Simple radioimmunological assay of anti-alpha-galactosyl antibody (anti-Gal): application to autoimmune thyroid diseases and thyroid-associated ophthalmopathy.

Two radioimmunological techniques for the measurement of anti-alpha-galactosyl (anti-Gal) antibodies were developed and we demonstrated that this antibody is present in the serum of most untreated patients with Graves' disease, thyroid-associated ophthalmopathy (TAO) and autoimmune thyroiditis. Thyrotropin-binding inhibiting immunoglobulins (TBII) and Thyroid Stimulating Antibodies (TSAb) are frequently found in serum of patients with Graves' disease but not with autoimmune thyroiditis. Although TBII and anti-Gal were found sometimes simultaneously in the same serum samples, we demonstrated that anti-Gal could be present in the absence of TBII and reciprocally.

Specific stimulation of Graves' disease thyrocytes by the natural anti-Gal antibody from normal and autologous serum.

Anti-Gal is a natural Ab abundantly produced in humans. It interacts specifically with the carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope). This epitope is expressed in large amounts on thyrocytes of nonprimate mammals, but not of humans. We have previously found that binding of anti-Gal to alpha-galactosyl epitopes on porcine thyrocytes results in stimulatory effects similar to those exerted by thyroid-stimulating hormone (thyrotropin). In the present study, we tested the hypothesis that anti-Gal may contribute to Graves' disease (GD) pathogenesis by stimulation of the thyrocytes of patients with this autoimmune disorder. Anti-Gal binding and stimulatory effects were assessed in primary thyrocyte cultures. Anti-Gal specifically bound to GD thyrocytes and induced an increase in cAMP synthesis, 125I uptake, and DNA synthesis in these cells. Furthermore, the stimulatory effects of autologous sera on GD thyrocytes were greatly reduced after specific depletion of anti-Gal from these sera. No binding and no stimulatory effects of anti-Gal were observed, however, with normal human thyrocytes and with thyrocytes from thyrotoxic patients who lack thyroid-stimulating Igs or thyrotropin binding inhibiting Igs. These in vitro stimulatory effects of anti-Gal on GD thyrocytes suggest that this natural Ab may contribute to the in vivo continuous stimulation of thyrocytes in GD patients. The possibility that anti-Gal may stimulate GD thyrocytes via interaction with aberrantly expressed alpha-galactosyl epitopes on the thyroid-stimulating hormone receptor is discussed.

Xenogeneic thyroid-stimulating hormone-like activity of the human natural anti-Gal antibody. Interaction of anti-Gal with porcine thyrocytes and with recombinant human thyroid-stimulating hormone receptors expressed on mouse cells.

Anti-Gal is a natural polyclonal antibody that constitutes 1% of circulating IgG in all humans and that interacts specifically with the mammalian carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope). This epitope is abundant on thyrocytes, as well as, on other cells of nonprimate mammals, prosimians and New World monkeys, but its expression is diminished in Old World monkey, ape, and human tissues. We hypothesized that anti-Gal may bind in vitro to alpha-galactosyl epitopes on xenogeneic TSH receptors (TSHR) and mimic the effect of TSH on xenogeneic thyrocytes. Assays performed with porcine thyrocytes have indicated that anti-Gal can mimic in vitro TSH effects in stimulation for cAMP synthesis, 125I uptake, and cell proliferation. Furthermore, depletion of anti-Gal from serum of patients with Graves' disease resulted in elimination of a large proportion of the thyroid stimulating immunoglobulin activity and half of the thyroglobulin binding inhibiting Ig activity, when the sera were assayed with porcine thyrocytes. The effect of anti-Gal binding to alpha-galactosyl epitopes on TSHR was further demonstrated by the antibody-mediated stimulation for cAMP synthesis in mouse 3T3 cells (cells expressing alpha-galactosyl epitopes), which were transfected with recombinant human TSHR. CHO cells (cells lacking alpha-galactosyl epitopes), transfected with recombinant human TSHR were not stimulated by anti-Gal. It is, therefore, suggested that in studies on antibodies in Graves' disease sera, the effect of anti-Gal may be excluded by using target cells that are devoid of alpha-galactosyl epitopes. Alternatively, anti-Gal could be removed from the tested sera, before the assay with xenogeneic thyrocytes.

