American Journal of Pathology, Vol. 137, No. 5, November 1990 Copyright © American Association of Pathologists
Rapid Communication Immunocytochemical Localization of Progesterone Receptors in Endocrine Cells of the Human Pancreas Claudio Doglioni,* Marcello Gambacorta,t Giuseppe Zamboni,t Guido Coggi, and Giuseppe Vialet From the Second Department of Pathology, University of Milan School of Medicine, Milan, and the Departments of Surgical Pathology, Ospedale Civile,* Feltre, Ospedale Borgo Roma,t Verona, and Ospedale Ca' Granda,j Milan, Italy
ing assays on tissue homogenates, in pancreatic adenocarcinomas, and in fetal pancreas, whereas the existence of such receptors in normal adult pancreas has long been controversial.12-14 These studies, based on the use of receptor-binding assays, could not assess any possible restriction of steroid hormone receptor expression to specific cell types. Using autoradiographic techniques, however, selective estrogen and progesterone binding to pancreatic islet cells recently was documented in ba-
boons.15'16 Progesterone receptors (PgR) have been immunocytochemically localized in the nuclei of several (40% to 75%) endocrine cells of the human pancreas and in a more variable number of neoplastic cells of 7 of 18 endocrine pancreatic tumors. Conversely the exocrine epithelial cells of the pancreas did not exhibit any PgR immunoreactivity in normal as well as in different pathologic conditions, including pancreatic adenocarcinomas. Estrogen receptors were not detected in any of the pancreatic samples investigated. Double immunocytochemical experiments have documented that PgR immunoreactivity in normal Langerhans islets is a consistentfeature of most (75%) glucagon-producing A cells, of approximately 5% to 20% of insulin-producing B cells, and of a variable percentage ofpancreatic polypeptide (PP)-producing cells, ranging from 5% to 70%. Thesefigures were not affected by the sex, age, or underlying disease of the patients. The reportedfindings corroborateprevious clinical and experimental evidence indicating that sex steroid hormones may have some regulatory effects on thefunctional activity of the endocrinepancreas. (Am J Pathol 1990, 13 7:999-1005)
The pancreas is not usually included among the sex steroid hormone target organs, although existing clinical'-4 and experimental5-11 data indicate that steroid hormones exert some effects at least on its endocrine functions. In humans, receptors for estrogen, progesterone, and androgen hormones have been detected by ligand-bind-
Because of the availability of specific monoclonal antibodies (MAbs), it is now possible to immunocytochemically localize estrogen (ER) and progesterone receptors (PgR) in hormone-responsive cells.17-19 This prompted us to investigate the possible expression of these receptors in the human pancreatic cells, both in normal and diverse pathologic conditions. The present report documents the immunocytochemical localization of PgR in a major subpopulation of normal islet cells as well as in some insular neoplasms. Double immunocytochemical staining techniques for the simultaneous localization of PgR and islet cell hormones, chromogranin, and synaptophysin also have been performed to precisely define the nature and the functional activity of the PgR-immunoreactive cells.
Materials and Methods Tissues Pancreatic tissue samples were obtained from 95 patients undergoing surgery for different pathologic conditions, as detailed in Table 1. Fifty-eight patients were men, aged 10 to 82 years, and 37 women, aged 24 to 79 years; none of the patients was known as diabetic. Menstrual data from the 10 premenopausal women were not recorded. Accepted for publication August 22, 1990. Address for reprint requests to G. Viale, MD, Department of Pathology, Via Di Rudini 8, 20142 Milano, Italy.
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Table 1. Tissues Enclosed in the Current Investigation
Samples Normal pancreas* Heterotopic pancreast Chronic pancreatitis Pancreatic adenocarcinoma Ampullary adenocarcinoma Microcystic cystadenoma Papillary-cystic neoplasm Endocrine tumorst
Number of cases (male/female)
21 (12/9) 5 (3/2) 11 (8/3) 22 (17/5) 15 (8/7) 2 (1/1) 1 (0/1) 18 (9/9)
* Normal pancreatic tissue removed at surgery for technical reasons during gastrectomy or colectomy. Residual normal pancreas also was present in all the remaining cases under study. t Located in the stomach (two cases) or in Meckel's diverticulum (three cases). t Predominant hormone content (as revealed by immunocytochemical staining): insulin (3 cases), glucagon (3 cases), somatostatin (3 cases), gastrin (3 cases), and pancreatic polypeptide (3 cases). No pancreatic hormones were detected in the remaining three cases. Two tumors had metastasized at the time of surgery (one producing somatostatin and one lacking immunoreactivity for the different hormones).
