A Study on Anti-Pituitary Serum

Transcription

1 Immunology, 1969, 16, 311. A Study on Anti-Pituitary Serum W. PIERPAOLI AND E. SORKIN Schweizerisches Forschungsinstitut, Medizinische Abteilung, 7270 Davos-Platz, Switzerland (Received 28th June 1968) Summary. Rabbit anti-mouse pituitary serum was prepared. Its affinity for the acidophilic, growth hormone producing cells was demonstrated by the fluorescent antibody technique. The anti-pituitary serum also had some anti-growth hormone activity. Besides the changes described earlier in the thymus and lymphoid tissue injection of anti-pituitary serum into young mice also produced an alteration in the thyroid gland. INTRODUCTION Previous investigations demonstrated the feasibility of producing antisera containing specific antibody against cells of the anterior pituitary gland (Anigstein, Rennels and Anigstein, 1960; Beutner, Djanian and Witebsky, 1964; Milgrom, Maide Tuggac and Witebsky, 1965). These authors repeatedly injected homologous or heterologous hypophysis tissue homogenates into animals and the sera were used to study the existence of pituitary specific antigens. Our recent findings on the induction of a wasting syndrome and thymus atrophy in mice following injection with rabbit anti-mouse hypophysis serum (Pierpaoli and Sorkin, 1967) have prompted us to investigate in vitro the degree of specificity of our anti-pituitary serum for pituitary cells. The effect of anti-hypophysis serum on the thyroid and adrenal glands in vivo has also been investigated by histological examination. MATERIALS AND METHODS Animals Two groups of four rabbits weighing 3-4 kg were used for immunization with mouse pituitary or thymus cells. Hypophyses and thymus were removed from groups of young adult male albino mice of an outbred colony. The animals weighed g. They were killed by decapitation. The organs were taken under clean but not aseptic conditions. Preparation of cell suspensions Pituitaries were gently removed from the sella turcica after dissection of the invagination of the dura mater and of the other connections. The intact glands were washed in cold Gey's solution. The thymuses were decapsulated and similarly washed. The cells of both organs were gently dissociated in a glass grinder fitted with a loose Teflon pestle, filtered through three sheets of gauze and carefully washed three times at 40 with Gey's solution. Cells from the pituitaries and thymuses were collected and counted. The number of isolated pituitary 311

2 312 W. Pierpaoli and E. Sorkin cells per mouse ranged from 4x 105 to 7x 105 and included cells from the anterior, intermediate and posterior lobes. The total yield of cells obtained from the pituitaries of groups of mice ranged from 35x 106 to 84x 106. Immunization procedures A group of four rabbits was injected subcutaneously into the back of the neck and into two other sites with pituitary cells obtained from 130 mice. Every rabbit received about 14x 106 cells in 1 ml Gey's solution incorporated into 2 ml of Freund's complete adjuvant. The quantity injected was equally distributed between the three sites. Another group of rabbits was similarly injected with thymus cells and the same procedure was followed in the subsequent injections (see also Grabar, 1969). Sodium penicillin (100,000 units) was injected intramuscularly in order to prevent infections. One week after the first injection the appropriate groups of rabbits received a further dose of about 15 x 106 of either pituitary or thymus cells which were again incorporated into Freund's complete adjuvant. At 2 weeks after the first inoculation, each rabbit received 21 x 106 pituitary or thymus cells suspended in Gey's solution, and at 3 weeks after the first inoculation 16 x 106 cells were injected. Half of the cells were injected intramuscularly and half of them intravenously. One week after this fourth injection all rabbits received, on 3 consecutive days, an intravenous dose of about 16 x 106, 13x 106 and 9x 106 pituitary or thymus cells. Five and 7 days after the last injection all rabbits were bled from the ear marginal vein. On. the 1st and 2nd day after the bleeding, the rabbits were injected again with 20x 106 and 15 x 106 pituitary or thymus cells. Final bleedings from all rabbits were carried out 4-6 days after these two last injections. Treatment of antisera Separate pools of all anti-hypophysis and anti-thymus sera were prepared from the first two bleedings and from the final bleeding. The antisera were inactivated at +560 for 30 minutes, absorbed once with 0-25 volume of fresh, washed and packed mouse erythrocytes and finally stored in small aliquots at -20. Acetone powders For absorption of cross-reacting antibodies acetone powders were prepared from pooled fresh lungs, kidneys and brains of NMRI mice (Colowick and Kaplan, 1955). Preparation of globulins The total globulin fraction of the antisera was precipitated by ammonium sulphate at 50 per cent final saturation and after dialysis, redissolved to the original volume of antiserum. The protein content was determined. The globulins were then concentrated four times by ultrafiltration in the cold. They were absorbed with mouse kidney, brain and lung acetone powders for 2 hours at room temperature, centrifuged at 30,000 g for 1 hour at 40 and finally stored in small aliquots at In vitro tests with anti-pituitary and thymus serum (a) Fluorescent antibody technique. The direct staining and the double layer technique were employed (Nairn, 1962). For the direct staining technique, globulins from the anti-thymus and anti-pituitary antisera were repeatedly precipitated and re-dissolved, the protein concentrations were measured and the globulins conjugated to fluorescein

