Effect of thyroxine-binding globulin (TBG) on thyroxine (T4) uptake by human peripheral mononuclear cells: evidence of TBG-dependent uptake of T4

The effect of sialylated TBG and desialylated TBG on thyroxine (T4) uptake by human peripheral mononuclear cells was investigated in vitro. [125I]-T4 uptake was observed when the cells were incubated with free [125I]-T4. The uptake was inhibited in a concentration dependent manner when TBG was added. During the incubation, [125I]-T4 binding to TBG was observed. [125I]-T4 incorporation into cells was also observed when the cells were incubated with [125I]-T4-sialylated TBG or with [125I]-T4-desialylated TBG complex. The uptake was related to the temperature and length of time of the incubation. The amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-sialylated TBG was greater than that into the cells incubated with [125I]-T4-desialylated TBG during the early 0-20 min. incubation, whereas the amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-desialylated TBG became greater than that into the cells incubated with [125I]-T4-sialylated TBG after 20 min. of incubation. Pretreatment of the cells with methylamine blocked [125I]-T4 uptake in both cases, i.e. incubated with [125I]-T4-sialylated TBG and incubated with [125I]-T4-desialylated TBG. The results suggest that TBG plays a role not only as a carrier protein for T4 in circulation but also as a protein which can transport T4 from the extracellular into the intracellular space, so that the mechanism of T4 transport mediated by desialylated TBG is different from that mediated by sialylated TBG, and that the T4 transport system in both cases, mediated by sialylated TBG and by desialylated TBG, may be related to the internalization of T4-TBG-TBG receptor complex or of T4-T4 receptor complex if TBG receptors are present in the outer surface of the cell membrane.     

TBG-dependency of age related variations of thyroxine and triiodothyronine.

Thyroxine (T4), triiodothyronine (T3) and thyroxine-binding globulin (TBG) were determined in healthy individuals ranging in age from newborn to 95 years. T4: 10.25 +/- 1.62 microng/100 ml, T3: 1.62 +/- 0.35 ng/ml and TBG: 1.34 +/- 0.15 mg/100 ml, were found elevated until puberty compared to a middle age group with T4: 7.27 +/- 2.26 microng/100 ml, T3: 1.15 +/- 0.24 ng/ml and TBG: 0.98 +/- 14 mg/100 ml. T4 and T3 followed almost TBG concentration. In old age is dissociation between T4: 5.79 +/- 1.56 microng/100 ml, T3: 0.79 +/- 0.21 ng/ml and TBG: 1.28 +/- 0.15 mg/100 ml was found. Except for old age the ratio T4/TBG and T3/TBG minimized the age dependent variation of T4 and T3 and reduced the coefficient of variance from 26% to 17.7% for T4 and from 26.5 to 25% for T3. Age reduction of T4/TBG is 15% and of T3/TBG 13% respectively more pronounced than for T4 and T3 alone. These data indicate: 1) age related variations of T4 and T3 due to age dependency of TBG, 2) deviation of T4 and T3 values in old age from that expected by their TBG levels and 3) the importance of the routine use of hormone/TBG ratio.     

Surface plasmon resonance assay of inhibition by pharmaceuticals for thyroxine hormone binging to transport proteins

We developed a surface plasmon resonance (SPR) assay to estimate the competitive inhibition by pharmaceuticals for thyroxine (T4) binding to thyroid hormone transport proteins, transthyretin (TTR) and thyroxine binding globulin (TBG). In this SPR assay, the competitive inhibition of pharmaceuticals for introducing T4 into immobilized TTR or TBG on the sensor chip can be estimated using a running buffer containing pharmaceuticals. The SPR assay showed reproducible immobilization of TTR and TBG, and the kinetic binding parameters of T4 to TTR or TBG were estimated. The equilibrium dissociation constants of TTR or TBG measured by SPR did not clearly differ from data reported for other binding assays. To estimate the competitive inhibition of tetraiodothyroacetic acid, diclofenac, genistein, ibuprofen, carbamazepine, and furosemide, reported to be competitive or noncompetitive pharmaceuticals for T4 binding to TTR or TBG, their 50% inhibition concentrations (IC₅₀) (or 80% inhibition concentration, IC₈₀) were calculated from the change of T4 responses in sensorgrams obtained with various concentrations of the pharmaceuticals. Our SPR method should be a useful tool for predicting the potential of thyroid toxicity of pharmaceuticals by evaluating the competitive inhibition of T4 binding to thyroid hormone binding proteins, TTR and TBG.     

