Thyrocytes isolated from autoimmune-diseased thyroids secrete soluble tumor necrosis factor-R1 that is related to their elevated protein kinase C activity.

Soluble tumor necrosis factor (TNF)-alpha receptors have the potential to modulate TNF-alpha activity during autoimmune thyroiditis. In this study we examined cell-surface TNF-alpha receptors and soluble TNF-alpha receptor production by thyrocytes from normal and MRL-lpr(-/-) (diseased) mice, which spontaneously develop autoimmune thyroiditis. We found that murine thyrocytes possess the 55-kd receptor (TNF-R1). Examination of soluble TNF-R1 production revealed that diseased thyrocytes produced sevenfold more soluble TNF-R1 than normal thyrocytes. Furthermore, basal protein kinase C (pKC) activity in diseased thyrocytes was 67% higher than that found in normal murine thyrocytes. The elevated basal pKC activity in diseased thyrocytes was related to their enhanced production of soluble TNF-R1 because inhibition of pKC activity with calphostin C caused soluble TNF-R1 production to decrease significantly. Additionally, soluble TNF-R1 production by murine thyrocytes was not a result of cell-surface receptor shedding but through secretion of a truncated version of TNF-R1. This was evident when cell-surface TNF-R1 levels were unchanged after treatment of diseased thyrocytes with calphostin C. Also, the 28-kd form of TNF-R1, which corresponds to the soluble receptor, was present in the intracellular membranes of the diseased thyrocytes.

Elevation of protein kinase C in thyrocytes isolated from a Lewis rat model of autoimmune thyroiditis prevents assembly of immunodetectable connexin43 gap junctions and reduces intercellular communication.

In the Lewis rat model of experimental autoimmune thyroiditis (EAT), decreased immunodetectable connexin assembly into gap junctions and diminished intercellular communication are associated with the loss of thyroid function (hypothyroidism) that occurs prior to significant tissue destruction. The current study explores the hypothesis that the loss of connexin 43 (Cx43)-mediated intercellular communication in these cells is caused by upregulation of protein kinase C (pKC) activity. Thyrocytes isolated from EAT rats exhibited a 78% increase in basal pKC activity; whereas, basal protein kinase A (pKA) activity was unchanged. Increased pKC activity was a result of increased isozyme protein levels. Thyroid cells expressed pKC isozymes gamma and lambda and had elevated levels of alpha (40%), beta (30%), delta (31%), and epsilon (25%) as quantified by western blot analyses. Furthermore, modulation of pKC activity inversely altered Cx43 assembly and function in monolayer thyrocytes. For example, octoacetyl glycerol (OAG) treatment of normal thyrocyte monolayers to increase pKC activity resulted in deficient Cx43 gap junction assembly and reduced intercellular communication indistinguishable from the deficits in EAT thyrocytes. Conversely, calphostin C inhibition of pKC activity in EAT thyrocyte monolayers restored these parameters to normal. Thus, pharmacological modulations of pKC activity in cultured thyrocytes support a causal relation between the changes in pKC activity and Cx43-mediated intercellular communication. Abnormalities in autoimmune diseased thyroid tissue (eg, increased pKC) appear to contribute to reduced intercellular coordination of thyroid follicles and thereby can affect subsequent thyroid function. The persistence of target cell abnormalities in the absence of infiltrating lymphocytes and their products supports an alternative mechanism by which thyroid function can be affected that does not depend on the loss of thyroid glandular epithelium.

Reduced cell-cell communication in experimentally induced autoimmune thyroid disease.

