Evidence supporting the identity in Graves' disease of thyroid-stimulating antibody and thyroid growth-promoting immunoglobulin G as assayed in FRTL5 cells.

This paper addresses the question: in Graves' disease is there a thyroid-growth stimulating IgG (TGI) separate from thyroid-stimulating antibody (TSAb)? Using the functioning rat thyroid line (FRTL5) cells for TGI (incorporation of [3H]-thymidine into DNA) and TSAb (increase in cAMP concentration) assays, we tested IgG from 30 Graves' patients. Positive TGI assay occurred only if cAMP increased in the cells and responses correlated, i.e., r = 0.95, P less than 0.001. With one very potent TSAb-IgG we showed that Fab was active as TGI and TSAb, IgG with pI of 8.5-9.0 was the most potent fraction in both systems and an inhibitory IgG prevented the action of both TSAb-IgG and TSH in both the TSAb and TGI assays. In the last example, the action was on the cell membrane and not on the TSH or IgG. These data are entirely compatible with the view that in Graves' disease, at least as tested in FRTL5 cells, the same IgG is active in stimulating both growth and adenylate cyclase.

Studies on multiple thyroid cell membrane-directed antibodies in Graves' disease.

Immunoglobulin G was obtained from the serum of a woman who had given birth to three children with a delayed onset of hyperthyroidism; the clinical events were due to the coexistence of thyroid-stimulating antibody (TSAb) and an inhibitor of TSAb in the maternal serum. The current studies explore the possible existence of additional thyroid membrane-directed antibodies. Human thyroid slices, cells in monolayer culture, and functioning rat thyroid cells (FRTL5), with measurement of cyclic AMP concentration, were used for TSAb assays. Assays of the inhibition of binding of 125I-thyrotropin (TSH) to its receptor used human thyroid and FRTL5 cells, and human thyroid and guinea pig fat cell membranes as receptors. All activities were associated with IgG kappa. Fractions of IgG kappa obtained by adsorption to and the desorption from human thyroid and guinea pig fat cell preparations and F(ab')2 and Fab fragments of the parent IgG were tested. Results indicated that there were three activities in the IgG, namely, TSAb; an inhibitor of TSH-binding that was active in all species and preparations tested, and was effective as Fab and F(ab')2 on both particulate and solubilized thyroid membranes; and an enhancer of TSH-binding (e.g., approximately equal to 220% increase in binding) that was relatively specific for human thyroid membranes only in particulate form, was not adsorbed by fat, and was active as F(ab')2, but minimally as Fab. The concept is developed that dilution of the total IgG, experimentally in vitro or by metabolic clearance in vivo in neonates, determines the effect on either thyroid stimulation or TSH-binding. The incidence of such multiple antibodies and their interaction remains to be determined.

Immunoglobulin G inhibitor of thyroid-stimulating antibody is a cause of delay in the onset of neonatal Graves' disease.

Studies were carried out with the serum IgG from a mother and her two children who developed neonatal Graves' disease several weeks after birth. The maternal IgG: (a) stimulated the human thyroid in vitro, but maximal stimulation was found only with dilution of the IgG; (b) was very potent in the long-acting thyroid stimulator (LATS)-protector assay, but only when an inhibitor of the system was diluted out; (c) inhibited a standard preparation of LATS in the mouse bioassay; (d) was biphasic in the thyrotropin-binding inhibition (TBI) assay, i.e., enhanced binding at low concentrations of IgG and inhibited binding at high levels. Enhancement in the TBI assay was found only with particulate preparations of human thyroid membranes as receptor and not when that material was solubilized, nor with guinea pig fat cell membranes as receptor. Serial blood samples from the second child were obtained at birth and until 3 mo of age. In the thyroid slice (cyclic AMP) assay system there was a negative dose-response relationship in testing IgG until age 45 d when it became positive, coinciding with the clinical recognition that hyperthyroidism had developed. The data are compatible with a concept that this mother's IgG contained thyroid-stimulating antibody (TSAb) and another moiety that inhibited TSAb through an action on the thyroid cell membrane, thus delaying the onset of hyperthyroidism in the neonate until the inhibiting IgG was metabolically cleared to an ineffective concentration.