Polymorphism of human thyroxine binding serum globulin (TBG); existence of a new form of serum protein without detectable triiodothyronine binding power

Two kinds of TBG polymorphism are described in human, one found in deglycosylated TBG from individual blood donors, the other is a genetically determined polymorphism. TBG from plasma samples from a patient with toxic goiter, not autoimmune, (p)TBG, from the patient's mother (m)TBG and from individual donors (n)TBG, were labeled with [125I]T4 or [125I]T3 and submitted to isoelectric focusing (IEF), followed by autoradiography. Three faint [125I]T4 radiolabeled bands were detectable in (p)TBG while four strong [125I]T4 radiolabeled bands were detectable in (m)TBG and (n)TBG), respectively. IEF of the [125I]T3 incubated serum samples resulted in no detectable isoelectric radiolabeled band for (p)TBG while a normal pattern was found in (m)TBG and in (n)TBG, respectively. These data suggest a new intraindividual not linked to sexual chromosome X polymorphism characterized by a loss in hormone binding.     

Effect of specific placental proteins--trophoblastic beta 1-glycoprotein chorionic alpha 1-microglobulin--on the proliferation of lymphocytes and malignant fibroblasts in vitro

A study was made of the action of trophoblastic beta 1-glycoprotein (TBG) and chorionic alpha 1-microglobulin (CAG1) on proliferation of malignant fibroblasts (transplanted L line) and on phytohemagglutinin-stimulated lymphocytes. TBG depressed proliferation of the stimulated lymphocytes and transformed fibroblasts (according to 3H-thymidine incorporation). A dose-response dependence was ascertained. CAG1 did not affect cell division. The inhibitory effect of TBG was seen to be reversed or decreased provided the lymphocyte culture was supplemented with CAG1. The decreased inhibitory effect of TBG in the presence of CAG1 was also noted in the L cell culture. It is likely that in vivo protection of intensely proliferating fetal tissues or tumor from the inhibitors is effected just in this way since placental proteins are synthesized both by embryonic and tumorous cells.     

Placental proteins as regulators of immunological reactions in pregnancy

The effect of two specific placental proteins, trophoblastic beta 1-glycoprotein (TBG) and chorionic alpha 1-microglobulin (CAG), on the immunological reactions was studied in vitro. TBG in physiological doses suppressed the proliferation of lymphocytes induced by plant mitogens and allogenic cells in the unidirectional mixed cultures, strengthened the effect of concanavalin A upon the induction of cells-suppressors in the culture and, in low concentrations, decreased the percentage of E- and EAC-rosette-forming cells. In none of the tests used CAG was effective. But when studying the effect of TBG and CAG mixture on PHA-induced proliferation of lymphocytes the inhibiting effect of TBG was weakened and, in some cases, completely relieved.     

Comparison of the action of the sera of pregnant women and of trophoblastic beta 1-glycoprotein in a number of immunological tests

The effects of blood sera of pregnant women in the I, II and III trimesters (SPWI, SPWII, SPWIII) were compared with those of purified preparations of trophoblastic beta 1-glycoproteid (TBG) on phytohemagglutinin (PHA) induced proliferative response of lymphocytes of the intact donors and on the production of a factor inhibiting the migration of leucocytes (FIM-L). SPWI and purified TBG preparations in doses corresponding to its level in SPWI were shown to produce similar effects. With the increase of TBG concentration, however, the inhibiting effect of its preparations on the proliferation of lymphocytes increased much more intensively than that of SPWII and SPWIII containing the same dose of this protein. The stimulating effect of TBG on the production of FIM-L increased with the dose as well while SPWII and SPWIII did not exert any effect on the migration of leucocytes. A suggestion is put forward that SPWII and SPWIII contain factors modifying the effect of TBG.     

