EVIDENCE THAT BOTH LONG-ACTING THYROID STIMULATOR AND LONG-ACTING THYROID STIMULATOR-PROTECTOR STIMULATE THE HUMAN THYROID GLAND S. D. HOLMES, S. M. DIRMIKIS, T. J. MARTIN AND D. S. MUNRO * and Department of Pharmacology Department of Chemical Pathology, The University, Sheffield, S10 2TN
(Received 27 April 1978) SUMMARY
Thyroid-stimulating immunoglobulins were prepared from two potent sera, one contained long-acting thyroid stimulator (LATS) and the other contained both LATS and LATS\x=req-\ 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\p=n-\ IgG; LATS-P\p=n-\IgG was inactive. In contrast, both LATS\p=n-\IgG and LATS-P\p=n-\IgG were equally effective in slices of human thyroid. INTRODUCTION
Two types of thyroid-stimulating autoantibodies have been detected in the serum of patients with Graves's disease. These are known as the long-acting thyroid stimulator (LATS; Adams & Purves, 1956) and LATS-protector (LATS-P; Adams & Kennedy, 1967). The first has been shown to stimulate thyroid activity in the mouse (McKenzie, 1958), guinea-pig (Adams «fe Purves, 1956) and monkey (Knight & Adams, 1973) whereas LATS-P is probably specific to the human thyroid gland (Adams, Fastier, Howie, Kennedy, Kilpatrick «fe Stewart, 1974a). In a series of untreated patients with Graves's disease, 75% had only LATS-P activity, 14-5% had both LATS and LATS-P activity and only 10-5% contained no detectable activity of either type (Dirmikis «fe Munro, 1976). Influenced by the much greater prevalence of LATS-P activity and the lack of clear correlation between the concentration of LATS and the uptake of 131I in their patients with Graves's disease, Adams, Kennedy & Stewart (l91Ab) proposed that LATS-P is the sole factor responsible for the thyroid overactivity character¬ istic of Graves's disease. It has also been proposed that LATS (which is usually detected by its effects in mice) should be renamed the mouse thyroid stimulator (Adams, Dirmikis, Doniach, El Kabir, Hall, Ibbertson, Irvine, Kendall-Taylor, Manley, Meholi, Munro, Purves, Smith & Stewart, 1975). In this study an alternative hypothesis has been examined: that both LATS and LATS-P belong to a single population of human thyroid-stimulating autoantibodies which sometimes act upon the glands of other species, including mice. We have measured the concentrations of LATS and LATS-P in two potent sera and have assayed their relative potencies in stimulating the accumulation of cyclic AMP in mouse or human thyroid tissue in vitro. MATERIALS AND METHODS
Units One unit of our internal laboratory standard for LATS is the activity in 1 µ of a potent LATS serum in which no LATS-P can be detected. In the McKenzie (1958) bioassay this is
equivalent to 467 mu. of the Medical Research Council standard for LATS (fiducial limits 324, 684). One unit of LATS-P is defined as the activity which blocks the binding of one unit of LATS in the assay as conducted in this laboratory (Dirmikis, 1974). Immunoglobulin G of G Preparations immunoglobulin (IgG) were made by ion-exchange chromatography on columns of DEAE 52 cellulose as described previously (Holmes, Dirmikis, Martin «fe Munro, 1978). The concentration of IgG was measured by radial immunodiffusion (Diagnostics test kit, ICL Scientific). Assays of the LATS and LATS-P content of the two IgG preparations used in this study in vitro are shown in Table 1. Animals White Swiss male mice were obtained from Sheffield University animal breeding unit and maintained under controlled conditions of temperature and lighting. After weaning, they were fed on Diet 86 (Oxoid Ltd) for 2 weeks, then kept on a low iodine diet of dried bread for a further 2 weeks (Broadhead, Dirmikis, Humphries, Justice, Loy & Smith, 1975). At the end of this period each mouse weighed 24-30 g. mice
Thyroid tissue -
Mice were killed by dislocation of the neck and the thyroid gland was dissected out, still adherent to the segment of trachea to which it was bound by fibrous tissue. The glands were kept in 0-9% saline (4 °C) until the dissections were complete. All experiments were performed between 09.00 and 12.00 h. When the thyroid glands were left adhering to the trachea there was a marked increase in the responsiveness to thyroid-stimulating hormone (TSH) compared with the lobes dissected free of tissue. This difference was probably due to the unavoidable tissue damage caused when removing lobes from the trachea.
Effects of diet on response of mouse thyroid glands Two groups of mice (same age and weight range) were fed on Diet 86 (Oxoid Ltd) or a diet of low iodine content (dried bread) for 2 weeks after weaning. A comparison of the influence of the two diets on the time course of accumulation of cyclic AMP by the thyroid gland in response to TSH (2 mu./ml) was made. After 20 min (the time of peak effect for TSH) there was over a twofold difference in the concentration of cyclic AMP accumulated by tissue in the two groups. Mice given a low iodine diet were used in all further studies (Broadhead et al.
