Histochemical demonstration of steroid hormone binding sites in the lung

Journal oJ Steroid Biochemwry. Vol. 13, pp. I I21 to 1124 Pergamon Press Ltd 1980. Printed in Great Britain

HISTOCHEMICAL DEMONSTRATION OF STEROID HORMONE BINDING SITES IN THE LUNG LOUIS P. F'ERTSCHUK, MARY F. A. C. DI MAIO*and ERIC GAETJENS Department of Pathology and the School of Medicine*, State University of New York, Downstate Medical Center, Brooklyn, NY 11203, U.S.A. (Received 9 January 1980) SUMMARY Steroid hormone binding sites were localized anatomically by histochemical techniques utilizing steroid covalently bound to bovine serum albumin labeled with fluorescein isothiocyanate. In a study of lungs from 64 rats, specific binding was limited to the lining epithelium of the respiratory tract. Similar findings were present in other species including man. These results suggest that steroids may modulate growth and differentiation of bronchial epithelium, bronchial secretion, and pulmonary cleansing.

INTRODUCTION The presence of sex steroid hormone receptors has been demonstrated in the lungs of several adult and

fetal animals through biochemical methods [l-4]. However, biochemical steroid receptor analyses, with the exception of autoradiography, necessitate mechanical disruption of the tissue and cell fractionation. This process results in cytosol and nuclear fractions of cellular origin permitting multiple, pulmonary, quantification but the actual anatomical location and tissue distribution of the steroid hormone receptor proteins cannot be appreciated. This laboratory has applied fluorescent histochemical techniques to visually detect and localize estrogen, progesterone and androgen binding sites in intact rat lung tissue sections. MATERIALS AND METHODS

Lung tissue was obtained from 64 Sprague-Dawley and Stanley-Gumbreck rats. Thirty-nine were adults who varied with regard to their hormonal status. Included were pregnant animals, male and female castrates and rats with testicular feminization syndrome (TFM). A select number of the castrated females, males and TFM were estrogen or androgen primed respectively. In addition 25 immature rats ranging in age from 3 weeks gestation to 6 weeks postnatal were also studied. After each animal was sacrificed, the lung tissue was immediately frozen in liquid nitrogen. The histochemical method for steroid hormone binding analysis utilized ligand-conjugates consisting of 17gestradiol hemisuccinate, 1la-hydroxyprogesterone hemisuccinate or 17fi-testosterone hemisuccinate covalently bound to bovine serum albumin (BSA) labeled with fluorescein isothiocyanate (FITC) [S]. Correspondence to Dr Pertschuk, Box 25, Downstate Medical Center, SUNY, 450 Clarkson Avenue, Brooklyn, NY 11203, U.S.A.

The respective ligand-conjugates in a concentration of 50pmol were then applied to frozen sections, 4 pm thick, mounted on gelatin coated slides. Parallel hematoxylin and eosin stained sections were prepared as an aid to structural orientation [6]. In order to ensure the specificity of each ligandconjugate, competition binding studies with CI-628, R5020 and cyproterone acetate or MK316 were routinely employed [S]. A control of BSA covalently bound to FITC, but not to steroid was used to monitor non-specific binding. Slides were incubated in a room temperature humidifying chamber for 2 hr. This was followed by a 10 min fixation in acetone-ethanol and three lo-min washes in phosphate buffered saline (PBS). Buffered glycerol and coverslips were applied to the processed slides. Examination was performed by incident light ultraviolet microscopy. Cytoplasmic or nuclear fluorescence was interpreted as positive demonstration of binding and the degree of staining intensity assessed by gross visual observation. However, it was necessary that coincubation with the respective competitor result in diminished fluorescence as evidence of ligand conjugate specificity. It was also required that the BSA-FITC controls show no evidence of staining. In addition the controls served as a measure of non-specific background binding against which specific fluorescent intensity could be graded. These criteria had to be fulfilled before positive identification of steroid hormone binding could be accepted. RESULTS Specific fluorescent staining was confined to the epithelium lining the intrapulmonary bronchi and bronchioles with occasional perilumenal accentuation (Fig. 1). In most positive specimens, the entire bronchial/bronchiolar lining was fluorescent but in a few instances there was only focal uptake of the ligand-conjugate. Non-specific uptake of the ligand-

1121

LOUIS P. PERTSCHUK et al.

