TSAb Modulates Microglia M1 Polarization via Activation of the TSHR-Mediated NF-κB Signaling Pathway.

INTRODUCTION: Thyrotropin receptor-stimulating antibody (TSAb) is a pathogenic antibody in the serum of patients with Graves' disease. The binding of TSAb to thyroid-stimulating hormone receptor (TSHR) in non-thyroid tissue may be associated with the occurrence and development of Graves' disease-related complications. However, only few studies have been conducted on the effects of TSAb on the brain, and the pathogenesis of acute hyperthyroidism myopathy (ATM) is unclear. Therefore, this study aimed to explore the effect of TSAb on the polarization of BV-2 cells in the brain and its possible mechanism and provide a basic experimental basis for ATM. METHODS: BV-2 cells were treated with different concentrations of TSAb. The relative survival rate of BV-2 cells was determined using the CCK-8 assay; the migration ability of BV-2 cells was detected using the Transwell migration assay; and the expression levels of M1/M2 polarization markers (CD86, inducible nitric oxide synthase [iNOS], CD206, and arginase 1 [Arg-1]), TSHR, tumor necrosis factor-alpha (TNF-α), and nuclear factor-kappa B (NF-κB) protein in BV-2 cells were measured using WB. RESULTS: Compared with the negative control group, the proliferative activity of BV-2 cells was significantly increased in the 20, 50, and 100 ng/mL TSAb groups, and the migration ability of BV-2 cells was significantly enhanced in the 50 and 100 ng/mL TSAb groups. The expression levels of M1 polarization markers (CD86 and iNOS), TSHR, TNF-α, and NF-κB protein in BV-2 cells treated with 50 and 100 ng/mL TSAb for 24 h were significantly upregulated, whereas those of M2 polarization markers (CD206 and Arg-1) significantly decreased. CONCLUSIONS: TSAb can induce abnormal activation of microglia, polarize to the M1 phenotype, and promote the inflammatory cascade reaction, in which TSHR plays a key role in NF-κB activation and proinflammatory cytokine release.

In vitro stimulation by long-acting thyroid stimulator of thyroid glucose oxidation and 32P incorporation into phospholipids.

Long-acting thyroid stimulator (LATS) increased glucose oxidation and (32)P incorporation into phospholipids in in vitro experiments with dog thyroid slices. The time course of the response was different from that obtained with thyroid-stimulating hormone (TSH), but was very similar to the delayed effect observed in vivo. During a 45 min incubation, TSH, but not LATS increased glucose oxidation, whereas in longer experiments up to 6 hr, both substances augmented (14)CO(2) production. Amounts of pooled human gamma globulin equivalent to LATS were inactive. Although TSH stimulated (32)P incorporation into phospholipids during a 2 hr incubation, LATS was ineffective. In longer incubations, from 4(1/2) to 8 hr, LATS did increase (32)P incorporation. The stimulatory effect of LATS was not abolished by anti-TSH antibody capable of neutralizing human TSH. Effects of LATS were also obtained with beef and pig thyroid slices. In addition to stimulation of glucose oxidation in dog thyroid slices, LATS occasionally also stimulated glucose oxidation in dog spleen and liver slices. Despite a 54-fold increase in LATS concentration, a satisfactory dose-response curve could not be demonstrated when (14)CO(2) production was measured.

Effects of long-acting thyroid stimulator on thyrotropin stimulation of adenyl cyclase activity in thyroid plasma membranes.

Both thyroid-stimulating hormone (TSH) and long-acting thyroid stimulator (LATS) stimulated adenyl cyclase activity in plasma membranes obtained from bovine thyroid glands. The stimulation induced by LATS was much less than that obtained with maximal amounts of TSH. LATS inhibited TSH stimulation of adenyl cyclase activity while an equivalent amount of normal human gamma-globulin did not influence basal or TSH-stimulated activity. The inhibition by LATS appeared to be noncompetitive and was greatest when the plasma membranes were initially exposed to LATS for 30 min at 0 degrees C before being incubated with TSH for 10 min at 37 degrees C. Inhibition could still be demonstrated when the plasma membranes were incubated for 30 min at 0 degrees C with TSH before the addition of LATS. Prolonging the period of incubation of plasma membranes with LATS from 30 to 60 min did not augment the stimulation of adenyl cyclase or increase the inhibition of the effect of TSH. Papain digests of LATS also increased adenyl cyclase activity of thyroid plasma membrane and inhibited the stimulation induced by TSH. The inhibitory effect of LATS was not completely specific for TSH and thyroid plasma membranes since glucagon stimulation of adenyl cyclase in hepatic plasma membranes was also inhibited, but to a lesser extent. In contrast to the results obtained with thyroid plasma membranes, LATS did not influence basal adenyl cyclase activity in hepatic plasma membranes. Furthermore equivalent amounts of normal human gamma-globulin also decreased glucagon stimulation of adenyl cyclase activity in plasma membranes obtained from liver. The present data suggest that LATS stimulation of adenyl cyclase in thyroid plasma membranes might be due to a change in the membrane configuration rather than binding to a specific receptor site. Such modification of the membrane structure could interfere with the binding of TSH to specific receptors or to the subsequent stimulation of adenyl cyclase. However, the results do not exclude the possibility that some component in the preparation other than LATS might be responsible for the inhibition of the stimulation by TSH.