Characterization of human thyroxine-binding globulin. Evidence for a single polypeptide chain.

Thyroxine-binding globulin (TBG) was purified from fresh human plasma by affinity, anion exchange, and gel filtration chromatography. The protein gave a single band in overloaded analytical disc gel electrophoresis. The molecular weight was 54,000 and E1%/1 cm at 280 nm, corrected for thyroxine (T4) absorbance, was 6.17. Six preparations of TBG contained from 0.09 to 0.64 mol of T4/mol; the TBG used in this study contained 0.19 mol of T4 and was able to bind an additional 0.85 mol. The carbohydrate composition was determined and accounted for 23% of the molecular weight. Four lines of chemical and physical evidence failed to demonstrate subunits. These included quantitative COOH-terminal amino acid analysis, peptide mapping and amino acid composition, treatment with sodium dodecyl sulfate, and denaturation of the reduced, alkylated protein with guanidine. From these data, we conclude that TBG is a single polypeptide chain.     

Short and long term effects of radioiodine and antithyroid drugs on T4 binding proteins, free T4 and T3, during Graves' disease therapy.

Ninety five patients with Graves' disease were studied before and at three months intervals after antithyroid drugs (ATD) (31 cases) or radioiodine (64 cases) therapy until recovery. Before treatment, the T4 maxima binding capacity of TBPA was significantly decreased 253.5 +/- 11.4 mug/100 ml)(mean + se) (control values: 287 +/- 10.4 mug/100 ml) (alpha = 0.04), especially in 53.7% of patients (m = 177 +/- 8 mug/100 ml). The mean of TBG (m = 20.7 +/- 0.9 mug/100 ml) was not different from euthyroid subjects (m = 19.7 +/- 1.7 mug/100 ml) except in 51.2% of patients who had a low TBG (m = 14.3 +/- 1.1 mug/100 ml). An inverse linear correlation was found between TBG-DFT4 (alpha = 0.05) and DF T 3 (alpha = 0.002), TBPA-log DF T4 (alpha = 0.05) but not between TBG and TBPA. The physiological relationship between DFT3, DFT4, TT3, TBG and TBPA was studied in vitro; after adding increased quantities of T4 to a pool of sera collected from eu, hypo or hyperthyroid patients, DFT4, DFT3, FT3 index increased in linear positive relationship with TT4 concentrations, the kinetic of this phenomena was inversely correlated with T4 maximal binding capacity of TBG or TBPA for T4. Addition of T3 to the same sera did not show any effect on the previous parameters. DFT3 depended on the level of T4 in serum more than T3 concentration and was in inverse relationship with the maximal binding capacity of TBG. This data might explain the paradoxal normal or slightly increased values of DFT3 found in T3 thyrotoxicosis. In patients treated with ATD or radioiodine, TBPA but not TBG increased significantly on year after. However, in subjects with an initial very low TGB or TBPA, this phenomenon occurred on the third month after radioiodine or ATD. During the same period, DF T4 and DF T3 were inversely correlated to TBG and TBPA. In conclusion, important changes in T4 binding proteins and free fractions of thyroid hormones were observed in Graves' disease but were corrected by antithyroid therapy. All these data were in good agreement with the normalisation of thyroid function.     