3 Anti-Pituitag Serum 313 isothiocyanate (SIGMA, Chemical Company, 3500 Dekalb Street, St Louis, Mo , U.S.A.) as described by Holborow (1964). The conjugated globulins were dialysed at 40 against phosphate buffered saline until no fluorescence was detectable in the dialysate. A portion of both the anti-thymus and anti-pituitary fluorescein conjugated globulins (4 volumes) was absorbed with one volume of well-washed packed thymus cells at 370 for 30 minutes. The absorption procedure was repeated four times. No agglutinating antithymus antibody could then be detected in the globulin solutions. The other portion of anti-hypophysis and anti-thymus globulins was submitted to the same incubation procedure but without thymus cells. All unabsorbed and absorbed globulin solutions were then centrifuged at 12,000 g for 2 hours at 40. The same absorption procedure as used for the globulins was also performed with antithymus, anti-hypophysis and normal rabbit serum. Suspensions of cells from hypophyses and thymuses were prepared as described above. Smears of thymus or pituitary cells were made on glass slides, left to dry at room temperature for a few hours, then fixed to the glass by quickly heating in a flame. The slides were stored at 40 and used within 2 days. For direct fluorescent staining, a few drops of undiluted fluorescein-conjugated antithymus, anti-pituitary and normal rabbit serum globulins, absorbed or not absorbed with thymus cells, were added to the thymus and hypophyseal cell smears. The slides were left overnight at 370 in a damp chamber. They were then repeatedly washed with phosphatebuffer, ph 7-2, for 20 minutes, left to dry and finally examined. As a further control, a 'blocking-test' was performed by pretreating thymus and hypophysis cells with unconjugated anti-hypophysis, anti-thymus and normal rabbit serum and subsequently with conjugated fluorescent globulins. For the double layer test, the cell smears were covered with a few drops of anti-pituitary, anti-thymus or normal rabbit serum, which were unabsorbed or absorbed with thymus cells as described above. The smears were left for 2 hours at 370, washed several times with phosphate-buffer, ph 7*2, and finally covered with undiluted fluorescein-isothiocyanate conjugated sheep anti-rabbit globulin serum (Pentex, Inc., Kankakee, Illinois, U.S.A.) They were then incubated in a damp chamber for 5-6 hours at 370. After repeated washing with phosphate-buffer, ph 7-2, they were left to dry. A Leitz Ortholux microscope equipped with an Osram XBO 200 Xenon lamp and adapted for fluorescence microscopy and microphotography was used. (b) Direct agglutination test. Agglutination tests were carried out in small test tubes. Suspensions of thymus and pituitary cells containing about 7 x 106 cells/ml were prepared in phosphate solution, ph 7*35. One drop of the suspensions was added to serial dilutions (1: 1 to 1: 2048) of the antisera and normal rabbit serum. These sera were absorbed with thymus cells prior to the test or remained unabsorbed. The tubes were agitated, incubated 1 hour at 370 and left 2-3 hours at room temperature. The sedimentation pattern was then read with the naked eye and under the microscope. The reading was repeated after 12 hours and the presence of agglutinated cells was also controlled after gentle agitation. (c) Cytolytic test. Suspensions of pituitary and thymus cells, containing about 4x 106 cells/ml were prepared at 40 in Gey's solution. The cells were incubated for 30 minutes at 370 with undiluted and inactivated anti-thymus, anti-pituitary and normal rabbit serum. The cells were then washed three times and resuspended with Gey's solution to 0S5 ml.