Fig. 1. Adult rat lung after incubation in 50 pmol of estradiol ligand-conjugate. nuclear binding limited to the bronchial lining epithelium. Fluorescence

conjugate by histiocytes was observed infrequently. Coincubation with the competitors decreased epithelial fluorescence but not histiocytic fluorescence, and BSA-FITC binding never appeared in the epithelium but was seen in histiocytes. In general the intracellular distribution of fluorescence was cytoplasmic but occasional nuclear ligand binding was apparent. In prenatal life specific ligand binding was found in 78% of the specimens and was totally limited to the cytoplasm. During post-natal development, binding could be demonstrated in only 25% of the animals and was either cytoplasmic or nuclear. Eighty percent of adult rats were positive for the presence of steroid binding, often displaying a mixture of nuclear and cytoplasmic staining. The overall results are summarized in Table 1. The degree of fluorescent intensity varied between specimens and often within the same specimen. This was most evident in castrated and TFM animals and

There is cytoplasmic microscopy x 100.

and

those treated with steroids. The intensity escence was greater in the former cases. DISCUSSION

Another technique that permits localization of steroid binding sites in intact tissue is autoradiography. However, this method is tedious and time consuming and requires special technology [7]. In addition difficulty in obtaining a satisfactory background is often encountered and may possibly contribute to imprecise localization. Fluorescent histochemical assay may be completed within a few hours, and involves a simple methodology requiring equipment normally found in histopathology laboratories. High background staining is seldom a problem (Fig. 1). The major disadvantage that histochemistry presents at this time is difficulty in quantification. Advantages of the method include a precise, visual,

Table 1. Cytoplasmic and nuclear steroid hormone ligand binding sites in rat lung by histochemistry Age 21 Days gestation 1442 Days neonatal 6-8 Months adult

of fluor-

Number positive Progesterone Testosterone

Number studied

Estradiol

9

6

4

5

787;

16

3

0

2

25%

39

26

28

28

80%

% Positive

Steroid hormone binding sites in lung localization of bound steroid hormone, subcellular localization to nucleus or cytoplasm, avoidance of contamination by other tissue types present in the specimen, as well as by steroid binding globulin fractions. The fluorescent histochemical method implemented in this study for the detection of steroid hormone binding sites has been previously applied to an extensive array of human and animal tissues. Guinea-pig, rabbit, rat and ferret were among the animal species examined and representative sections of most organs both human and animal were taken [SJ. Histochemical tissue analyses have only demonstrated steroid binding where receptor positivity has previously been determined biochemically. The specificity of each ligand-conjugate was assessed by extensive competition binding studies with multiple steroidal and anti-steroidal agents. A substantial decrease in fluorescence was evidence of competition for the binding site. At the concentrations employed in this survey, only estrogens and anti-estrogens competed with the estradiol ligandconjugate, androgens and anti-androgens with the testosterone ligand-conjugate, and progestins with the progesterone ligand-conjugate [8]. Of importance was the high correlation of histochemica1 assay results of breast and prostate tumors with parallel dextran-coated charcoal (DCC) and sucrose gradient assays (SGA). In the latter estrogen receptors correlated in 927; of 314 cancers [9], progesterone in 86% of 86 neoplasms [9], and androgen receptors in 89% of 54 specimens [6]. In this present study fluorescent histochemistry demonstrated estrogen, androgen and progesterone binding sites to be almost exclusively bronchial in rat lung. Althou~ the role of steroid binding sites in the lung has not been elucidated, their presence in the bronchi of the majority of neonates studied suggests a role in pulmonary differentiation and development. Their presence in bronchial epithelium of adults may imply a continuing role of sex steroids in pulmonary function. The abrupt post-partum drop of endogenous maternal hormones might be a significant factor in the lower percentage of neonates demonstrating binding sites (Table I). Pasqualini et aI.[2,3] using SGA, DCC and autoradiography, detected estrogen receptors in the lungs of guinea-pigs ranging from 34 days gestation to 4 weeks post-partum. The general trend was a progressive increase in cytoplasmic binding during fetal development which continued into the newborn period. Although there was an accompanying increase in nuclear binding with increasing fetal age, a continuous decline was found post-partum. Diminished steroid binding noted histochemically in rat neonates is at variance with Pasqualini’s results. This may be due to a species difference, study of an insufficient number of neonates by fluorescent microscopy, or vbidely divergent methodologies. Morishige et a!.[lO] detected cytosol and nuclear morphological