Suppression of peripheral blood natural killer cell activity by excess thyroid hormone.

Natural killer (NK) cells were assessed in patients with hyperthyroxinemia due to Graves' disease or treatment with thyroxine (T4). Cytolytic activity was measured with 51Cr-labeled K562 tumor cells and NK enumeration was by flow cytometry using NKH-1 monoclonal antibody to identify the relevant surface marker. Activity was uniformly decreased in association with hyperthyroxinemia, regardless of the underlying pathology; however, there was no reduction in the number of NKH-1+ cells. NK activity was enhanced by addition of interleukin 2 (IL-2) in both control and patients' cells although the value in the latter instance failed to reach the basal control level. Production of IL-2 by lymphocytes from hyperthyroxinemic subjects, in response to phytohemagglutinin, was also reduced. Since NK cells are thought to act as a defense against viral infections and some malignancies and may play a role in autoregulation of the immune system, this effect of T4 may have significant biological implications.

Evidence of hyperthyroidism in apparently euthyroid patients treated with levothyroxine.

A concentration of serum thyroxine (T4) above the accepted normal range has recently been recognized to result from commonly prescribed replacement dosages of levothyroxine sodium. To determine if the high levels of serum T4 have undesirable metabolic effects, despite the fact that the subjects are accepted as euthyroid, we studied 28 patients receiving long-term levothyroxine therapy; 19 patients were considered to be receiving replacement dosages and nine suppressive dosages of levothyroxine. To assess the effect of levothyroxine on target tissue, we measured the thyrotropin response to protirelin and systolic time intervals obtained by simultaneous electrocardiography and echocardiography. Thyrotropin response to protirelin was suppressed in patients with elevated serum T4 levels and normal serum triiodothyronine levels. These patients also had shortened systolic time intervals typical of hyperthyroidism. Our data indicate that commonly given replacement dosages of levothyroxine may induce undesirable metabolic consequences and that these patients perhaps ought to be seen as having "subclinical hyperthyroidism." The prescribed dosage of levothyroxine as therapy for hypothyroidism is still frequently excessive.

Influences of cholera toxin on thyroid stimulation by thyrotropin and thyroid-stimulating antibody.

Thyroid-stimulating antibody (TSAb) is an immunoglobulin G (IgG) occurring in the blood in hyperthyroid Graves' disease patients; it stimulates the thyroid in a manner analogous to the action of TSH, i.e. by activation of adenylate cyclase. Since cholera toxin is also known to stimulate thyroid adenylate cyclase, we studied possible interaction of the enterotoxin on effects of TSAb and TSH using slices of canine thyroid in vitro and an increase in the concentration of cAMP as endpoint. Normal human IgG, known to inhibit the binding of [125I]-iodo-TSH to thyroid membranes, decreased stimulation of thyroid slices by TSH; this inhibitory effect occurred also with preparations of TSAb (inevitably comprised mainly of normal IgG) that were themselves stimulatory. The cholera toxin effect was not prevented by normal IgG and, by factorial analysis of variance, was shown to potentiate the action of subsequently added TSAb or TSH. There was also positive interaction of the effect of TSAb with the combination of TSH and cholera toxin. The data indicate that responses of thyroid tissue to TSAb and TSH are readily influenced by effects of other membrane-active agents (in the present context, normal IgG and cholera toxin).

Variations in the culture medium for FRTL5 cells: effects on growth and iodide uptake.