We have recently described a spontaneous murine model of autoimmune thyroid disease. The disorder was in part characterized by reduced thyroid epithelial cell-cell communication that was associated with abnormalities in three major connexins. To compare whether this finding was a common secondary occurrence in autoimmune thyroid disease, or unique to the spontaneous development in the MRL mice, we induced thyroiditis in Lewis rats. Immunization with thyroid extract and thyroglobulin resulted in extensive lymphocytic infiltration and increased expression of major histocompatibility gene complex (MHC) class II surface antigen in the diseased thyroid. Both experimental and control rat thyroid tissues produced gap junction proteins connexin 43, connexin 32, and connexin 26. The connexins in nondiseased tissue was located in the plasma membrane at points of cell-cell contact and labeled as discrete arrays of punctate fluorescence. The quantity of all three connexins were reduced in the diseased thyroid tissue. More importantly, the connexin proteins were not distributed as gap junctions at contacting cell interfaces. Both nondiseased and diseased thyroid tissue expressed messenger RNA (mRNA) for the three connexins, but the diseased tissue had reduced levels of mRNA for connexin 43 (45%), and to a lesser extent, connexin 26 (25%) and connexin 32 (20%). The reduced connexin mRNA, protein, and lack of assembled gap junctions measured in the diseased tissue were obtained under conditions where the infiltrating cells and their potent cytokine products were continuously present. To determine if this difference persisted when these inflammatory components were absent, primary cultures of thyroid cells from control and experimental rats were established and connexin localization experiments repeated. The diseased thyroid cells, like the diseased tissue, lacked plasma membrane associated connexin protein. The lack of gap junction assembly in the thyrocytes cultured from the diseased tissue was accompanied by a loss of functional coupling. Collectively, the data document that autoimmune diseased thyroid tissue from both the spontaneous mouse and induced rat models have reduced plasma membrane assembled gap junctions and deficient intercellular communication as determined by the inability to transfer lucifer yellow dye to contiguous cells. Nondiseased cultured thyrocyte monolayers and follicles transferred dye to second and third order neighboring cells in 80 and 95% of trials, respectively. In contrast, only 5-10% of the diseased thyrocytes transferred microinjected dye, and in these cases the transit was limited to primary contacting cells. Culturing removed inequities introduced by the infiltrating cells and their products. However, the established cultures of diseased thyroid cells retained their communication deficiency. This suggests that the loss of communication may be a common abnormality in autoimmune disease, and furthermore, this uncoupling could contribute to the loss of coordinated hormonal regulation (hypothyroidism) in the diseased thyroid gland in the absence of thyroid cell destruction.

Reduced cell-cell communication in a spontaneous murine model of autoimmune thyroid disease.

MRL-lpr/lpr mice manifest a systemic lupus-like autoimmune disease. As part of this syndrome, the mice spontaneously develop autoimmune thyroiditis, which is morphologically and biochemically similar to human autoimmune thyroiditis. In this study we investigated whether thyroid tissue obtained from sites of chronic inflammation had altered gap junctional communication. Fresh tissue sections revealed that thyroid from the nondiseased mice (MRL-(+)/+) had connexins (Cx) localized to the plasma membrane at points of thyroid cell-cell contact. In contrast, the Cx in diseased mouse (MRL-lpr/lpr) thyroid tissue were not localized to the plasma membrane, and the fluorescent intensity was reduced for Cx43 and Cx26. Northern analysis confirmed that murine thyroid tissue expressed messenger RNA for these Cx. However, the diseased tissue expressed lower levels of Cx32 and Cx26 messenger RNA. The infiltrating cells and their biologically active products present in the diseased thyroid tissue may mediate the reduced Cx expression and aberrant gap junctional assembly. We established primary thyrocyte cultures to determine whether these differences persisted when the inflammatory factors were removed. The nondiseased thyroid cells were communication competent, with fluorescent dye transfer proceeding from the injected cell to primary contacts (95%) and to second and third order neighboring cells in 75% of the trials. Thyroid cells from the diseased mice were communication incompetent, in that 80% of microinjections failed to result in dye transfer to cells in direct contact. Immunocytochemistry indicated that the functional coupling in the normal mouse thyroid cells was associated with Cx43 located in the plasma membrane as assembled gap junctional plaques. The communication-deficient diseased thyroid cells had internalized Cx43 predominantly localized to perinuclear regions of the cells. Collectively, these data document altered Cx-protein distribution in the autoimmune diseased thyroid. The diseased thyroid tissue was devoid of plasma membrane identifiable gap junctions and deficient in intercellular communication. Culturing removed the inflammatory mediators; however, the disease cells retained their communication incompetence. These results suggest that if this deficiency was initiated by components of the inflammation process, then protracted changes must have occurred so that the continued presence of these factors was no longer required to sustain this difference.

Characterization of autoimmune thyroiditis in MRL-lpr/lpr mice.

MRL-lpr/lpr mice are genetically predisposed to develop a systemic lupus erythematosus-like syndrome that is clinically very similar to the human disease. The results presented here demonstrate, for the first time to our knowledge, that MRL-lpr/lpr mice also develop thyroiditis as part of their systemic autoimmune disorder. The thyroid gland was infiltrated by immunocomponent cells with defined lymphoid follicular centers and extensive interstitial lymphocytes dispersed throughout the thyroid epithelium. All the diseased mice were hypothyroid with reduced, relative levels of thyroid hormone (free T4) and elevated levels of thyroid-stimulating hormone (TSH). They also had high concentrations of circulating IgG class autoantibodies directed against thyroglobulin, thyroperoxidase and double-stranded DNA. The MRL-+/+ age-matched allelic counterpart mice had relatively few lymphocytes in their thyroid tissue, and normal levels of thyroxine and TSH. The non-diseased mice also had undetectable levels of thyroid reactive autoantibodies tested for by enzyme-linked immunosorbent assays. Collectively these findings document that the MRL-lpr/lpr mice spontaneously develop autoimmune thyroiditis and can be used as a model for the study of thyroid-specific autoimmunity.