Reexpression of Blood Group ABH Antigens on the of Human Thyroid Cells in Culture Surface

Using indirect immunofluorescence (IFL) on viable human thyroid cultures, it has been shown that, although adult follicular cells do not express blood group ABH antigens in vivo, they invariably reexpress the corresponding antigens on the cell surface when cultured in monolayers, even for very short periods. The absence of blood group antigens on noncultured thyroid cells was confirmed by negative IFL on cell suspensions obtained after enzymatic digestion of the glands, whereas these antigens were readily demonstrable on cell suspensions obtained by trypsinization of established monolayers. The quantitative expression of ABH antigens on individual thyroid cells was variable and the cell-surface IFL pattern due to binding of blood group isoantibodies was different from that given by organ-specific thyroid autoantibodies on viable cultures. Reexpression of blood group antigens by cultured thyroid cells could not be related to the secretor status of the donors, the presence of a particular source of serum in the culture medium or cell division in vitro. After 2-3 wk in culture, thyroid cells became morphologically dedifferentiated and no longer displayed blood group antigens, though they still expressed cell-surface fl2-microglobulin. Fibroblasts present in the primary thyroid cultures were invariably negative for ABH antigens. These results demonstrate that the surface antigenic repertoire of cultured human cells is not necessarily identical to that present on the same cells in vivo. Furthermore, the possibility that blood group natural isoantibodies bind to the cell surface must be taken into account in experiments in which cultured thyroid cells are exposed to human sera. The expression of blood group antigens of the ABH system by cells other than erythrocytes has been extensively studied in adult and fetal human tissues, either by mixed agglutination of erythrocytes and tissue cell suspensions (4, 15, 35) or indirect immunofluorescence (IFL) on tissue sections (9, 10, 15, 28, 29). In these reports, thyroid follicular cells were shown to be consistently negative for ABH cell-membrane antigens (9, 29). However, using early human embryos, Szulman (30) was able to detect blood group antigens by IFL on the thyroid primordium, still lacking acinar structure, up to the 12th week of gestation. The loss of ABH antigens at this stage takes place when the thyroid begins to show the histological appearance and functional properties of the differentiated gland (12). Expression of ABH surface antigens has also been investigated on cultured human fetal tissues, though not thyroid, by means of specific adherence of erythrocytes to cell monolayers (13) and on established cell lines of human origin (3, 36). Although the exact identity of cells showing positive reactions in the primary cultures of fetal organs was not determined, epithelioid cells ofteh displayed the antigens on their surfaces, whereas fibroblastoid cells were usually negative. The presence of these surface isoantigens on epithelioid cells decreased in cultures grown for more than 3 wk or repeatedly subcultured (14). On the other hand, several established human cell lines were shown to include a variable proportion of cells expressing blood group H, but not A or B, surface antigens for indefinite periods of culture (36). An increase in the expression of isoantigens on these cloned cell lines was related to the rate of ceU division (20) or the addition of blood group precursors to the culture medium (3). Viable human thyroid cell monolayers have been used for immunological studies in which they are exposed to human sera or immunoglobulin fractions to detect either cytotoxic (6, 16, 19, 25) or stimulating (32) organ-specific autoantibodies. In THE JOURNAL OF CELL BIOLOGY • VOLUME 94 JULY 1982 193-200 © The Rockefeller University Press • 0021-9525/82/07/0193/08 $1.00 193 on A uust 8, 2017 jcb.rress.org D ow nladed fom the course of IFL studies on these cell-surface reactive autoantibodies it was found that some normal sera stained monolayers from blood group A or B, but not O, donors. This finding led to the investigation of the reexpression of ABH surface antigens under these conditions. MATERIALS AND METHODS Forty-three human thyroids were studied. 41 were from adult individuals (18 were blood group O, 16 A, 6 B, and 1 AB) and 2 were fetal glands, from a 14-wkold blood O and a 17-wk-old blood group A fetus, respectively. The adult glands were normal in 12 cases (from patients undergoing radical surgery for carcinoma of the larynx), the other 29 being thyrotoxic (10), multinodular colloid goiter (9), simple goiter (2), Hashimoto's thyroiditis (3), dyshormonogenetic goiter (1) and peri-adenomatous thyroids (4). Culture of Human Thyroid Cells The procedure has already been reported in detail (18). Briefly, the thyroid tissue was chopped into small pieces, washed in culture medium (Flow 199 plus NaHCO3 0.35 mg/ml, Penicillin 300 IU/ml and Streptomycin 300/~g/ml Flow Laboratories, Inc., Rockville, MD), and incubated for 60-90 min at 37°C with either collagenase (Worthington type IV, 2 mg/ml Worthington Biochemical Corp., Freehold, NJ) or trypsin (Difco, 2.5 mg/ml Difco Laboratories, Detroit, M1). In some experiments no enzyme was used, and ceils were obtained by shaking the small tissue fragments in culture medium. Cell viability was assessed by differential staining with an acridine orange/ethidium bromide mixture (AO/ EB) using fluorescence microscopy (21), being >90% in all cases. An accurate cell count at this stage was not possible as most thyroid cells were in clumps (23). In some experiments, cells were resuspended and cultured in 15% blood group O or AB normal human serum (NHS) or blood group O fetal human serum (FHS) instead of FCS. 100 bd of the cell suspension were plated onto round glass cover slips, placed in multiwell plates (Linbro), and 400 btl of culture medium containing 15% FCS was added to each well. The cultures were kept at 37°C in a 5% CO2 humidified incubator for various periods, with periodic changes of culture medium, until they were stained.