Abstract
Chronic lymphocytic thyroiditis characterized by multifocal follicular lymphoid cell infiltrates with germinal centers, thyroid acinar atrophy and pituitary cell hyperplasia/hypertrophy of the adenohypophysis was detected in a vehicle control, 4-year-old female Cynomolgus macaque in a routine toxicology study. Lymphoid cells of germinal centers were positive for the B-cell marker CD20 by immunohistochemistry (IHC), while remaining lymphocytes were positive for the T-cell marker CD3. Hypertrophied/hyperplastic pituitary cells were positive for thyroid stimulating hormone (TSH) by IHC, consistent with an adaptive response due to removal of hormonal negative feedback from the diseased thyroid gland. Features of this case are similar to chronic lymphocytic thyroiditis in humans, an autoimmune disorder also known as Hashimoto’s disease. Chronic lymphocytic thyroiditis with compensatory pituitary changes may occur spontaneously in young, clinically normal cynomolgus macaques and its presence in drug treated animals should be interpreted with caution.
Keywords
Chronic lymphocytic thyroiditis is the most common inflammatory condition of the thyroid gland in humans and the most common cause of goiter and hypothyroidism in iodine-sufficient areas of the world. Thyroid deficiency results from gradual loss and replacement of glandular tissue by an intense mononuclear cell infiltrate composed of lymphocytes and fewer plasma cells usually arranged in distinct follicular structures. The disease is caused by an autoimmune-mediated process involving the activation of helper (CD4) T-lymphocytes against thyroid antigens with tissue recruitment of cytotoxic (CD8) T-lymphocytes and B-cells capable of producing thyroid auto-antibodies (Dayan and Daniels, 1996; Slatosky et al., 2000). There are two recognized clinical forms of chronic autoimmune thyroiditis: a goitrous form also known as Hashimoto’s disease and a less common atrophic form called atrophic thyroiditis. Spontaneous inflammatory disease of the thyroid gland has been reported in laboratory animal species including rats and dogs, however to the best of the author’s knowledge, naturally occurring disease in non-human primates has not been reported in the literature. Here we report a spontaneous case of lymphocytic thyroiditis with compensatory pituitary alterations in a Cynomolgus monkey with features similar to chronic autoimmune thyroiditis in humans.
The animal was a captive bred 4-year-old female, Cynomolgus macaque (Macaca fascicularis) assigned to the vehicle control group of a 1-year oral toxicity study and was negative for simian retrovirus types 1, 2, and 5 by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The monkey was pair housed in stainless steel cages and fed Purina Certified Hi Fiber Primate Diet (PMI Nutritional International Inc) with free access to water. Clinical examinations, body weights, electrocardiograms, clinical hematology/biochemistry and urinalysis were periodically performed throughout the conduct of the study.
At study termination, the animal was sacrificed by exanguination under intravenous barbiturate anesthesia and necropsied. Representative tissue samples were collected, fixed in 10% buffered formalin and routinely processed to H&E slides for microscopic examination. To further characterize microscopic lesions, immunohistochemical stains for CD3 (T-cell) and CD20 (B-cell) were applied to sections of thyroid gland while stains for thyroid stimulating hormone (TSH) were performed in sections of pituitary gland.
All the parameters assessed during the in-life portion of the study remained within the historical limits of normal. Body weight and absolute thyroid weights at terminal sacrifice was 6.1 kg and 0.47 g, respectively. Absolute thyroid weight and ratios were comparable to other animals on study and were within the historical reference range. There were no gross pathology observations. Microscopic alterations were evident only in the thyroid and pituitary glands. The thyroid gland was extensively infiltrated by multifocal lymphoid cell aggregates arranged into distinct, often confluent follicular structures (Figure 1A). Many lymphoid follicles had well-defined germinal centers and mantle zones formed by large immature lymphoid cells (Figure 1B). A uniform population of small, mature lymphocytes was present around and between the lymphoid follicles. There was a generalized decrease in the size of the remaining thyroid glandular acini (atrophy), which contained small quantities of colloid and were lined by tall columnar epithelial cells with abundant basophilic cytoplasm.
The larger immature lymphoid cells in germinal centers and mantle zones were strongly positive for the B-cell marker CD20 (Figure 1C), while the surrounding small mature lymphocytes were strongly positive for the T-cell marker CD3 (Figure 1D). Additional microscopic changes were detected only in the pituitary gland and consisted of epithelial cell hypertrophy and hyperplasia of the adenohypophysis. Hypertrophied cells had an abundant pale basophilic, sometimes vacuolated cytoplasm and were strongly positive for TSH by immunohistochemistry (Figures 1E, F).