The subcellular localization of the long-acting thyroid stimulator inhibitor in bovine thyroid gland.

To characterize further the nature and site of the receptor for the IgG stimulator in Graves' disease, we have developed a preparative scheme to provide a bovine thyroid subfraction rich in plasma membranes. Pellets (800 X g) obtained from bovine thyroid homogenates were layered on a 30-64% continuous sucrose gradient (SG). The centrifuged gradient was divided into four portions (SG1, 2, 3, and 4); and these along with the 800 X g pellet were characterized in terms of their capacity to neutralize (bind) the biological activity of LATS, their specific binding of [125I]TSH, and their subcellular marker content. Subfraction SG1 contained the highest adenyl cyclase specific activity, the highest [125I]TSH binding specific activity, and vied with the 800 X g pellet and SG3 for the highest specific activity of LATS neutralization. Electron microscopy of SG1 showed a predominance of plasma membrane structures contaminated with a modest amount of cellular debris. Adenyl cyclase activity in SG1 was enhanced by TSH, LATS, and sodium fluoride. Although a dose-response curve could be established for TSH, a similar relationship could not be established for LATS. We conclude that activation of plasma membrane-bound adenyl cyclase is associated with neutralization of the biological activity of LATS. Further, the difference between [125I]TSH binding and LATS neutralization activity observed in the various thyroid membrane subfractions suggests that either LATS and TSH interact at different sites or have different mechanisms of binding at a common site.

Characterization of monoclonal antibodies raised against solubilized thyrotropin receptors in a cytochemical bioassay for thyroid stimulators.

Selected clones of monoclonal antibodies from mice immunized with solubilized preparations of bovine TSH receptors have been characterized in a cytochemical bioassay (CBA) for thyroid stimulators. This assay is based upon quantification of changes in naphthylamidase activity of sections of guinea pig thyroids with use of a chromogenic substrate. Monoclonal 22A6 is a thyroid-stimulating antibody directed at a site within the TSH receptor. Thus, although it is a weak inhibitor of 125I-TSH binding to thyroid membranes, 22A6 is inhibited from binding to membranes by TSH, exhibits a more than additive agonist effect on adenylate cyclase activity when tested at low TSH concentrations in thyroid cells, and is a competitive antagonist of TSH enhancement of adenylate cyclase activity at high TSH concentrations. In the CBA, 22A6 is a stimulator whose maximal activity is obtained with 77 pg/ml (3-min exposure). Dose-response curves of a long acting thyroid stimulator (LATS)-B standard and 22A6 have slopes which are not significantly different; as anticipated, the response to LATS-B is inhibited by antihuman immunoglobulin G (IgG) and that due to 22A6 by antimouse IgG. In contrast to 22A6, monoclonal 11E8 is a relatively potent inhibitor of 125I-TSH binding as well as TSH stimulation of adenylate cyclase activity, while failing to act as a stimulator itself. 11E8 is itself inactive as a stimulator in the CBA over a wide dose range; it does, however, inhibit TSH stimulation in the CBA. This inhibition is abolished by antimouse IgG. The transient peak of response observed in time courses to TSH occurs later in the presence of 11E8. Unlike its effect on TSH 11E8 shows relatively low potency (greater than 10,000-fold lower) when inhibiting stimulation by the thyroid stimulating antibodies, 22A6 or LATS-B. Since this difference cannot be explained by quantitative differences in the ability of 22A6 or 11E8 to bind to thyroid membranes, the CBA data suggest that the stimulating antibodies, 22A6 and LATS-B, may interact with different determinants on TSH receptors then either TSH or the blocking antibody, 11E8. This also implies that in Graves' disease blocking antibodies may be incompletely expressed in the presence of stimulating antibodies, although they may be potent inhibitors of TSH binding, as measured in receptor assays.