The alpha-galactosyl epitope on mammalian thyroid cells.

Studies on the distribution of alpha-galactosyl epitopes with the structure Gal alpha 1----3Gal beta 1----4GlcNac-R on mammalian thyroid cell membranes are of interest, since a natural antibody interacting with this carbohydrate antigen (i.e. the natural anti alpha-galactosyl IgG antibody) was found to increase in its titre in patients with autoimmune thyroid disorders. By using a radioimmunoassay for quantification of the alpha-galactosyl epitope, we found variable concentrations of the alpha-galactosyl epitope, we found variable concentrations of this epitope on thyroid cell membranes of all nonprimate mammals and New World monkeys studied, but not in Old World monkeys and human thyroid. The absence of the identifiable alpha-galactosyl epitopes on human and Old World monkey thyroid cells correlates with diminished activity of the enzyme, alpha 1-3 galactosyltransferase, which, in other species, synthesizes the alpha-galactosyl epitopes within the Golgi apparatus. It is argued that a proportion of anti-thyroid reactivity in human normal and pathologic sera, when assayed with mammalian thyroid cells, may be attributed to natural anti alpha-galactosyl IgG antibody, which interacts with alpha-galactosyl epitopes on thyroid tissues used for the bioassay.

The alpha-galactosyl epitope on human normal and autoimmune thyroid cells.

As much as 1% of circulating IgG in man (the natural anti-Gal antibody) is directed against the alpha-galactosyl epitope, with the structure Gal alpha 1----3Gal beta 1----4GlcNAc-R. The alpha-galactosyl epitope is abundantly expressed on cells of nonprimate mammals, prosimians, and New World monkeys. Its expression is diminished in Old World monkeys, apes, and humans. It has been previously suggested that interaction between anti-Gal and aberrantly expressed alpha-galactosyl epitopes on thyroid cells may contribute to the initiation of autoimmune thyroid disorders. To study this possibility, alpha-galactosyl epitope expression on thyroid cell membranes of normal individuals and patients with Graves' disease was assessed by a sensitive radioimmunoassay. alpha-Gal-actosyl epitopes were found both on normal and diseased thyroid cells. Whereas the concentration of these epitopes on Graves' disease thyroid membranes was somewhat higher than that observed in normal glands, the difference was not significant. The activity of the enzyme, alpha 1-3-galactosyltransferase, which synthesizes the alpha-galactosyl epitope, was higher in microsomal fractions obtained from some patients as compared with healthy controls, but not significantly different. In view of the abundance of anti-Gal antibody in the circulation, it is argued that, under physiologic conditions, the interaction of this antibody with alpha-galactosyl epitopes does not elicit pathologic effects. However, aberrant expression of the alpha-galactosyl epitope may result in effective anti-Gal binding to thyroid cells (e.g., rearrangement of this structure on the cell membrane or its increased expression).(ABSTRACT TRUNCATED AT 250 WORDS)

Original acquisition in the pathogenesis and the treatment of endocrine ophthalmopathy.

In the present work, we demonstrate in the serum of patients with progressive exophthalmos, the presence of circulating immunoglobulin which is able to induce an experimental exophthalmos in goldfish. This immunoglobulin disappears when exophthalmos becomes residual. In parallel, we describe an easy technique for the measurement of delayed hypersensitivity which is positive for thyroid or retroorbital antigens in progressive exophthalmos but negative in residual exophthalmos. We also demonstrate in patients with exophthalmos an increase in urinary glycosaminoglycans. We propose a new therapeutic approach in treatment of acute exophthalmos which consists of intensive plasma exchange followed during a few months, by the administration of small amounts of corticosteroids. The plasma exchange mechanism consists of the extraction of the antibody directed against retroorbital eye muscle antigen. In addition, plasma exchange produces the extraction of glycosaminoglycans from retroorbital tissue. These hygroscopic molecules are responsible for the marked oedema in retroorbital tissue.

Thyroid growth modulating factors in the sera of patients with simple non-toxic goitre.