We evaluated the composition of the medium in which FRTL5 cells were incubated for effects on basal and stimulated growth and differentiated function. Variables included the concentration of calf serum (CS) from 0% (0.2% BSA) to 5% and the addition of insulin or insulin-like growth factor-I (IGF-I); stimulation was by TSH, with some experiments using thyroid-stimulating antibody-immunoglobulin G. The basal rate of incorporation of [3H]thymidine and the content of DNA per well were enhanced progressively by the increasing concentration of CS, whether in a pretreatment period of 3 days or over a subsequent 2 days of incubation. Concomitantly, I- uptake was progressively inhibited. Stimulation of growth by TSH required serum and was greatest with 5% CS; TSH effect on I- uptake was progressively decreased by increments of CS. Insulin and IGF-I had effects comparable to each other, but much smaller concentrations of IGF-I were required; they both augmented growth and enhanced growth stimulation by TSH. These actions were progressively annulled by increasing concentrations of CS. I- uptake (basal and stimulated) was inhibited by insulin and IGF-I, but this inhibitory effect was abolished by CS, particularly at 5% concentrations. Overall, the influences of varying the concentrations of CS and the effects of insulin or IGF-I were consistent; if there was enhancement of growth stimulation, there was inhibition of the TSH effect on I- uptake. Underlying mechanisms for these observations have yet to be elucidated.

Influence of cytokines on growth and differentiated function of FRTL5 cells.

A functioning rat thyroid cell line (FRTL5) was used to study interactions of TSH with interferon-gamma (IFN gamma) and tumor necrosis factor-alpha (TNF alpha). We examined effects on growth and differentiated function. Growth was assessed by DNA, incorporation of [3H]thymidine ([3H]Tdr) into DNA, and cell number; uptake of 125I (I- uptake) and the concentration of cAMP were measured simultaneously with growth assessment. IFN gamma stimulated the 30-min I- uptake and enhanced the effect of TSH. TNF alpha had minimal effects on growth indices (slight increase in [3H]Tdr incorporation) and had no influence on I- uptake; it inhibited TSH stimulation of both growth and I- uptake. When combined, IFN gamma and TNF alpha synergized in inhibiting TSH-stimulated growth. By itself TNF alpha inhibited stimulation of I- uptake by TSH, but augmented the enhancement seen with IFN gamma. The influence of calf serum (CS) was to increase the rate of incorporation of [3H]Tdr, but a similar qualitative pattern for the actions of the cytokines remained. A reverse profile (stimulation by IFN gamma, inhibition by TNF alpha, and stimulation by the combination) was seen for I- uptake, with CS increasingly diminishing all values. TSH stimulation of growth was progressively effective with increments of CS in the medium, but consistently there was inhibition that was greater with IFN gamma than with TNF alpha and was almost total with the combined cytokines. Stimulation of I- uptake by TSH was enhanced by IFN gamma, reduced by TNF alpha, and, when serum was present, increased to a degree that was greater than additive by the combined cytokines. Growth stimulation by insulin or insulin-like growth factor-I was inhibited partially by the individual cytokines and completely by the combination. Both insulin and insulin-like growth factor-I inhibited TSH stimulation of I- uptake, but similar stimulation by the cytokines was not affected. Simultaneous with inhibition of TSH-stimulated growth, both IFN gamma and TNF alpha enhanced cAMP accumulation. The mechanism of these multiple effects of IFN gamma and TNF alpha, especially on the actions of TSH, may not currently be fully explained, but they almost certainly reflect differing modes of action. The relevance to thyroid function in man is conjectural. Especially in Graves' disease, where thyroid infiltration with cells that secrete these cytokines is common, it seems probable that both IFN gamma and TNF alpha will have significant influences on both growth and differentiated cell function.

Influence of cholera toxin on in vitro refractoriness to thyrotropin of thyroids from rats fed propylthiouracil.

Using an increase in the concentration of cAMP as an index of stimulation, we previously reported that in vitro thyroid tissue of rats fed a goitrogenic diet (0.1% propylthiouracil in Purina) were unresponsive to TSH. We now show that the addition of cholera toxin (0.1-1.0 microM) to fragments of normal thyroid enhanced the concentration of cAMP. Subsequent exposure of the tissue to TSH resulted in a further increase in the concentration of cAMP, and there was statistical evidence of interaction or potentiation between the effects of TSH (20 mU/ml) and cholera toxin (1.0 microM). With fragments of thyroid from animals fed propylthiouracil (i.e. unresponsive in vitro to TSH), an increase in the concentration of cAMP was effected by 1 or 9 microM cholera toxin, and prior exposure to 9 microM toxin caused the tissue to be responsive to TSH. Unresponsiveness of the goitrous tissue was associated with a reduced capacity of the membrane to bind [125I]iodo-TSH although the affinity of the binding sites was unaffected. Cholera toxin at 0.1 microM enhanced and at 1 microM diminished the binding of [125I]iodo-TSH to membranes from either normal or goitrous glands, and these effects also reflected influences on the capacity rather than on the affinity of the binding sites. It is postulated that the unresponsiveness to TSH of thyroid tissue from rats fed propylthiouracil represents a down-regulation of receptor capacity, and this is effected by the binding sites becoming relatively inaccessible, rather than nonexistent.