The biphasic stimulatory and inhibitory effects of concanavalin A on thyroid activation induced by thyrotropin.

Concanavalin A (Con A) was tested for its ability to affect thyroid activation induced by TSH in mouse thyroid lobes. Pretreatment of thyroid lobes with Con A at concentrations from 1.55--400 microgram/ml was found to have biphasic stimulatory and inhibitory effects of the TSH-induced accumulation of cAMP and formation of colloid droplets. Low concentrations of Con A potentiated TSH activation of thyroidal formation of cAMP and endocytosis. In contrast, higher concentrations of Con A markedly inhibited these TSH effects. The inhibitory effects observed after preincubation with Con A were abolished by the addition of alpha-methyl-D-glucoside to the incubation medium. A high concentration of Con A also inhibited cAMP formation induced either by prostaglandin E2 or the long-acting thyroid stimulator. However, the basal and TSH-stimulated glucose oxidation in mouse thyroid lobes was not depressed by a high concentration of Con A. Uptake of 125 I-labeled Con A by thyroid tissues increased with time up to 1 h and was directly proportional to tissue weight. These findings suggest that the specific interaction between Con A and its receptors may lead to conformational changes in the structure of the membranes of the thyroid follicular cells which facilitate TSH-induced thyroid hormone secretion via the adenylate cyclase-cAMP system.

Perchlorate ion enhances mouse thyroid responsiveness to thyrotropin, human chorionic gonadotropin and long acting thyroid stimulator.

Perchlorate treatment of mice increased by 1.5-2-fold the thyroid secretory response to TSH, hCG and LATS, in the McKenzie bioassay. Perchlorate alone did not increase basal plasma radioactivity. Perchlorate augmentation of the secretory response index was roughly proportional to the level of stimulation; it was similar for all three stimulators despite their different time courses of action which were unaltered by perchlorate; it was the same whether perchlorate administration preceded, coincided with or shortly followed injection of the stimulator, a finding in keeping with the slow clearance of this ion. The perchlorate effect was dose-related, although within a narrow range (6.25-12.5 microng/mouse). Near-maximal per chlorate effect was obtained with a dose (12.5 microng) which, when tested in different experimental conditions (MMI-blocked thyroid), discharged 80% of intrathyroidal radioiodide. Perchlorate exerted its augmenting effect by enhancing thyroid secretion: it increased plasma radioiodothyronines and radioiodide concentrations without decreaseing the blood disappearance rates of iodide and iodothyronines. The potentiating effect of perchlorate probably takes place at a step prior to cyclic AMP action since it did not affect dbcAMP-stimulated secretion. The perchlorate effect may be indirect, through mobilization of minute amounts of intrathyroidal iodide.

The inhibitory effect of acute administration of excess iodide on the formation of adenosine 3', 5'-monophosphate induced by thyrotropin in mouse thyroid lobes.

In a previous paper, we demonstrated that an inhibitory action of excess iodide on thyrotropin-induced thyroid hormone secretion occurs at a site subsequent to the generation of cyclic AMP. In the present study, however, we have found that thyroidal cyclic AMP formation induced by thyrotropin in vitro was markedly inhibited by the acute administration of excess iodide to mice fed a low iodine diet. In contrast, excess iodide failed to produce inhibition in animals fed a regular diet. In vitro stimulation by long-acting thyroid stimulator (LATS), prostaglandin E2, and 4-methylhistamine of cyclic AMP formation in mouse thyroid lobes was also significantly inhibited by the acute in vivo administration of excess iodide. The inhibition was completely relieved by the administration of methimazole prior to excess iodide. Furthermore, it has been shown that thyroid adenylate cyclase activity induced by thyrotropin was markedly depressed by excess iodide under similar experimental conditions. Therefore, it is suggested that one of the inhibitory actions of excess iodide is on the adenylate cyclase-cyclic AMP system and further, that iodide can elicit its inhibitory action after its conversion to some form of organic iodine.

Evidence for the existence of a histamine H2-receptor in the mouse thyroid.