1123

androgen and estrogen receptors in neonatal, pubertal, adult and castrated rats utilizing SGA, protamine sulfate and thin layer chromatography. Their results indicated that the level of the receptors was influenced by the age, sex and hormonal status of the animal. Receptor concentration appeared to be related to the level of the endogenous hormones, being increased in castrated-adrenalectomized males. The histochemical findings agree with Morishige’s results with regard to the demonstration of androgen and estrogen binding. In addition to cytoplasmic binding nuclear staining was also seen perhaps indicative of an ongoing process of nuclear translocation of receptor. There was a substantial increase in fluorescence noted in castrates. Granberg and Ballard[l l] demonstrated glucocorticoid binding by L-2 cell lines whose origin appears to be the alveolar Type II epithelial cell of the adult rat lung. It was postulated that in fetal development glucocorticoids stimulated the production of surfactant through a direct effect on the Type II cell or via the production of an oligopeptide intermediate by another cell type [ 121. Abdul-Karim et a/.[131 found evidence that estradiol-17fl causes the dilation of pulmonary vessefs in the neonate rabbit which the authors believed to be mediated through acetycholine. Nenci[14,15] implementing estrogen, progesterone, and androgen immunofluorescent and immunoperoxidase methods detected steroid binding in centrioles and basal bodies in several immature rat tissues including respiratory epithelium. He concluded that steroids may influence cellular proliferation, differentiation and regulate ciliogenesis. These findings are of interest in that the histochemical fluorescent assay revealed a perilumenal accentuation limited to bronchial lining epithelium. This would correspond to the ~stribution of basal bodies of the ciliated epitheliat cells. Boyd[16] found that ectrogens influenced ciliary activity in the buccoesophageal mucosa of frogs. If similar findings were applicable to the respiratory tract, estrogen might have an impact on the debrisclearing mechanism that ciliated epithelium provides for the lung. Swaneck ef a@173 demonstrated that estrogen and progesterone regulate the synthesis of ovalbumin and ovomucoid in chicken oviduct. Estrogen has also been shown to produce cell proliferation and differentiation of the oviduct epithelium to tubular glands. The presence of estrogen binding sites in rat bronchial epithelium, suggest that steroid hormones may control the synthesis of some proteins in the lung which contribute to the mucus or watery subphase that comprises bronchial secretions. Included in the histochemical investigation were testosterone treated and untreated TFM rats. Cytoplasmic and nuclear binding sites were evident in untreated TFM. In vivo testosterone administration did not result in a detectable decrease in fluorescence. This suggests that androgen binding sites were available but in some way defective as has been previously postulated [IS].

Louts P. PERTSCHUK et al

1124

Other species have been examined for lung steroid binding sites by histochemistry including guinea-pig, rabbit, ferret and human. In all cases specific fluorescent staining of the bronchial/bronchiolar lining epithelium was visible, and in humans binding was also seen in the bronchial glands. This may be of clinical significance since there is a correlation between onset and exacerbations of bronchial asthma and the hormonal status of some patients [19,20]. The histochemical technique may provide a relatively simple approach to the study and function of steroid hormone receptor proteins in the lung of man and experimental animals in health and disease. Acknowledgements-This study was supported in part by USPHS grants Nos CA23623 and CA25760 from the National Cancer Institute. Dr Lewis Krey, Rockefeller University, New York donated TFM tissues. Nitromifene citrate ((X-628) was a gift from Warner-Lambert/ParkeDavis, MK 316 from Merck Sharp & Dohme, and cyproterone acetate from Schering A. G. Dr Jean-Pierre Raynaud, Centre de Recherches, Roussel-UCLAF generously supplied R5020. MS E. A. Rainford and E. Jones provided technical expertise.