Affinity chromatography of thyroxine-binding proteins from human serum

The affinity matrix prepared by the attachment of L-thyroxine (T4) to epichlorohydrine-activated Sepharose 4B biospecifically absorbs the T4-binding globulin (TBG), 25K and 80/27K proteins, immunoglobulin G (IgG) and albumin (HSA) from human normal and retroplacental sera. The absorbed protein patterns were shown to depend on the immobilized T4 concentration, pH, temperature and incubation time. The potent eluents desorbing 85-100% of the protein are 1 mM NaOH, 3 M NH4SCN, 10(-5) M T4 or 3 mM 8-anilinonaphthalene-1-sulfonic acid (ANS) for TBG; NaOH, NH4SCN, 3 mM MgCl2 or 12mM sodium cholate for 25K protein and HSA; NaOH, NH4SCN or MgCl2 for the 80/27K and 25K proteins and IgG. Moreover, T4 desorbs small amounts (6-8%) of the 80/27K and 25K proteins, while sodium cholate elutes about 6% of TBG. The eluted from T4-Sepharose 4B and further purified TBG, 25K and 80/27K proteins display different [125I]T4-binding activities within the pH range from 2 to 9 and differ by their resistance to thermal inactivation at 50-80 degrees C. Double radial immunodiffusion analysis with the use of antisera to TBG, 25K, 80/27K, HSA and IgG demonstrated that the proteins share no common antigenic determinants. It was concluded that the novel 25K and 80/27K proteins represent endogenous components of the human blood thyroid hormone-binding protein system rather than fragments or aggregates of the known T4-binding proteins.     

Fractionation of mouse T and B lymphocytes by preparative cell electrophoresis. Efficiency of the method

Mouse lymphocytes have been fractionated in preparative cell electrophoresis into two functionally viable populations, a high mobility cell (HMC) and a low mobility cell (LMC) population. The distribution of HMC in CBA spleen, blood, and lymph node corresponds to known proportions of θ-positive cells in these organs. The HMC carry the θ-isoantigen, respond to phytohemagglutinin in vitro, and induce a graft-versus-host reaction in newborn F₁ hybrid mice. Nearly all spleen LMC have complement receptors on their surface. About 70% of spleen LMC are sensitive to anti-MBLA serum and form “caps” when incubated with FITC-conjugated anti-Ig. Only LMC respond to E. coli lipopolysaccharide. Thus, T cells localize in the HMC population and B cells in the LMC population. There is no detectable contamination of T lymphocytes among the LMC, nor of B lymphocytes among the HMC.     

Thyroxine-binding globulin, triiodothyronine, thyroxine and thyrotropin in newborn infants and children.

Thyroxine-binding globulin (TBG), triiodothyronine (T3), thyroxine (T4) and thyrotropin (TSH) have been determined by radioimmunoassay in plasma of newborn infants and throughout childhood until puberty. Mean maternal TBG concentration was 1.65 +/- 0.09 mg/100 ml (SEM) and significantly higher (p less than 0.01) than cord blood levels of TBG (1.16 +/- 0.08 mg/100 ml (SEM). Throughout infancy and childhood TBG remained significantly elevated (p less than 0.01) compared to a middle age control group of healthy blood donors. T3, T4 and TSH concentrations behaved postnatally as known from previous studies. The T3 and T4 increase observed immediately after birth was not a secondary phenomenon due to changes in TBG concentration since this globulin did not change significantly during this period.     

Thyroxine turnover and transport in viral hepatitis

Thyroxine turnover and transport studies were done on 20 patients in the acute phase of viral hepatitis. No significant changes in T4 degradation rate (k), the T4 distribution volume (V), and the metabolic clearance rate (MCR) were found. A significant increase of T4 serum concentration and TBG capacity, and decrease of TBPA capacity were observed. A negative correlation between TBG and TBPA capacities was found. Significant interrelationships between TBG capacity and T4 serum levels as well as T4 turnover parameters were shown. The values of T4/TBG ratio were within normal range.     