Up to 95% of cases of chronic lymphocytic thyroiditis occur in women (Vanderpump et al., 1995), and the disease is characterized by glandular enlargement with extensive lymphoid infiltrates arranged in follicular structures and well-developed germinal centers (LiVolsi, 1994). The gender of this monkey and microscopic alterations in the thyroid are consistent with the human disease. The lack of a grossly visible enlargement of the thyroid gland may be explained by the concurrent atrophy of the remaining glandular tissue. In humans, enlargement of the gland may be minimal and in a smaller fraction of the patient population the gland actually undergoes atrophy (Nordmeyer et al., 1990).
Similar to the human disease, lymphoid cells that formed distinct germinal centers with mantle zones were identified by immunohistochemistry as B-lymphocytes while the more abundant interfollicular small lymphocytes were identified as T-lymphocytes (Saxena et al., 2004). B-lymphocytes from the thyroid of human patients with the disease are known to produce antibodies against thyroid specific antigens such as thyroid peroxidase (Czarnocka et al., 1985). The presence of these thyroid specific antibodies in serum is considered the hallmark of the disease and together with serum TSH measurements are used to confirm a diagnosis (Slatosky et al., 2000).
Although none of these parameters could be evaluated in this monkey, the presence of pituitary lesions suggests a significant alteration in the pituitary-thyroid axis consistent with removal of the hormonal negative feedback due to decreased production of triiodothyronine and/or thyroxine (T3/T4) by the diseased thyroid gland. Hypertrophy and hyperplasia of TSH producing cells is likely secondary to increased stimulus by the hypothalamic thyrotropin releasing hormone (TRH) and is considered an adaptive and compensatory response in an attempt to maintain adequate thyroid function. Hypertrophy and/or hyperplasia of TSH producing cells of the pituitary has been reported in rats after surgical thyroidectomy (Ozawa, 1991) or treatment with antithyroid chemicals (Norford et al., 1993) and in humans suffering from long-standing primary hypothyroidism (Scheithauer et al., 1985).
In human patients, the clinical course of the disease is variable and may range from normal thyroid function, primary hypothyroidism with low T4 and high TSH concentrations or subclinical hypothyroidism with normal T4 and elevated TSH levels. Patients with subclinical hypothyroidism can develop overt hypothyroidism, but the progression is usually very slow (Dayan and Daniels, 1996). The normal clinical condition of this monkey and the hypertrophy and hyperplasia of pituitary TSH producing cells is consistent with normal output of T4 with elevated TSH, as expected in subclinical hypothyroidism.
An increased frequency of antiretroviral antibodies not specific for human immunodeficiency virus has been reported in humans patients with chronic lymphocytic thyroiditis, however a direct link to an infectious etiology remains unproven (Drabick et al., 1991). In Cynomolgus macaques, subclinical simian retrovirus (SRV) infection may be characterized by the presence of mononuclear infiltrates in the form of lymphoid follicles in various organ systems including bone marrow, salivary gland, and kidney (Guzman et al., 1999). The negative SRV status of this monkey and the absence of lymphoid infiltrates in additional organ systems suggest that other mechanisms played a role in the development of these thyroid lesions.
Among laboratory animal species, lymphocytic thyroiditis was reported in 26% of dogs in a closed breeding Beagle colony that were allowed to live to their full life span (mean = 12 years) (Benjamin et al., 1996). Rats of the Buffalo strain have a genetic predisposition for the development of the disease, which correlates closely with the presence of serum thyroid antibodies (Noble et al., 1976). In non-human primates the disease has been experimentally induced in rhesus macaques by the administration of heterogenous thyroglobulins (Andrada et al., 1968). Spontaneous lesions of biologic significance in the thyroid gland of non-human primates are rare in drug safety studies.
Nodular thyroid hyperplasia in a clinically normal Cynomolgus monkey was recently reported in a short-term toxicity study (Kouchi and Okimoto, 2004). This case represents the first description of spontaneous chronic lymphocytic thyroiditis with adaptive TSH pituitary cell hyperplasia/hypertrophy in a non-human primate. With the increased preclinical testing of biologics and immune modulating agents in non-human primates, our case illustrates that the presence of inflammatory lesions in the thyroid gland of drug-treated animals should be interpreted with caution.