Since 'Thyroid-Growth-Immunoglobulins' are implicated in the pathogenesis of some goitrous thyroid diseases, we have investigated the presence of thyroid growth modulators in the sera from patients with simple non-toxic goitre (diffuse non-toxic goitre and colloid nodular goitre). To detect growth effect, two procedures were employed, both using [3H]thymidine incorporation into DNA. In one procedure, porcine thyroid follicles in suspension were employed. Gamma-globulin (2 mg/ml) from 10/26 of patients with diffuse non-toxic goitre and 8/27 of patients with colloid nodular goitre were found to increase [3H]thymidine incorporation when compared to gamma-globulins from control group. The other procedure used sparsely plated thyroid cells isolated from normal or porcine thyroid gland and from human simple non-toxic goitre. No serum from patients with simple goitre showed greater growth stimulating activity than that of normal individuals. Moreover, using simple goitre cells as target, a lower serum activity was observed in 7/28 of patients with diffuse non-toxic goitre and in 9/30 of patients with colloid nodular goitre. Analogous results were obtained with normal porcine thyroid cells. The lower serum activity of these patients was observed in a wide range of serum concentrations (1 to 15%) and was associated with and inhibitory effect recovered in the gamma globulin fraction. By the two different procedures, we have therefore evidenced the presence of thyroid growth modulators in the sera of several patients with simple non-toxic goitre. The stimulatory effect observed with the thyroid follicles strengthens the implication of thyroid growth stimulating immunoglobulin in the pathogenesis of some simple sporadic non-toxic goitre.(ABSTRACT TRUNCATED AT 250 WORDS)

Some unexpected effects of thyrotrophin on the metabolism of thyroid cells isolated from non-toxic goitre.

In cultured cells of normal dog thyroid thyrotrophin (TSH) has been shown to have a mitogenic effect. The present study suggests an opposite action in cells from human non-toxic goitre. After incubation of these cells with [3H]thymidine, a decreased incorporation into DNA is observed in the presence of TSH. Whereas in dog thyroid cells the hormone enhances the incorporation of [3H]leucine and [14C]glucosamine into macromolecules, TSH inhibits the incorporation by human non-toxic goitre cells. Polyacrylamide gel electrophoresis analysis of the proteins present in the cell layer or released in the culture medium shows that the inhibition affects all the newly synthesized peptides, including the radioactive material co-migrating with the major band (330 000 daltons) observed with human purified thyroglobulin. While several hydrolase activities are not influenced by TSH in dog thyroid cells, the hormone markedly decreases the activities of some hydrolases of thyroid cells from human non-toxic goitre. Our results therefore suggest that, in cultured thyroid cells from human non-toxic goitre, TSH induces a resting state of the cells, characterized by reduced growth, reduced protein synthesis and reduced thyroglobulin hydrolysis.

A sensitive technique for determination of thyroid-stimulating immunoglobulin (TSI) in unfractionated serum.

A new sensitive and reproducible technique for the determination of thyroid stimulating immunoglobulins is described. This procedure employs cells obtained from primary culture of human thyroid gland. We measured the increase of cyclic AMP after addition of patient sera. The major advantages of this procedure are the improved sampling throughout with the ability to measure eighty samples simultaneously without fractionation of serum. This large sample allows us to test forty sera together and to assay in duplicate, thus increasing the validity of the results. The TSI activity is associated with the immunoglobulins G (IgG) fraction and we obtained a linear dose response curve with IgG as well as with whole serum. The results obtained in various thyroid diseases are discussed.

Effect of thyrotropin on the properties of messenger RNA from cultured human thyroid cells.

When added to the culture medium of thyroid cells isolated from diffuse nontoxic goiter, thyrotropin increased the poly(adenylic acid) content and the template activity of the unfractionated RNA. This increase was correlated with higher thyroglobulin messenger activity, as demonstrated by specific immunoprecipitation of the labeled peptides synthesized in two heterologous cell-free systems. When RNAs were separated in a sucrose gradient, thyrotropin was shown to enhance the poly(adenylic acid) content and template activity of fractions with sedimentation coefficients of 34, 23 and 15 S. Specific immunoprecipitation showed that a thyroglobulin messenger activity was present in these three fractions. Another way by which thyrotropin regulates the thyroid protein synthesis is suggested by the shift of poly(adenylic acid)-containing RNA to large polysomes when thyroid cells were cultured in the presence of the hormone.