4 314 W. Pierpaoli and E. Sorkin Fresh guinea-pig serum (0.1 or 0-2 ml) was added and the mixture incubated for 90 minutes at 370. The number of intact cells in suspensions was counted. (d) Antiglobulin test. Anti-thymus and anti-pituitary sera (pre-absorbed with thymus cells or not absorbed) were tested for the possible presence ofincomplete, non-precipitating anti-pituitary antibody. Suspensions of pituitary cells were preliminarily exposed to a 1:2 dilution of chicken serum (Mathews, 1959). After incubation for 30 minutes at 370 the pituitary cells were gently washed four times with cold Gey's solution. Sets of the packed pretreated cells were resuspended and incubated with the anti-thymus and antipituitary sera for 2 hours at 370, with occasional agitation. They were gently washed four times with cold Gey's solution and resuspended to the maximal concentration given by the pituitary cell preparation. One drop of the sensitized cell suspensions was added to 0-2 ml of undiluted sheep anti-rabbit globulin serum, and to each doubling dilution up to 1: The mixture was incubated for 10 minutes at 370 with occasional agitation. The tubes were finally centrifuged at low speed at 40, gently agitated, one drop from each tube was put on a glass slide and read under the microscope for the presence of agglutinated cells. (e) Passive haemagglutination tests for the detection of anti-growth hormone and anti-thyrotropic hormone antibodies. The passive haemagglutination technique with formalin-treated sheep erythrocytes was used. The red cells were tanned and coated either with Raben-type bovine growth hormone or with bovine thyrotropic hormone. The antibody content of rabbit anti-pituitary sera was measured. Anti-thymus sera, normal rabbit serum and high titre anti-somatotropic hormone and anti-thyrotropic hormone antisera were used as controls. Technical details are reported elsewhere (Pierpaoli and Sorkin, 1968). Histological examination of tissuesfrom mice injected with anti-pituitary serum Numerous litters of C57/BL inbred mice, days old, received one intraperitoneal injection of anti-pituitary or anti-thymus serum (0.15 ml/5 g body weight). These sera were preabsorbed with acetone powders of mouse lung, kidney and brain. Thymuses, adrenals, thyroids, lymph nodes and spleen of mice wasting after injection of antipituitary serum (Pierpaoli and Sorkin, 1967) and of control mice were removed for histological examination. Sections were stained with haematoxylin and eosin. To identify the pituitary cells which were positive in the fluorescent antibody technique, staining with acid fuchsin, which is specific for acidophilic cells, was performed. RESULTS EVIDENCE FOR THE PRESENCE OF ANTIBODY AGAINST MOUSE PITUITARY ACIDOPHILIC CELLS IN RABBIT ANTI-PITUITARY SERUM USING THE FLUORESCENT ANTIBODY TECHNIQUE Both the direct and the double layer technique showed that the rabbit anti-mouse pituitary sera or globulin preparations contained antibodies against cells of the anterior and probably of the intermediate lobe of the mouse pituitary gland (Fig. 1). The identity of the large brightly fluorescent cells was established by specific staining with acid fuchsin. These cells were recognized as acidophilic or alpha cells of the mouse anterior pituitary. Groups of small round cells resembling those of the intermediate lobe also stained strongly with fluorescein-conjugated antisera in the direct and double layer technique. These cells

5 Anti-Pituitag Serum 315 were not identified (Fig. 1). Thymus cells also showed fluorescent staining with antipituitary serum. Control tests with anti-pituitary serum which was preabsorbed three times with thymus cells still however showed strong fluorescent staining of the acidophilic cells. In contrast such a serum no longer stained thymus cells. Unabsorbed anti-thymus sera stain the pituitary cells and thymus cells. This indicates the presence of cross-reacting FIG. 1 (a), (b) and (c). Photomicrographs of mouse pituitary cells stained by the fluorescent antibody technique, double layer method. The big heavily stained cells are acidophilic cells; such cells can also be stained with acid fuchsin. The smaller fluorescent round cells (in b and c) were not identified. Bright field photomicrograph, oil immersion, x 570. anti-thymus and anti-pituitary antibodies in our antisera. However, the controls performed with anti-thymus sera which were absorbed three times with thymocytes showed that thymus-cross-reacting antibodies could be removed and that fluorescent staining of acidophilic cells disappeared in parallel. Therefore, we can conclude that rabbit anti-mouse pituitary sera contain antibody against the acidophilic cells of the mouse anterior pituitary. OTHER in vitro TESTS FOR ANTI-PITUITARY ANTIBODY The direct agglutination test, the cytolytic test and the antiglobulin test were carried out with hypophyseal cells and thymus cells and the corresponding antisera or globulin preparations. Both anti-thymus and anti-pituitary serum agglutinated thymocytes or pituitary cells to the same extent before absorption with thymus cells and failed to produce agglutination of both cell types after absorption with thymus cells. No cytolytic activity

