Action of peripheral or intrathyroidal lymphocytes on autologous thyrocytes cultured in follicles in collagen gel

Abstract

We have studied the action of peripheral blood lymphocytes (PBLs) and intrathyroidal lymphocytes (ITLs) on the biochemical and hormonal metabolism of autologous thyrocytes cultured in follicles in a collagen gel. The production of tumour necrosis factor alpha (TNF-alpha) in culture was also measured. Thyroid tissues and lymphocytes were obtained from ten patients with Graves' disease and from five control subjects. Lymphocyte-induced cytotoxicity was evaluated in autologous thyrocytes cultured in a collagen gel by several tests; neutral red uptake, lactate dehydrogenase activity and glutathione level. Hormonal metabolism was assessed by evaluating tri-iodothyronine (T3) and total cAMP production under TSH stimulation. TNF-alpha levels were measured in supernatants after 5 days of coculture. PBLs altered biochemical metabolism, T3 synthesis and cAMP production in autologous thyroid follicles. These inhibitions were greater than those obtained with ITLs. No difference was seen between cells obtained from patients with Graves' disease and those from normal subjects. TNF-alpha levels secreted by PBLs were higher than those secreted by ITLs. The concentrations of this cytokine decreased in coculture. Significant correlations were observed between the decrease in biochemical and hormonal parameters and TNF-alpha levels. Exogenous TNF-alpha and high doses of interferon gamma inhibited follicle metabolism, especially hormone secretion. In conclusion, thyrocytes cultured in follicles provide a more sensitive model than monolayer cultures for analysis of lymphocyte-induced interactions. Lymphocytes gradually inhibit the biochemical and hormonal metabolism of autologous thyroid follicles depending on the isolation method. These alterations may be particularly attributed to TNF-alpha secreted by lymphocytes. The cytokine-induced inhibition of thyroid hormonal function apparently involves the adenylate cyclase system.

Increased expression of tumor necrosis factor-alpha and decreased expression of thyroglobulin and thyroid peroxidase mRNA levels in the thyroids of iodide-treated BB/Wor rats

“…Studies suggest that increases in endogenous TNFa production may be involved in the regulation of thyroid function in ATD. That is, TNFa and TNFa mRNA are found in human thyroid tissues from patients with Graves' disease and Hashimoto's thyroiditis (1)(2)(3)(4). In FRTL-5 cells, we (5,6) and others (7)(8)(9)(10) have observed that TNFa inhibits type I 5 0 -deiodinase activity and thyroglobulin (Tg), thyroid peroxidase (TPO) and type I 5 0 -deiodinase gene expression.…”

Thyrocyte integration, and thyroid folliculogenesis and tissue regeneration: Perspective for thyroid tissue engineering

“…The interactions between thyrocytes and these cell types are thus expected to be involved in the stability and regulation of thyrocyte growth and differentiation. But the intercommunications within the thyroid are poorly defined 47–52 . The capturing ability of the 3‐D collagen gel culture system makes it easy to carry out stable cocultures of various cell types.…”

Thyroid Culture from Monolayer to Closed Follicles

“…Our results showed that if culture was begun with closed follicles then extracellular matrix support elements were not required [56,57,79,80] nor was TSH for maintaining follicular cavity, as has been described in most pseudofollicle or follicle cultures reconstructed from monolayers, or similar structures called "follicles" [57,50,60,61,62,79,96], thereby demonstrating that if closed follicles are used from the start of culture they conserve their morphology, having the correct polarity as that of their thyrocytes in culture and have a binding complex (tight junction, belt desmosome and spot desmosome) in lateral membranes' apical part as well as in vivo [9]. Such closed follicles in culture responded to TSH, like other in vitro models [42] or like gland follicles in normal in vivo to TSH stimulus [3,25], epithelium thickness, RER and GC becoming increased, and follicular cavities becoming notably reduced [11,83,90].…”

“…It is so important that specific genes have been described which govern follicle formation and maintain follicle architecture, the gene transcribing the thyroid-specific enhancerbinding protein (T/ebp or Nkx2.1) regulating the transcription of genes implicated in hormone synthesis: NIS, TPO, TSH receptor and Tg and re-organised transfecteds thyrocites in follicles [78]. Human goitre follicle structure has been covered with collagen to preserve it longer and cavities in human [79,80] and mouse thyrocyte [78] thin cell bilayers have persisted 2 days more. Collagen's importance in preserving epithelial cells' apical-basal polarity has been described for obtaining MDKC (Madin-Darby canine kidney epithelial cell line) cell 3D cultures, but hepatic growth factor (HGF) was added to cultures [81].…”

“…Monolayer growth should be avoided because this increases iodide accumulation values and interferes with analysis of iodide organification function; fresh enzyme dissociation must be carried out to recover the follicles [82,86]. When follicular cell fragments are cultured in collagen they do not reform follicles as has been described for human [79,80] and mouse thyrocytes [78]; some MDKC epithelial cells are organised in follicle-like structures requiring HGF [81]. The foregoing meant that the use of glass, plastic and collagen for follicle culture was rejected and agarose type I used instead.…”