Solubilization, purification, and partial characterization of thyrotropin receptor from bovine and human thyroid glands.

In an attempt to purify the thyroid receptor for TSH and to study interaction with the thyroid-stimulating antibody (TSAb) of graves' disease, we used both bovine and human thyroid glands. With either tissue, the 10,000 X g pellet of homogenized material was solubilized with 0.5% Triton N-101; excess Triton was removed by Amberlite XAD-2, and purification was effected by TSH affinity chromatography, followed by gel filtration on Sepharose 6B. Greatest purification was achieved with bovine tissue; this receptor preparation was 183-fold concentrated over the starting material, but contained only 6% of the original TSH-binding activity, due in part to spontaneous loss over the 4 days required for processing. On polyacrylamide gel electrophoresis, there were at least three protein bands, one of which was probably a subunit of thyroglobulin. Purified immunoglobulin G with thyroid-stimulating antibody activity inhibited the binding of TSH at all stages of purification of the receptor.

Thyrotropin binding to rat thyroid membranes: reduced capacity associated with goitrogenesis.

To investigate a possible role of TSH in the regulation of its own receptor, a sensitive assay of [125I]TSH tropin binding to rat thyroid membranes was used. With 1 mM MgCl2 in the buffer, Scatchard analysis of displacement of TSH gave a curvilinear plot with a high affinity, low capacity (K1, 3.4 nM; Q1, 3.1 pmol/microgram) and a low affinity, high capacity binding site (K2, 0.54 microM; Q2, 1.2 X 0.1 nmol/microgram). Feeding rats propylthiouracil led to a decrease in [125I]TSH binding (expressed either per U protein or per wet wt of tissue) that was related to the duration of treatment. Evaluation by Scatchard analysis showed that this was due to a loss of binding sites, e.g. a 50-60% decrease after 1 month of propylthiouracil treatment; affinity was actually slightly increased in the goitrous tissue. This change in the number of TSH-binding sites was readily reversed in association with the suppression of TSH in vivo either by injections of T3 for 3 days or more by feeding a normal diet for 1 month. Thus, the data are compatible with TSH, that is in high concentration in the serum of rats fed propylthiouracil, exerting a downregulatory influence on its own receptors.

Comparison of FRTL-5 cell growth in vitro with that of xenotransplanted cells and the thyroid of the recipient mouse.

The present work was designed to compare in vitro cell growth kinetics with in vivo growth under conditions as similar as possible using labeling with [3H]thymidine. To this purpose, FRTL-5 cells were cultured as monolayers and as three-dimensional spheroids embedded in collagen gels and transplanted simultaneously into nude mice treated with perchlorate and a low iodine diet. The growth of the transplants was compared to that of the thyroids in host mice. In the intact thyroid, the fraction of [3H]thymidine-labeled follicular cells (FLC; 24-h labeling) increased sluggishly to a maximum of 10% after 3 weeks of goitrogen exposure, with a subsequent autoregulatory decrease to 3% at 7 weeks. A 4-fold higher FLC was found in six adenomas, indicating focal failure of growth-restraining mechanisms. In nonconfluent monolayer cultures the FLC was as high as 90%, even within large individual clusters where cells are in tight mutual contact. Solid, highly cellular grafts growing from transplanted monodispersed cells showed an average FLC of 20%, which is 5 times higher than the FLC in the identically stimulated mouse thyroid. In collagen-embedded cells, forming three-dimensional spheroids, the mean FLC decreased from 70% at 1 week in vitro (40% in vivo) to 20% at 3 weeks both in vitro and in vivo, suggesting effective auto-regulation of excessive growth in both conditions. However, these FLC were again much higher than the 3% FLC in simultaneously assessed host thyroids. The difference remained throughout the 45-day period studied. We conclude that FRTL-5 cells growing as monolayers and as three-dimensional spheroids in vitro or after xenotransplantation in vivo invariably show much higher proliferation rates under comparable environmental conditions than the normal follicular epithelium in the thyroids of host mice. The one exception is the confluent monolayer with near-zero growth, while densely packed three-dimensional transplants still grow intensively. Although growth-retarding cell to cell interactions are also clearly operative in growing FRTL-5 cells, they are less effective than those dampening the replication rate of the thyrocytes within the monolayer hull of normal follicles. A local failure of these mechanisms, allowing growth rates comparable to those of grafted FRTL-5 cells results in adenoma formation in normal thyroids. These observations call for caution in the transfer of in vitro growth studies with FRTL-5 cells to in vivo conditions prevailing in the normal thyroid.

