cells with PI. Apoptotic cells will not take the stain because the phosphatidyl serine is on the inside of the cell membrane. However, necrotic cells will take the dye because the cell membrane does not change. With this method apoptotic cells could be separated from necrotic cells. But thyrocytes need to be incubated with CD45 magnetic beads two or three times to get better results. There is a chance of overestimation of both apoptotic and necrotic cells with this procedure (2).

Measurement of the expression of pro-apoptotic and anti-apoptotic genes

In this section we will examine the methods that are used to evaluate the expression of apoptosis-related receptor/ligand pairs on thyrocytes and intra-thyroidal lymphocytes. There are indications that Fas/FasL (ligand), expression on thyrocytes and intra-thyroidal lymphocytes and their ability to induce may determine the susceptibility of these cells to undergo apoptosis or their ability to kill other cells (2).

Immunohistochemistry

This test is based on the ability of inducing apoptosis by specific antigen antibody interaction. Frozen sections need to be fixed with and microwave in citric acid buffer to reveal the antigens before incubating with specific antibody for an hour at room temperature. A double staining technique can be done to identify antigen antibody by sequential use of streptadividin-peroxidase and streptavidin alkaline phosphatase. The advantage of this method is that it allows the direct in situ visualization and localization of gene expression within a particular subset of cells. However, this procedure relies heavily on the affinity and specificity of antibody and antigen expression (2).

Staining of thyroid cells in suspension and in culture

Suspensions of freshly cultured thyroid cells can be assessed for expression of apoptosis- related gene products such as death inhibiting bcl-2 or death promoting Bax and Bad. The cell suspensions are incubated for an hour on ice with the specific primary antibody and a secondary fluorochrome-conjugated antibody. If an intracellular molecule is to be stained, the cells should first be fixed and permeabilised for 20 minutes on ice. In order to identify the thyrocytes from lymphocytes, a double-staining technique can be carried out by using an antibody specific for a lymphocyte surface marker such as CD3 or thyroid peroxide, which is the surface marker of thyrocytes. The antibody is conjugated with fluorochrome. The samples were then analyzed by using a flow cytometry. The disadvantage of this procedure is the requirement of cell manipulation (2).

Immunoblotting

In this method, one can identify the proteins in the apoptotic thyroid cell. The cells lysed by suspending in lysis buffer containing proteinase inhibitors. After 30 minutes on ice, the lysates are centrifuged to remove cell debris and nuclei. The proteins will be separated on a 12% SDS polyacrylamide gel and transferred to a nitrocellulose or PVDF membrane by electroblotting. This membrane is blocked with non fat milk in TBS containing detergent for 1-2 hours at room temperature. Blots will then be incubated for 2 hour at room temperature with specific 1º antibody. After further washing the membrane is incubated with an enzyme-conjugated 2º antibody for 40-60 minutes at room temperature and visualized with enhanced chemiluminescent substrate. The antibody-protein complexes appear on the membrane as bands. The disadvantage of this procedure is that it requires a certain amount of expertise and training (2).

Conclusions

The hypothesis of autoimmune destructive thyroiditis is caused by apoptosis is supported by the use of the TUNEL technique and by in vitro flow cytometric assays in conjunction with DNA-binding fluorochromes. Fas staining has been found to be consistently positive or increased in thyrocytes from HT glands in all experiments performed by using paraffin-embedded sections, flow cytometry, and immunoblotting. Fas receptors on thyrocytes were inducible by IFN-γ and IL-β and that this induction could be inhibited by TSH. However, the Fas expression on intra-thyroidal lymphocytes is low in comparison to the follicular thyrocytes. The expression of Fas receptors and its role in apoptosis can be detected through FAC analysis and MTT assay. In situ and in vitro expression of anti-apoptotic genes on thyroid cells has been studied by immunochemistry on cryostat sections by immunoperoxidase and fluorescence techniques and by immunoblotting analysis. Decreased staining was observed particularly near the areas of lymphocytic infiltration. The availability of stronger and more specific antibody reagents would facilitate the correct evaluation not only of the apoptotic/anti-apoptotic gene products but also of the signaling cascade leading to thyrocyte death. The advancement of cDNA arrays technology contributed to study the clusters of genes involved in thyrocyte apoptotic-related pathways. Finally, the recent advance of microscope methodologies will facilitate the visualization of protein-protein interactions both on the cell surface and at the intracellular level. As one can see, a combination of many of the methods above can lead the conclusion on the detection of apoptosis in thyroid cells in Hashimoto’s thyroiditis.

Bibliography

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