Cloning of a complementary deoxyribonucleic acid coding for human thyroxine-binding globulin (TBG): existence of two TBG messenger ribonucleic acid species possessing different 3'-untranslated regions

An adult human liver cDNA library constructed in expression vector, bacteriophage lambda gt11, was screened with polyclonal antibody directed against human T4-binding globulin (TBG). TBG cDNA cloned in the present study was 944 nucleotides in length. It contained approximately 70% of the coding region and complete 3'-untranslated region. When the sequence was compared with that of TBG cDNA recently cloned by I. L. Flint, T. J. Bailey, T. A., Gustafson, B. E. Markham, and E. Morkin, the 3'-untranslated region of our cDNA was 231 nucleotides shorter than their cDNA. These results indicated that two TBG mRNAs with different length of 3'-untranslated regions may exist in human liver. Indeed, Northern blot analysis revealed that two TBG mRNAs differing in the length approximately 200 base pairs were present in normal human liver as well as in human hepatoma cell line (HepG2). It was demonstrated that this size difference was due to the length of 3'-untranslated region by hybridization with a probe specific to the longer 3'-end. Together with the sequence data, it was suggested that these two TBG mRNA species may be produced by alternative processing and polyadenylation at two different sites.     

Thyroid functions in patients with various chronic liver diseases

In order to clarify an alteration in thyroid functions in patients with chronic liver diseases, serum total and free thyroxine (T4, FT4), total and free triiodothyronine (T3, FT3), total reverse T3 (rT3), thyrotropin (TSH), thyroxine-binding globulin (TBG) concentrations, and T3 uptake (T3U) were measured by radioimmunoassays in 53 patients with chronic hepatitis (CH), 24 patients with compensated liver cirrhosis (LC), 17 patients with hepatocellular carcinoma associated with LC (HCC), and 40 normal subjects. Serum T4, T3, and rT3 in CH, and serum rT3 in HCC were significantly increased, while serum T4 in LC and serum T3 in HCC were significantly decreased. Serum TBG was increased and T3U was decreased in these patients. Serum TBG in CH and LC correlated positively with transaminase, and inversely with prothrombin time. FT4 and T4/TBG ratios in CH and LC and FT3 and T3/TBG ratios in LC and HCC were significantly decreased. Although T4/TBG ratios in HCC and T3/TBG ratios in CH were significantly decreased, FT4 in HCC and FT3 in CH were not decreased. The ratio of rT3/T3 in CH and LC correlated with various liver function tests. FT3 in LC and HCC correlated inversely with BSP (45') and positively with KICG. No differences in serum TSH values were found between chronic liver diseases and normal subjects. From these results, it was concluded that the thyroid functions in patients with chronic liver diseases were affected by the decrease in serum thyroxine, elevated serum TBG, the degree of which is in proportion to that of the liver cell damage, and impaired peripheral conversion of T4 to T3, the degree of which is in proportion to that of the hepatic dysfunction.     

Concentration of thyroxine-binding globulin: value of direct assay.

The concentration of thyroxine-binding globulin (TBG) in the serum can now be measured by direct assays that are simple and inexpensive. Comparison of a direct measurement of TBG concentration with a widely used indirect method (Thyopac-3) showed that the indirect method was inaccurate when TBG concentrations were high. This will result in an increase in the derived free thyroxine index (FTI), so that euthyroid patients with a raised TBG concentration may be at risk of being labelled thyrotoxic. Correction of serum total thyroxine (T4) concentration according to the actual TBG concentration (T4:TBG ratio) provided a better correlation with thyroid state than FTI.     

A labile antigen complex at the lymphocyte surface: the effect of the substratum on its expression