Evidence that adenosine 3',5'-monophosphate mediates stimulation of thyroid growth in FRTL5 cells.

Thyroid function, including growth, is TSH dependent, and most metabolic functions of TSH are thought to be mediated by cAMP. Recently, it has been suggested by several groups that growth may be an exception and that it may not be related to cAMP action. In addition, evidence has accrued indicating that the thyroid-stimulating antibody (TSAb) of Graves' disease, the metabolic actions of which are also cAMP mediated, may not be the goitrogenic agent in that syndrome. To evaluate these concepts, we used functioning rat thyroid cells (FRTL5) in monolayer culture and, as indices of growth, the incorporation of [3H]thymidine ([3H]Tdr) into DNA, the concentration of DNA measured directly, and the percentage of cells in S phase, as assessed by flow cytometry, all studied over 72 h of incubation. TSH, forskolin, and cholera toxin enhanced growth by each criterion and increased the concentration of cAMP in parallel; the effect on cAMP occurred rapidly and was maximal well in advance of influences on growth. In all instances, measures of growth promotion were minimal at 24 h and maximal at 48 h, except for [3H]Tdr incorporation, which was greater at 72 h than at 48 h. 3-Isobutyl-1-methylxanthine (IBMX) and (Bu)2 cAMP were also tested. Both enhanced all indices of growth and were as effective as TSH. Maximal responses to TSH were obtained at 100-200 microU/ml, maximal responses to both IBMX and (Bu)2cAMP occurred at 5 X 10(-4) M, and all three stimulators increased the DNA concentration and [3H]Tdr uptake and induced S phase in at least 20% of all cells in culture. The peak effect on DNA and S phase was consistently at 48 h. Epidermal growth factor (EGF) was shown to increase [3H]Tdr incorporation in a nondose-dependent fashion (10(-10) to 5 X 10(-9) M gave approximately 250% of control) over 1, 2, 3, 5, and 7 days, with no increase in DNA and a slight decrement in the concentration of cAMP. A laboratory standard TSAb-immunoglobulin G was shown to parallel TSH in both increasing cAMP (over 2 h of incubation) and growth stimulation (over 72 h). The data are entirely consistent with the view that TSH-stimulated thyroid growth is mediated by cAMP and that the established action of TSAb on adenylate cyclase is sufficient to explain goiter as well as hyperthyroidism in Graves' disease.

Clinical review 3: The clinical use of thyrotropin receptor antibody measurements.

There are relatively few circumstances wherein the assay of TRAb, as TSAb or TBIAb, is merited in the routine clinical management of patients with autoimmune thyroid disease. These include: 1. the prediction of neonatal hyperthyroidism; 2. the confirmation, by assay of maternal serum, and perhaps newborn blood, that neonatal hypothyroidism, identified on routine screening, may be transient and due to transplacental passage of TBIAb; 3. the prediction of relapse of hyperthyroidism at the end of a course of antithyroid drugs; this information should lead to more careful observation to identify relapse as early as possible; 4. the confirmation that adult agoitrous hypothyroidism reflects the effect of an antibody (TBIAb) blocking the action of TSH although, as discussed, there is little, if any, practical gain from this information. Additional applications of the routine use of TRAb assays will require the development of a procedure that combines the sensitivity, specificity, availability, and ease found variably in current TSAb and TBI procedures, and at much less cost than what is provided today.