In all the cases, formalin-fixed, paraffin-embedded samples including normal pancreatic tissue were available for immunocytochemical studies. In 25 cases (10 normal pancreas, 12 pancreatic adenocarcinomas, and 3 ampullary adenocarcinomas extending into the pancreas) frozen material also was investigated. Frozen and formalinfixed, paraffin-embedded sections of 10 breast carcinomas (with a content of ER and PgR more than 10 fmol/ mg of cytosol protein as evaluated by dextran-coated charcoal ligand-binding assays) and of a late proliferative endometrium of a 28-year-old woman served as positive controls for the immunocytochemical localization of both ER and PgR.
Immunocytochemical Staining The specificity, working dilution, and source of the antibodies used in the current investigation are detailed in Table 2.
Frozen tissue sections were immunostained for ER and PgR only, using the peroxidase-anti-peroxidase (PAP) complex staining technique.22 With the rat MAbs, the reaction was performed using all the reagents in kit form (ER-ICA and PR-ICA, Abbott Laboratories, North Chicago, IL) and following the manufacturers' instructions. Formalin-fixed, paraffin-embedded tissue sections were immunostained for ER and PgR, pancreatic hormones, chromogranin, and synaptophysin using the avidin-biotinylated peroxidase complex (ABC) method,' as previously reported.24 Double enzymatic pretreatment with trypsin and DNAse was performed before immunostaining for ER, according to Hiort et al.25 Control sections for specificity were incubated with the immunoglobulin fraction of nonimmune rat, mouse, or rabbit sera, and were constantly unstained. Double immunocytochemical staining experiments were performed on 20 formalin-fixed, paraffin-embedded tissue samples containing normal islets from both the pancreatic polypeptide (PP)-rich and the PP-poor pancreatic areas. Briefly, the immunoreactions for insulin, glucagon, PP, somatostatin, chromogranin A, and synaptophysin were performed first, on consecutive serial sections, according to the PAP staining technique and using diaminobenzidine as a chromogenic substrate to give a brown end product. On the very same sections, PgR then were immunolocalized using rat KD68 MAb, 1/200 dilutions of species-specific biotinylated anti-rat IgG antiserum (Amersham, Buckinghamshire, UK) and 1/200 dilution of alkaline phosphatase-labeled streptavidin (Dakopatts, Glostrup, Denmark). Alkaline phosphatase activity was developed with the McGadey reagent, as reported by Unger et al,26 to produce a blue end product. The results of the immunoreactions for ER and PgR were expressed as the percentage of the cells showing nuclear immunostaining in at least 10 normal Langerhans islets, or more that 2000 neoplastic cells from different areas of the endocrine neoplasms. Similarly, in double immunocytochemical experiments, the percentage of cells showing simultaneous immunoreactivity for the endocrine
Table 2. Specificity, Working Dilution, and Source of the Antibodies Used in the Study Clone or Working Reagent code number dilution Source Anti-ER (MAb) H222 Undiluted Abbott Lab. Anti-PgR (MAb)* KD68 1/5 Abbott Lab. Anti-PgR (MAb) LET126 1/50 Transbio Anti-PgR (MAb) Li417 1/25 Transbio Anti-chromogranin A (MAb) LK2H10 1/5000 Hybritech Anti-synaptophysin (MAb) Sy38 1/20 Boehringer Anti-glucagon (poly) L1813 1/10 Dakopatts Anti-insulin (poly) K512 1/5 Dakopatts Anti-somatostatin (poly) L1840 1/20 Dakopatts Anti-PP (poly) K532 1/10 Dakopatts
Ref. 17 19 18 18 20 21
ER, estrogen receptors; PgR, progesterone receptors; PP, pancreatic polypeptide; MAb, monoclonal antibody; poly, polyclonal antiserum. *
Used undiluted on frozen tissue sections.
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Figure 1. Almost all the) eoplastic cells of this control breast carcinoma show immunoreactitvity forER(A) and PgR (B, KD68AMAb). (Formalin-fixed, paraffin-embedded sections with no couniterstain, X400.)
markers and for PgR over the number of cells immunoreactive for the endocrine marker was recorded.