Human T lymphocytes carry a surface antigen, detectable by a monoclonal antifibronectin antibody, which appears to consist of 150 and 55 kd components as revealed by SDS-PAGE. After in vitro culture of the lymphocytes on a plastic substratum for 48 hr comparatively few cells (40 +/- 18% in separate individuals) express the antigen. In contrast, the vast majority of lymphocytes cultured on a collagen matrix for the same time period maintains surface expression of the antigen (76 +/- 14% in separate individuals). Conditioned media from lymphocytes on plastic contain substantial amounts of antigenicity detectable by the same antibody, whereas conditioned media from lymphocytes on collagen are devoid of such antigenicity. The expression at the cell surface of other T lymphocyte antigens (Leu-4, Leu-3, and OKT8) is identical during culture on plastic and collagen for 48 hr. Collagen does not activate the cells to DNA synthesis or expression of IL 2 receptors, and consequently the potentiation of antigen expression by this substratum cannot be attributed to a mitogenic effect. The composition of subsets of T lymphocytes and the viability of the cells are the same on plastic and collagen, which excludes that the substratum-dependent variations in antigenicity reflect selection or loss of antigen-bearing cells. Thus, substratum-dependent regulation of the expression at the cell surface appears to be a unique property of the 150/55 kd T cell surface antigen. Culture on collagen substrata augments the number of lymphocytes showing motile behavior two to four times compared with culture on plastic.     

A thyroxine-binding globulin of major biological significance in the serum of mice: demonstration and ontogenesis

We demonstrate in the mouse serum a hitherto unrecognized major thyroxine binding globulin (TBG), analogous to human TBG or to the recently discovered rat TBG. Our demonstration is based on equilibrium dialysis, electrophoresis, immunoelectrodiffusion and autoradiography techniques. Mouse TBG displays a remarkable ontogenic pattern, with 2-3 times higher activity in foetal than in maternal serum, and a further dramatic increase after birth. Between 1 and 5 days, the T4 binding to serum reaches peak levels 7-10 times more elevated than those measured in normal or pregnant adults. We also present for the first time the ontogenesis of the thyroxine binding prealbumin (TBPA), considered until now as the only specific T4 carrier of the murine species. We show that throughout development it is the TBG, not the TBPA, which crucially governs the level of the T4-serum interactions.     

ACTH receptor distribution and modulation among murine mononuclear leukocyte populations

Murine mononuclear leukocytes express adrenocorticotropin (ACTH) receptors that were recognized by a monospecific antiserum to the ACTH receptor on Y-1 adrenal cells. The antiserum was utilized in an immunofluorescence (IF) assay to characterize the distribution of ACTH receptors on resting murine mononuclear leukocyte populations. Forty-seven percent of spleen cells, 32% of lymph node cells, and 1% of thymocytes constitutively expressed ACTH receptors. Separation of lymphocytes into purified B cell and T cell populations, followed by IF analysis revealed that 47% of B cells and 23% of T cells possessed ACTH receptors. Helper T cells (CD4+ T cells) constituted the majority of ACTH receptor-positive T lymphocytes. Furthermore, 47% of resident peritoneal macrophages, purified by adherence to plastic, expressed ACTH receptors. The T-lymphocyte mitogen, concanavalin A, interferon gamma, and ACTH enhanced ACTH receptor expression. The differential distribution of ACTH receptor-positive cells among specific leukocyte populations explains in part why differential cellular responses are observed and implies important regulatory functions for these receptors in the generation or regulation of immune responses.     

Fc receptors for IgA on human B, and human non-B, non-T lymphocytes.

Recently, receptors for IgA were demonstrated on subpopulations of human T lymphocytes. In this report, TNP-modified ox erythrocytes coated with the IgA myeloma MOPC-315 were used to detect IgA receptor-bearing lymphocytes within the human non T cell lymphocyte population. A mean of 5.3% (range 2.9 to 12.4%) of E-rosette negative human lymphocytes bound IgA-coated indicator cells. Blocking studies with soluble IgA, IgG, and IgM demonstrated that the IgA receptors on the non-T cell populations were separate and distinct from the Fc-receptors for IgG and IgM. Fractionation of the non-T lymphocytes on anti-human (Fab)2 columns into sIg+ and sIg- populations or by rosetting with EAC to provide CRL+ and CRL- populations demonstrated that Fc-IgA receptors were present on a subpopulation of sIg+, CRL+ lymphocytes, and also on sIg- (non-T, non-B) lymphocytes.