Adsorption of thyroid-stimulating antibody (TSAb) of Graves' disease by homologous and heterologous thyroid tissue.

Immunoglobulin G purified from the serum of patients with Graves' disease, stimulated the thyroid of man, calf, and guinea pig (cAMP accumulation as the end-point) in vitro, and the thyroid of the mouse in vivo (LATS bioassay). The stimulatory effect on the thyroids of all four species was removed by adsorption of the immunoglobulin G to human thyroid membranes and was diminished to a proportionately similar degree by adsorption with bovine thyroid membranes. All activity, as assessed by stimulation of the human thyroid in vitro. was recovered from both human and bovine membranes by elution with 2 M NaSCN solution. The data support the concept that the thyroid-stimulating antibody of Graves' disease is homologous to a human thyroid antigen and, in some instances, cross-reacts with a similar antigen in the thyroid of a distant species and so stimulates the heterologous gland.

Zoological specificity of human thyroid-stimulating antibody.

The zoological specificity of human thyroid-stimulating antibody (TSAb) that occurs in the blood in Graves' disease was examined by assessing its effect on the thyroid of the dog, guinea pig, calf, and mouse as well as that of man. With all but murine glands, thyroid stimulation was assayed by measuring the increase in the concentration of cAMP in the thyroid slices or fragments after 2 h of incubation in buffer containing TSAb. Effects on the thyroid of the mouse were monitored by the in vivo bioassay for LATS. Sera from 33 patients with Graves' disease were obtained and concentrates of TSAb were prepared by precipitation of IgG with 1.64 M (NH4)2SO4. These all stimulated the human thyroid, 13 were LATS-positive, and they variably affected the tissues of other species; of 27 tested, 14 stimulated the thyroid of the dog, 8 out of 23 stimulated the thyroid of the guinea pig, and 12 out of 16 stimulated the gland of the calf. The more potent the TSAb as assayed with human tissue, the more likely was it to stimulate other species of thyroid; however, frequent exceptions occurred. In a separate analysis of 35 LATS-positive preparations of TSAb, correlation between the responses in the LATS and human thyroid slice assays was statistically significant (P less than 0.001). The data are compatible with the view that stimulation by TSAb of nonhuman thyroids, including the murine as in the LATS bioassay, reflects cross-reaction of this immunoglobulin with an antigen that has sufficient similarity to the human molecule to be recognized by the human antibody.

A reconsideration of a thyroid-stimulating immunoglobulin as the cause of hyperthyroidism in Graves' disease.

Several assays for thyroid-stimulating immunoglobulins in the blood in patients with Graves' disease have been described recently; depending upon the method, different names have been used and distinct entities thus implied. Using an increase in cyclic AMP in the human thyroid slice after 2 h of incubation as an index of thyroid stimulation, we identified thyroid-stimulating activity in all of an unselected series of sera from 11 patients with hyperthyroidism of Graves' disease, but long-acting thyroid stimulator, by mouse bioassay, in only three. The theory is proposed that the thyroid-stimulating immunoglobulin is probably present in all such patients; it may be seen as a polyclonal antibody to a single human antigen that has a variable cross-reaction with a corresponding thyroid antigen in the mouse and in other species.

Pregnancy-associated changes in the thyroid-stimulating antibody of Graves' disease and the relationship to neonatal hyperthyroidism.