Tissue culture of normal rat glomeruli: glycosaminoglycan biosynthesis by homogeneous epithelial and mesangial cell populations.

The biosynthesis of glycosaminoglycans (GAG) by cultivated rat glomerular epithelial and mesangial cells was studied by incorporation of [35S]sulfate or [14C]glucosamine for 48 h. After dialysis, the incubation medium was subjected to digestion with papain. Labeled GAG were isolated from the digests by precipitation with cetylpyridinium chloride and ethanol. Results of cellulose acetate electrophoresis of the isolated 'epithelial' GAG fraction revealed the presence of two [14C] spots and one [35S] spot. The [35S] spot was identified as heparan sulfate, because it comigrated with the heparan sulfate standard and it was insensitive to testicular hyaluronidase. One [14C] spot comigrated with the [35S] spot and with the heparan sulfate standard. This GAG fraction did not contain galactosamine. The second [14C] spot was identified as hyaluronic acid, since it comigrated with the hyaluronic acid standard and since it was sensitive to testicular hyaluronidase. Results of cellulose acetate electrophoresis of the isolated 'mesangial' GAG fraction revealed the presence of one [14C] spot only. No [35S] spot was detectable. The [14C] spot comigrated with the hyaluronic acid standard and was sensitive to hyaluronidase. The data therefore suggest that the glomerular epithelial cells synthesize and secret both sulfated GAG (heparan sulfate) and nonsulfated GAG (hyaluronic acid) into the culture medium, whereas the glomerular mesangial cells synthesize and secrete nonsulfated GAG (hyaluronic acid) only into the culture medium.

Effects of thyrotropin on iodine metabolism of dog thyroid cells in tissue culture.

When grown in the presence of thyrotropin, dog thyroid cells in culture from follicle-like structures, take up labeled iodide, and iodinate macromolecular components in the cell. When grown in the absence of thyrotropin, dog thyroid cells in culture form a monolayer, take up only 6% of the iodide of follicular cells, and do not iodinate macromolelcular components in the cell. The iodide uptake in monolayer cells does, however, reflect an incorporation process unique to thyroid cells because hepatocytes and fibroblasts do not have the capacity of the monolayer cells to take up iodide. Thyrotropin stimulation of monolayer cells for a prolonged period (3-8 days) causes the cAMP levels of these cells to return to levels identical to those in follicular cells. The increased cAMP levels are not due to the induction of an adenylate cyclase enzyme, because homogenates of monolayer cells have a thyrotropin-stimulable adenylate cyclase activity. The low level of cAMP, thus, seems to be a problem of receptor coupling to the adenylate cyclase enzyme. The return of cAMP to normal levels is accompanied by an increase in iodide uptake and by macromolecular organification; the return of cAMP levels to normal values is not accompanied by follicular development. The majority (75%) of the iodinated macromolecular product accumulated by follicular thyroid cells, by monolayer thyroid cells stimulated with thyrotropin for a prolonged period, or by thyroid cells treated with dibutyryl cAMP from the onset of culture has the characteristics of 19 S thyroglobulin. The remainder appears to be low mol wt material which may be thyroglobulin-related i.e., be either precursor or biodegraded material.

Hormone responsiveness of adenylate cyclase activity of cultured myogenic cells.

We studied the effect of catecholamines on the adenylate cyclase activity of myogenic cells of the L6 line during the differentiation process. The enzyme of mononucleated myoblasts was found to be activated by adrenaline L, noradrenaline L, and to a small extent, by adrenaline D and dihydrocymandelic acid. The adenylate cyclase of the differentiated cells responded to adrenaline L and noradrenaline L but not to adrenaline D or dihydroxymandelic acid. This activation could be inhibited by the addition of DOPA and propranolol, a specific beta adrenergic blocker. alpha adrenergic compounds such as phentolamine did not have any effect. It is concluded that beta adrenergic receptors are present on myogenic cells before and after differentiation.