Thyroid tissue human Human thyroid tissue was obtained at the time of operation from patients undergoing partial thyroidectomy for non-toxic goitres. The tissue was transported on ice to the laboratory and was sliced and incubated within 1 h. Goitrous tissue was preferred to thyrotoxic tissue because of its greater sensitivity to thyroid-stimulating immunoglobulins (TSIg) and TSH (Holmes et al. 1978). -
Assay of cyclic AMP accumulation Human tissue The method described
by Holmes et al. (1978) was used.
gland (still adherent to its trachéal segment) was pre-incubated for 45 min in KrebsRinger bicarbonate buffer (5 ml) with glucose (1 mg/ml) in a metabolic shaker at 37 °C. The
gas phase was 95% oxygen, 5% carbon dioxide. The solution was aspirated and replaced with fresh medium (0-4 ml) plus 400 µg of the phosphodiesterase inhibitor, Ro 20-1724 (Roche Products Ltd) for 15 min. Thyroid stimulators were added in 0-1 ml Tris-buffered saline (0-15 M-NaCl, 0-01 M-Tris-HCl, pH 7-4) for the final incubation which lasted 20 min for studies on the action of TSH and 80 min for LATS-IgG. The thyroid lobes were quickly dissected free from the trachea, blotted dry and weighed before homogenization in 80% ethanol (2 ml chilled below 5 °C) for 30 s with a Teflon-glass power-driven homogenizer (Baird & Tatlock). The ethanolic extract was evaporated to dryness at 50 °C in a stream of air and dissolved in distilled water, and the cyclic AMP was measured by the proteinbinding assay of Brown, Albano, Ekins, Sgherzi «fe Tampion (1971). —
Statistics of the difference between means was established by Student's r-test. A analysis of value less than 0-05 taken indicative of a significant difference between was as probability means. Estimates of relative potency and 95% fiducial limits were calculated from parallel line bioassays as described by Finney (1964). Statistical
Reagents AMP (sp. act. 27 Ci/mmol) was supplied by The Radiochemical Centre, Amersham; DEAE 52 was obtained from Whatman Chemicals; cyclic AMP from Sigma. All chemicals were of analytical reagent grade.
Thyroid-stimulating hormone (1 i.u./mg) portions of Tris-buffered saline (pH 7-4) 1 serum bovine containing mg albumin/ml. Bovine TSH
stored at —20 °C in
Time-course of action of TSH and LATS A significant increase in the accumulation of cyclic AMP in mouse thyroid glands was observed after incubation for 5 min with TSH or 10 min with LATS-IgG. The peak effect for TSH was observed after 20 min and a plateau was reached after 80 min with LATS-IgG. These results are in good agreement with earlier reports (Kendall-Taylor, 1972; Zakarija «fe McKenzie, 1973). There is one report, however (Williams, 1972) in which the maximum cyclic AMP response to TSH occurred after 60 min. Preparations of IgG solely with LATS-P activity had no effect during the time studied.
Comparison of LATS and LATS-P potency hypothesis, two sera were chosen for the preparation of TSIg. The first
investigate (A) was rich in LATS and contained no detectable LATS-P. This is the serum used as our
reference in the McKenzie (1958) assay for LATS and for the reference mixture of LATS and binding protein in the detection of LATS-P; this serum requires a minimum amount of binding protein for absorption of its LATS activity (Dirmikis, 1974). The second serum (B), when assayed by the McKenzie system for its content of LATS and LATS-P, had 75% in the form of LATS-P and only 25% as LATS (Table 1). A serum with such high potency containing only LATS-P activity has not been found previously, although there are many in the lower activity range which contain only LATS-P. Preparations of IgG from less potent serum, made by methods used in this study, would not yield a sufficiently active preparation for studies in vitro. The effects of immunoglobulin G prepared from sera A and on the accumulation of cyclic AMP in mouse thyroid glands are shown in Fig. 1. The potency of relative to A was determined from these lines and was found to be 0-37 with fiducial limits of 0-60 and 0-32. our
This relative potency estimate was in close agreement with the relative LATS potency of the immunoglobulin G prepared from serum relative to the immunoglobulin G prepared from A, obtained in the McKenzie assay, which was 0-34 (Table 1). By comparison, Fig. 2 shows the dose-response lines for the effect of preparations A and relative to A on the accumulation of cyclic AMP in human thyroid tissue. The potency of
of the long-acting thyroid stimulator (LATS) and LATS-protector (LATS-P) of two immunoglobulin G (IgG) preparations in the McKenzie (1958) assay (mean and 95% fiducial limits). For units and experimental details see text Table 1. Assay contents
Immunoglobulin G preparations
Serum A Serum
2264(653,5176) 501 (299, 867)
1510(778,4091) Serum A contained only LATS activity:
had both LATS and LATS-P activities.