6 316 W. Pierpaoli and E. Sorkin FIG. 2. (a) Section of the thyroid gland of a young male mouse, 10 days after a single intraperitoneal injection of rabbit anti-mouse thymus serum. Normal structure of follicles and cuboidal secretory epithelium. (b) Section of the thyroid gland of a wasting young male mouse, 10 days after a single intraperitoneal injection ofrabbit anti-mouse hypophysis serum. Accumulation ofcolloid in the distended follicles which are lined with flattened, squamous epithelium. H & E. x 70.

7 Anti-Pituitag Serum 317 was observed in anti-pituitary and anti-thymus serum. The antiglobulin test was positive with both cell types after incubation with anti-thymus or anti-pituitary serum, and negative when both sera were absorbed with thymus cells. PASSIVE HAEMAGGLUTINATION TECHNIQUE FOR DETECTION OF ANTIBODIES AGAINST HORMONES This test showed the absence of anti-thyrotropic hormone antibodies in the anti-pituitary serum while cells coated with Raben-type bovine growth hormone were agglutinated at 1: 20 to 1: 40 serum dilutions. Controls with anti-thymus serum and normal rabbit serum were negative. HISTOLOGY The histological examination of lymphoid tissues of all mice dying with the symptoms of a wasting syndrome after injection of anti-pituitary serum gave essentially the results previously described (Pierpaoli and Sorkin, 1967, 1969). Microscopic examination of adrenal glands of these mice did not reveal any evident alteration of the adrenal cortex such as hyperactivity or hypofunction, when compared with adrenals of control mice. In contrast, examination of the thyroid glands of mice dying with symptoms of a wasting syndrome revealed a clear condition of inactivity or hypofunction. The follicles were enlarged and distended by the colloid content and the secretory epithelium was flat and squamous (Fig. 2a and b), recalling the similar appearance in hypophysectomized animals (Turner, 1966). DISCUSSION Our results indicate that rabbit anti-mouse pituitary serum contains antibodies which can react with the acidophilic cells of the mouse pituitary (Fig. 1). These cells are known to produce growth hormone. The anterior lobe of the pituitary gland is composed mainly of acidophilic or alpha cells. In females, some of the acidophilic cells are mammotrophs which fluctuate in number and size in relation to the reproductive cycle, pregnancy and lactation. These cells are rarely seen in the pituitary gland of males. Other types of cells populate the anterior pituitary gland of males, but in normal physiological conditions their number is quite low in comparison to that of acidophilic cells (Yoshida, 1966). We can assume, therefore, that antibodies directed against mouse pituitary cells are to a significant extent directed against cellular constituents or secretions of acidophilic, growthhormone-producing cells. The fluorescent antibody technique has shown that acidophilic cells are stained strongly with fluorescein-conjugated anti-pituitary antiserum, this also after removal of nonspecific fluorescence or fluorescence due to cross-reacting antibodies by repeated absorptions with thymus cells. It is not certain whether our anti-mouse hypophysis antiserum contains antibodies against cytoplasmic or membrane antigens of acidophilic and other cells and also against the mouse growth hormone. It is possible that this hormone is released from the suspensions of pituitary cells which were injected into rabbits. This possibility is supported by the positive haemagglutination reaction using sheep red cells coated with bovine growth hormone. These considerations have some bearing on our previous results with anti-hypophysis serum in vivo (Pierpaoli and Sorkin, 1967). The prolonged inhibition of body growth produced by a single injection of rabbit anti-mouse pituitary serum and the effects on the