REFERENCES

1. Pasqualini J. R., Sumida C. and Gelly C.: Cytosol and nuclear [3-H]-oestradiol binding in the foetal tissues of guinea-pig. Acta Endocr., Copenh. 83 (1976) 81 l-828. 2. PasquaiiG J. R., Sumida C.; Gelly C., Nguyen B. L. and Tardy J. : Specific binding of estrogens in different fetal tissues of guinea-pig during fetal development. Cancer Res. 38 (1978) 42464250. 3. Pasqualini J. R., Sumida C., Gelly C. and Nguyen B. L.: Specific [“-HI-estradiol binding in the fetal uterus and testis of guinea-pig. Quantitative evolution of [3H]-estradiol receptors in the different fetal tissues (kidney, lung, uterus and testis) during fetal development. J. steroid B&hem. 7 (1976) 1031-1038. 4. Dube J. Y., Chapdelaine P. and Tremblay R. R.: Further evidence for the presence of androgen receptors in the lungs and in the head appendages of the cock. Cancer J. Biochem. 55 (1977) 263-265. 5. Gaetjens E. and Pertschuk L. P.: Synthesis of fluorescent labeled-steroid hormone-albumin conjugates for the histochemical detection of hormone receptors. J. steroid Biochem. 13 (1980) (MS 891). 6. Pertschuk L. P., Zava D. T.,‘Tobin E. H., Brigati, D. J., Gaejens E., Macchia R. J., Wise G. J., Wax S. H. and Kim D. S.: Histochemical detection of steroid hormone receptors in the human prostate. In Progress in

Clinical and Biological Research. Prostcte Cancer and Hormone Receptors (Edited by G. P. Murphy and A. A.

Sandberg). Alan R. Liss. New York, Vol. 33 (1979) pp. 113-132. 7. Stumpf W. E. and Roth L. J.: Freeze-drying of small tissue samples and thin frozen sections below -60°C. J. Histochem. Cytochem. 15 (1967) 243-51.

8. Pertschuk L. P.. Tobin E. H.. Brieati D. J. and Gaetjens E.: Morphologic assay of ste;oid hormone receptors in human neoplasia. In Pathology Annual 1980 (Edited by S. G. Sommers and P. P. Rosen). AppletonCentury-Crofts, New York, in press. 9. Pertschuk L. P., Tobin E. H., Gaetjens E., Carter A. C., Degenshein G. A., Bloom N. D., and Brigati D. J.: Histochemical assay of estrogen and progesterone receptors in breast cancer: correlation with biochemical assays and patient response to endocrine therapies. Cancer, in press. 10 Morishige W. K. and Uetake C. A.: Receptors for androgen and estrogen in the rat lung. Endocrinologg 102 (1978) 1827-1837.

11 Granberg J. and Ballard P. L.: The role of sulfbydryl groups in the binding of glucocorticoids by cytoplasmic receptors of lung and other mammalian tissues. Endocrinology 100 (1977) 116&l 168. 12. Smith B. T. and Fletcher A. W.: Pulmonary epithelialmesenchymal interactions; beyond organogenesis. Human Pathol. 10 (1979) 2488250. 13 Abdul-Karim R. W., Marshall R. D. and Nesbitt R. E.: Influence of estradiol 178 on the acetylcholine content of the lung in rat neonate. Am. J. Obstet. Gyncc. 107 (1970) 641644. 14 Nenci I.: Receptor and centriole pathways of steroid action in normal and neoplastic cells. Cancer Res. (1978) 42044211. 15 Nenci I. and Marchetti E.: Concerning the localization of steroids in centrioles and basal bodies by immunofluorescence. J. Cell Biol. 76 (1978) 255-260. 16 Boyd E.. Clark J. W. and Perry W. F.: Estrogens and their effect on ciliated mucosa. Arch. Oto[aryngol. 33 (1941) 909-915. 17 Swaneck G. E., Tsai S. Y., Tsai J. J., Nordstrom J. L.. Roop D. R. and O’Malley B. W. : The ovalbumin gene: transcriptional regulation by estrogen. In Advances in Experimental Medicine and Biology. Steroid Hormone Receptor Systems (Edited by W. W. Leavitt and J. Clark). Plenum Press, New York (1979) pp. 461485. 18 Bullock L. P. and Bardin C. W.: The presence of estrogen receptors in kidneys from normal and androgen insensitive tfm/y mice. Endocrinology 97 (1975) 1106-1111. 19. Takino M., Takino Y. and Sugahara K.: Pathogenesis und Therapy of Bronchial Asthma with Special Reference to Organ Vagotonia. University Park Press. Balti-

more (1976) pp. 1666167. 20. Crofton J. and Andrew D.: Respiratory Diseases. William Clowes, London (1975) p. 432.