Assays for the thyroid-stimulating antibody (TSAb) of Graves' disease were performed with serum obtained from 17 women with 20 pregnancies who either had or had had Graves' disease, or who had delivered a child with neonatal hyperthyroidism. Ten of the children, of eight women, were diagnosed as being hyperthyroid; all eight mothers had high concentrations of TSAb, as measured by adenylate cyclase stimulation. In the infants with neonatal hyperthyroidism, a minimum 500% increase in cAMP was found on assay of maternal immunoglobulin G, and no such high values were associated with a euthyroid infant. Blood samples for TSAb assay were available from 11 mothers on both the day of delivery and at least 1 other time in the pre- or postpartum period. In 7 mothers, the lowest value (negative in 4) was obtained coincident with delivery, and in the remaining 4, there was no pregnancy-associated change. Thus, a pattern, related to pregnancy, of a decline in TSAb concentration or a subsequent postpartum increase was observed in the majority of subjects. Apparently, neonatal hyperthyroidism due to transplacental passage of TSAb occurred only when this decline did not reduce the concentration to a low value.

Prediction and therapy of intrauterine and late-onset neonatal hyperthyroidism.

These studies in a mother and child describe the effects of multiple antibodies directed against the TSH receptor that influenced thyroid function in the fetus and infant. Blood was taken periodically for 6 months from a child (C3) whose mother (M) was known to have in her serum immunoglobulin G (IgG) that contained thyroid-stimulating antibody (TSAb), an inhibitor of TSAb and TSH binding and action, and an enhancer of TSH binding to its receptor, the last activity presumed to enhance both TSH and TSAb action. We correctly predicted that C3 and an older sibling, C2, would have delayed onset of hyperthyroidism (approximately 45 days of age) due to interaction of these antibodies. In addition, in both C2 and C3, fetal hyperthyroidism in the second trimester was postulated, and therefore, M was given propylthiouracil from then until term (C2) or 8 months (C3), with associated return of the fetal heart rate to normal in the one fetus (C3) in whom this was monitored. IgG was purified from C3's serum samples and tested for TSAb activity using human thyroid cells in monolayer culture and its ability to alter binding of [125I]TSH to human thyroid cell membranes. In the TSAb assays, samples obtained from birth to 4 months of age produced a negative dose response, and those obtained from 4-6 months showed a positive dose response. The effect on the binding of [125I]TSH was enhancement with all IgG obtained for 6 months, except that for the first 52 days a high concentration was inhibitory. The combined clinical and assay data are compatible with the following interpretations. M's IgG contained TSAb and both an inhibitor and an enhancer of that activity, the total effects varying at different times. In the second trimester, the net effect of transplacentally transferred IgG was to induce hyperthyroidism. At birth and perhaps during the third trimester, the inhibitor of TSAb prevented hyperthyroidism; from about 45 days of life, the enhancing effect facilitated TSAb action to produce hyperthyroidism until maternal IgG was completely cleared from the infant's circulation at about 7 months of age.

Transient neonatal hypothyroidism: characterization of maternal antibodies to the thyrotropin receptor.

Transient neonatal thyroid disease is known to occur as a result of transplacental passage of maternal immunoglobulin G (IgG) that contains antibodies to the TSH receptor (TRAb). Thyroid-stimulating antibody (TSAb) produces hyperthyroidism, and antibody that blocks TSH binding (TBIAb) results in hypothyroidism. We have analyzed in detail the IgG from four women who gave birth to children with transient neonatal hypothyroidism and have shown stimulating and inhibiting antibodies to coexist in three. Human and/or rat thyroid (FRTL5) cells were used to show stimulatory effects in vitro. Inhibition was assessed as prevention of stimulation of these cells (by TSH or TSAb) or by the blocking of binding of [125I] TSH to its receptor. The IgG from two mothers was tested to identify whether the inhibitory and stimulating bioactivities resided in molecules characterized by either or both kappa- or lambda-light chains. Evidence for restricted heterogeneity (implying oligoclonality) was obtained, in that with one, purely inhibitory IgG all activity was with IgG kappa. With the other, stimulating and inhibitory activities were predominantly in IgG kappa and IgG lambda, respectively. In addition, the latter IgG contained a second stimulator that was not suppressed by either its own or other inhibitory IgG. Despite the presence of stimulatory antibodies in these IgG, the clinical effect was neonatal hypothyroidism, reflecting the greater potency of the inhibitory IgG in all instances. Based on the histories of these four women and their offspring it is apparent that TRAb, and in particular TBIAb, can develop at any point in the course of autoimmune thyroid disease, i.e. either at the onset or long after the autoimmune process has been established.