8 318 W. Pierpaoli and E. Sorkin thymus and spleen lymphoid cell population cannot be construed to be due solely if at all to an anti-growth hormone activity. Rather we suppose that the anti-hypophysis serum contains antibodies which in some way interfere with the normal activity of acidophilic cells thus producing a temporary inhibition of the synthesis of growth hormone or of its release from the acidophilic cells. Our previous experiments showed that a single injection of anti-pituitary serum into young mice can inhibit body growth for a long time and, if some of the animals do not die with symptoms of a wasting syndrome and thymus atrophy, the weight is kept astonishingly constant as in hypophysectomized animals. Recovery of these animals is very slow. This indicates that interference with the activity of acidophilic cells in young mice can produce a kind of dwarfism if a wasting syndrome does not occur. Further evidence in favour of a specific pituitary gland inhibition by anti-pituitary serum is provided by the aspect of the thyroid gland which shows marked hypofunction (Fig. 2). It is possible that this condition derives from interference with the activity of the acidophilic cells and/or basophilic cells of the pituitary. Therefore we suggest that our rabbit anti-mouse hypophysis serum probably contains specific antibodies against acidophilic cells and that the observed in vivo effects on body growth and lymphatic tissue development (Pierpaoli and Sorkin, 1967, 1969) are the result of inhibition of the physiological influence exerted by growth hormone on thymus and lymphatic tissue development during perinatal life and in the period of growth (Pierpaoli and Sorkin, 1969). ACKNOWLEDGMENTS The valuable technical assistance of Miss Laurence Schmocker is gratefully recognized. This work was supported by the Swiss National Foundation for Scientific Research, Grant No W.P. is a Research Fellow of the Italian National Research Council, Rome. ANIGSTEIN, L., RENNELS, E. G. and ANIGSTEIN, D. M. (1960). 'Inhibitory effects of antirat pituitary serum (APS) on hypophysectomized rats injected with pituitary hormones.' Acta endocr. (Kbh.), 35, 139. BEUTNER, E. H., DJANIAN, A. and WITEBsKY, E. (1964). 'Serological studies on rabbit antibodies to the rabbit anterior pituitary.' Immunology, 7, 172. COLOWICK, S. P. and KAPLAN, N. 0. (1955). Methods in Enzymology, Vol. I, p. 34. Academic Press, New York. GRABAR, P. (1969). 'Specific anti-thymocytes antibodies activities.' Symposium on: The Immune Response and its Suppression, Davos, Switzerland, March 1968 (Ed. by E. Sorkin). Karger, Basel. HOLBOROW, E. J. (1964). 'Fluorescent antibody techniques.' Immunological Methods, p Blackwell Scientific Publications, Oxford. MATHEWS, K. P. (1959). 'Adaptation ofthe antiglobulin test for use with tannic acid treated erythrocytes.' J. Immunol., 82, 279. MILGROM, F., MAIDE TUGGAC, Z. and WITEBsKY, E. (1965). 'Organ-specific antigens of liver, testicle and pituitary.'_j. Immunol., 94, 157. NAIRN, R. C. (1962). Fluorescent Protein Tracing. Livingstone, Edinburgh. PIERPAOLI, W., BARONI, C., FABRIS, N. and SORKIN, E. (1969). 'Hormones and the immunological capacity. REFERENCES II. Reconstitution of antibody production in hormonally deficient mice by somatotropic hormone, thyrotropic homone and thyroxin.' Immunology, 16, 217. PIERPAOLI, W. and SORKIN, E. (1967). 'Relationship between thymus and hypophysis.' Nature (Lond.), 215, 834. PIERPAOLI, W. and SORKIN, E. (1969). 'Effect of growth hormone and anti-growth hormone serum on the lymphatic tissue and the immune response.' Symposium on: The Immune Response and its Suppression, Davos, Switzerland, March 1968 (Ed. by E. Sorkin). Karger, Basel. PIERPAOLI, W. and SORMIN, E. (1968). 'Hormones and the immunological capacity. I. Effect ofheterologous antigrowth hormone (ASTH) antiserum on thymus and peripheral lymphatic tissue in mice. Induction of a wasting syndrome.' J. Immunol. (In press). TURNER, C. D. (1966). General Endocrinology, 4th edn. Saunders, Philadelphia. YOSHIDA, Y. (1966). 'Electron microscopy of the anterior pituitary gland under normal and different experimental conditions.' Meth. Achievm. exp. Path., (Ed. by E. Bajusz, and G. Jasmin) Vol. I, p Karger, Basel.