N.D., Not detectable. 50
Immunoglobulin G (mg/ml) Fig. 1. Effects of thyroid-stimulating immunoglobulins containing long-acting thyroid stimulator (LATS) or LATS and LATS-protector (LATS-P) activities on the accumulation of cyclic AMP in mouse thyroid lobes. Immunoglobulin G (IgG) was prepared from two serum samples. Serum A (O) contained only LATS and the IgG preparation contained 107 units/mg. Serum ( ) contained both LATS and LATS-P and the IgG preparation contained 36 units LATS/mg and 108 units LATSP/mg. The mean effects ±s.e.m. (n 3) are shown, D, Basal concentration. =
determined from these dose-response lines was found to be 1-20 with fiducial limits of 1-94 and 0-63, which was in good agreement with the estimate of their relative potencies made by the McKenzie assay when the sum of the LATS and LATS-P activities in the immunoglobulin G preparation from serum was used. Although there is intersection of the two lines of best fit, there was no significant deviation from parallelism. 80
Immunoglobulin G (mg/ml)
Fig. 2. Effects of thyroid-stimulating immunoglobulins containing the long-acting thyroid stimulator (LATS) or LATS and LATS-protector (LATS-P) activities on the accumulation of cyclic AMP by slices of human thyroid. Immunoglobulin G (IgG) was prepared from two serum samples. Serum A (o) contained only LATS and the IgG preparation contained 107 units/mg. Serum ( ) contained both LATS and LATS-P and the IgG preparation contained 36 units LATS/mg and 108 units LATS-P/mg. The mean effects ± s.e.m. (n 3) are shown, n, Basal concentration. =
In another experiment with the IgG preparations from sera A and
there was, again, close
agreement between the McKenzie B/A ratio for the sum of the LATS and LATS-P contents
preparation (1-35) and the B/A ratio from the cyclic AMP assay (1-51). The results imply that the accumulation of cyclic AMP in slices of human fluenced by both LATS and LATS-P.
thyroid is in¬
Initially, the conclusion by Adams, Kennedy & Stewart (1976) that only LATS-P stimulates the human thyroid gland was questioned because, in our own studies of the activity of LATS and LATS-P in the sera of untreated thyrotoxic patients, there was usually a variable excess of LATS-P activity over LATS, so that in LATS-rich samples the greater proportion of the total serum activity was in the form of LATS-P (Dirmikis & Munro, 1975, 1976). Thus, as reported by Adams et al. (19746,1976), thyroid hyperactivity might be expected to correlate less well with LATS than with LATS-P. It has been found that a high concentration of LATS usually indicates a high LATS-P activity but that the amount of LATS is not a constant proportion of the total. On the other
serum of very high activity in which only LATS-P was detectable has not yet been assayed. As might be anticipated from our experience of the variable ratio of LATS to LATS-P activities in various sera, some previous reports have failed to establish a positive correlation either between LATS activity (measured by mouse bioassay) and stimulation of the adenylate cyclase/cyclic AMP system in the human thyroid gland (McKenzie «fe Zakarija, 1976; Orgiazzi, Williams, Chopra «fe Solomon, 1976) or with inhibition of 125I-labelled TSH bind¬ ing to human thyroid membranes (Smith & Hall, 1974). In the present experiments the stimulation of the accumulation of cyclic AMP in mouse thyroid lobes was directly related to the LATS content ofthe IgG preparation. As anticipated, LATS-P had no effect on the mouse thyroid gland. Stimulation of human thyroidal cyclic AMP accumulation by IgG preparations containing LATS has been reported previously (Kendall-Taylor, 1973; Holmes, Dirmikis, Martin & Munro, 1976) and from the data presented here, LATS and LATS-P would appear to be equipotent in activating the human thyroid gland. Therefore, our results agree with those of McKenzie «fe Zakarija (1976) who found thyroidstimulating immunoglobulins in sera from the majority of patients with Graves's disease and that these immunoglobulins are polyclonal antibodies to a single human antigen that have a variable cross-reactivity with an analogous thyroid antigen in the mouse or in other species.
This work was supported by grants from the Yorkshire Cancer Research the Medical Research Council.
Adams, D. D., Dirmikis, S., Doniach, D., El Kabir, D. J., Hall, R„ Ibbertson, H. K., Irvine, W. J., KendallTaylor, P., Manley, S. W., Meholi, S. Q., Munro, D. S., Purves, H. D., Smith, B. R.