The existence of a histamine H2-receptor in the thyroid was investigated. Histamine in vitro stimulated the formation of cyclic AMP and colloid droplet formation in mouse thyroid lobes. Stimulation by histamine of cyclic AMP formation in mouse thyroid lobes was significantly inhibited by metiamide, a histamine H2-receptor antagonist. 4-Methylhistamine, a histamine H2-receptor agonist, markedly stimulated cyclic AMP formation, whereas 2-methylhistamine, a histamine H1-receptor agonist, was ineffective. The stimulation by 4-methylhistamine of cyclic AMP formation was markedly inhibited by metiamide, but not by chlorpheniramine, a histamine H1-receptor antagonist. In contrast, metiamide did not affect cyclic AMP formation induced either by TSH or by the long-acting thyroid stimulator. Therefore, it is suggested that there exists a histamine H2-receptor in the membranes of the thyroid follicular cells which facilitate thyroid hormone secretion via the adenylate cyclase-cyclic AMP system.

Radioimmunoassay and the hormones of thyroid function.

Radioimmunoassay (RIA) has provided the tools for wide-reaching investigations that have changed and continue to change many important concepts of thyroid physiology and pathosphysiology. The RIA vor human thyrotropin (TSH) was developed in 1965; development of the RIA for triiodothyronine (T3), thyroxine (T4), thyroxine-binding globulin (TBG), and, recently, thyrothropin-releasing hormone (TRH) and thyroglobulin (Tg) followed. The capacity to measure nanogram and picogram concentrations with relative ease and speed has permitted the demonstration of dynamic relationships of the intrathyroidal and circulating thyroid hormones to each other and to the pituitary and hypothalamic regulating hormones. Evidence for the presence of cross-influences between TRH and other hypothalamic regulating hormones on the secretion of pituitary hormones has accumulated. The impact of the new information on clinical practice is now becoming evident. There is new appreciation of the value of assaying serum T3 and TSH concentrations in the clinical management of patients with disturbed function of the thyroid, pituitary, or hypothalamus. The necessary components for RIA performance can be purchansed separately or in kit form from commercial sources. With appropriate quality-control procedures, precise, sensitive, and reliable data can be generated. Awareness of the specific technical problems relating to the RIA of these hormones is absolutely necessary to assure reliable results. The availability of kits or their components permits the performance of these studies in the community hospital and in reliable commercial-service laboratories.

Evidence that both long-acting thyroid stimulator and long-acting thyroid stimulator-protector stimulate the human thyroid gland.

Thyroid-stimulating immunoglobulins were prepared from two potent sera, one contained long-acting thyroid stimulator (LATS) and the other contained both LATS and LATS-protector (LATS-P). The potencies of the immunoglobulin G (IgG) preparations were estimated in the McKenzie assay. The accumulation of cyclic AMP in mouse thyroid lobes was stimulated only by LATS--IgG; LATS-P--IgG was inactive. In contrast, both LATS-IgG and LATS-P--IgG were equally effective in slices of human thyroid.

Effects of human thyroid-stimulating hormone and immunoglobulins on adenylate cyclase activity and the accumulation of cyclic AMP in human thyroid membranes and slices.

The activation of adenylate cyclase and the accumulation of cyclic AMP resulting from the action of human thyroid-stimulating hormone (TSH), long-acting thyroid stimulator (LATS) or LATS-protector (LATS-P) have been investigated in preparations of human thyroid membranes and slices. Human TSH significantly increased adenylate cyclase activity in membranes from non-toxic goitres whereas LATS and LATS-P had no consistent effect. However, pre-incubation of goitrous membranes with LATS--immunoglobulin G inhibited the effect of TSH on adenylate cyclase. When thyroid membranes were prepared from the glands of patients with Graves's disease neither TSH nor thyroid-stimulating immunoglobulins (TSIg) stimulated adenylate cyclase significantly. Whether from non-toxic goitres or thyrotoxic tissue, the concentration of TSH needed to induce half of the maximum response was lower in thyroid slices than in membranes. Both LATS and LATS-P significantly stimulated the accumulation of cyclic AMP in slices of goitrous tissue but thyrotoxic tissue slices did not respond. In goitrous slices, submaximum concentrations of TSH and TSIg caused additive responses in the accumulation of cyclic AMP but TSIg did not increase the maximum response to TSH.

Characterization of thyroid-stimulating immunoglobulin-induced cyclic AMP accumulation in the rat thyroid cell strain FRTL-5: potentiation by forskolin and calibration against reference preparations of thyrotrophin.

A clonal strain of rat thyroid cells (FRTL-5) has been used to investigate the biological activity of a Research Standard preparation of long-acting thyroid stimulator (LATS-B). Using the accumulation of intracellular cyclic AMP as a response parameter, significant stimulation was attained at a LATS-B dose of 0.75 mu./ml. The inter-bioassay coefficient of variation in response to a fixed dose of LATS-B (1.25 mu./ml) was 20.5%, as determined using eight sequential subcultures. Cells cultured directly from frozen stocks responded to both bovine TSH and LATS-B in a manner indistinguishable from cells subjected to regular subculturing. Cyclic AMP responses to incremental doses of LATS-B were potentiated after the inclusion of a low dose of forskolin (0.1 mumol/l). However, forskolin addition had no effect on the time-course of LATS-B-stimulated cyclic AMP accumulation, half-maximal responses being attained after 60 min in either the presence or absence of the diterpene. In the presence of 0.1 mumol forskolin/l, intracellular cyclic AMP responses to LATS-B were demonstrably parallel with those to human TSH (Second International Reference Preparation, 80/558), whilst parallel incremental cyclic AMP responses were also observed in respect of TSH and serial dilutions of a potent thyroid-stimulating immunoglobulin (TSIg) preparation, indicating that for this particular Graves' disease patient, TSIg bioactivity may be expressed in terms of a convenient and reproducible standard, as TSH microunit equivalents.

Studies on the action of thyroid stimulation blocking antibody (TSBAb) on thyroid cell membrane.

The effect of thyroid stimulation blocking antibody (TSBAb) on stimulated cyclic AMP (cAMP) production induced by adenylate cyclase stimulators in porcine thyroid membrane (PTM) and porcine thyroid cells (PTC) has been studied. Ten TSBAbs with high TSH binding inhibitory immunoglobulin (TBII) activities significantly blocked TSH-stimulated cAMP production in PTC. The blocking effect of TSBAb on the cAMP increase induced by forskolin or GTP-gamma S stimulation in PTC was found in a few cases. However, there was no blocking action of TSBAb on the cAMP increase stimulated by forskolin, GTP gamma S, or NaF in isolated PTM. When TSBAb-globulin was absorbed with PTM or guinea pig epididymal fat membrane (GPFM), the TBII activity in TSBAb-globulin was significantly absorbed by these membranes. A decrease of TSBAb activity (blocking activity for TSH-stimulated cAMP production in PTC) by PTM absorption, but no decrease by GPFM absorption, was found in six cases. This suggests that the potent TSBAb-neutralizing component may be associated with a non-TSH receptor site in the thyroid membrane. The other four cases showed a decrease of TSBAb activity by absorption with both PTM and GPFM. This suggests that the TSBAb-neutralizing activity may be associated with the TSH receptor site of both PTM and GPFM. The results of the present study suggest that TSBAb may block TSH action either via the TSH receptor itself or via a non-TSH receptor component of the thyroid membrane and not at a postreceptor level.

Demonstration of thyroactive smaller components released from TSAb-IgG by protease digestion.

Thyroid stimulating (TS) activity (cAMP production in thyroid cells) and TSH binding inhibition (TBI) activity (determined by TSH receptor assay) in fragments released from TSAb-IgG by protease digestion were examined. The unbound fraction (UF) and the bound fraction (BF) were separated using a protein A-Sepharose column after papain hydrolysis (more hydrolysis at pH 5.0 than pH 7.5) of TSAb-IgG. When both fractions were gel filtrated on a Sephadex G-100 column, the TS and TBI activity were found in both Fab fraction (Mr 50 kDa) and the retarded fraction (between Mr 50 and 20 kDa) in the UF, and also in the first fraction (undigested IgG, Mr 160 kDa), the second fraction (Fc with tracer amounts of Fab, Mr 50 kDa), and the retarded fraction (between Mr 50 and 20 kDa) of the BF. The biological activity in the second fraction was suggested as being derived from Fab, because the activity bound to the anti-F(ab')2 column but did not bind to the anti-Fc column. Anti-Tg and anti-TPO activities were found in Fab, but were not found in the retarded fraction that consisted of Mr 20-30 kDa. In pepsin hydrolysis the UF from the protein A column consisted of both F(ab')2 (Mr 100 kDa) and pF'c (CH3) (Mr 25 kDa), and the BF consisted of only the undigested IgG. The biological activities were found in both the F(ab')2 fraction and the retarded fraction (between Mr 100 and 25 kDa). Anti-Tg and anti-TPO activities were found in F(ab')2, but no activity was observed in the Mr 25-kDa fraction. The present study showed that the biological activity of TSAb is distributed in not only the Fab or F(ab')2 fragment, but also in thyroactive smaller components (TSC) (Mr 20-30 kDa) without antigen-binding activity such as anti-Tg and anti-TPO. We suggest that TSC may be released from the Fab fragment region of TSAb-IgG by protease hydrolysis.