Results In frozen and paraffin sections of the control breast carcinomas, ER and PgR were detected in the nuclei of a variable percentage (from 40% to 100%) of the neoplastic cells (Figure 1), while in the endometrium nuclear immunostaining for ER and PgR was observed in more than 75% of the epithelial cells and in more than 60% of the stromal cells. These results were not affected by the use of the different MAbs to PgR or by the staining of frozen or paraffin sections. In all the cases under study, ER immunoreactivity was never identified on either frozen and paraffin-embedded sections. On the contrary, a definite nuclear immunoreactivity for PgR was a consistent feature of the pancreatic endocrine cells. The number of immunostained cells did not significantly differ in frozen and fixed sections of the same specimens or when different MAbs to PgR were used, although KD68 rat MAb usually resulted in more intense nuclear staining and cleaner background than obtained with the remaining MAbs. PgR immunoreactivity was confined strictly to endocrine cell subpopulations of the normal pancreas and to a variable percentage of the neoplastic cells of some endocrine pancreatic tumors. Indeed in normal pancreatic islets, including those of heterotopic pancreas, 40% to 75% of the endocrine cells showed nuclear PgR immu-
noreactivity. This was particularly intense in the peripheral islet cells, while it was weaker in the more centrally located cells (Figure 2). Double immunocytochemical stainings demonstrated that PgR-immunoreactive cells simultaneously expressed chromogranin A and synaptophysin. Furthermore these experiments allowed us to ascertain that almost 75% of the glucagon-containing A cells showed intense PgR immunoreactivity (Figure 3A), while only a minor percentage (varying from approximately 5% in most cases to almost 20% in very few islets) of the insulin-producing B cells displayed a weaker PgR immunoreactivity (Figure 3B). In the PP-poor regions, approximately 5% of the PP-immunoreactive cells showed intense nuclear immunostaining for PgR, but in the PP-rich regions this percentage increased to 70% (Figure 3C). Finally somatostatin-containing D cells lacked any PgR immunoreactivity (Figure 3D). Progesterone-receptor-immunoreactive cells were not identified outside the Langerhans islets, except for scattered extrainsular endocrine cells (Figure 2A) and occasional basal cells of larger ducts, which were simultaneously immunoreactive for PgR, chromogranin A, synaptophysin, and in the vast majority of the cases, for PP. The distribution and staining intensity of PgR-immunoreactive cells were not affected by the sex, age, preor post-menopausal status of the female patients, or by the underlying disease. Seven (2 insulinomas, 3 glucagonomas, and 2 PPomas) of the 18 endocrine pancreatic tumors showed a variable percentage (from 5% to 80% of the neoplastic cells) of PgR-immunoreactive cells (Figure 4). Weak immunoreactivity also was observed in most neoplastic cells
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Figure 2. Progesterone-receptor immunoreactivity in several islet cells and in a few extrainsular endocrine cells in a normal pancreas (A). All the immunoreactive cells showed endocrine markers in double labeling experiments. In the control section for specificity (B), no staining of the endocrine and exocrine pancreatic cells is apparent. (A: KD68 MAb, differential interference contrast optics, X250; B: differential interference contrast optics, X600.)
in the single case of papillary-cystic tumor. Progesteronereceptor-immunoreactive neoplastic cells were never identified in the pancreatic and ampullary adenocarcinomas, although scattered chromogranin- and synaptophysin-immunoreactive cells were present in three cases.
Discussion The current investigation demonstrates the occurrence of nuclear PgR immunoreactivity in a major subpopulation of normal human pancreatic endocrine cells and in a certain percentage of endocrine tumors of the pancreas as well. Because very similar results have been obtained with three different MAbs recognizing diverse epitopes on the receptor molecule,18'27'28 false-positive staining due to unwanted cross-reactivity with nuclear protein(s) unrelated to PgR is very unlikely. The endocrine nature of the immunostained cells also has been documented by their simultaneous immunoreactivity, in double-labeling experiments, for general neuroendocrine markers, as synaptophysin and chromogranin. The immunocytochemical localization of PgR in pancreatic endocrine cells has not been previously documented. After completion of this investigation, however,
report on ER and PgR immunoreactivity in both endocrine and exocrine pancreatic cells of humans and monkies appeared in abstract form only.29 At variance with the results of the latter study and of previous investigations using ligand-binding assays, we could not immunocytochemically localize ER in normal pancreatic cells. The reasons for these discrepancies may be many and possibly include the use of different MAbs to ER or the synthesis by pancreatic cells of an estrogen-binding protein in which the epitope recognized by H222 MAb is lacking or inaccessible. Although in sex steroid hormone target tissues the synthesis of PgR is considered to be induced by estradiol,V' the PgR-immunoreactive endocrine cells of the pancreas could well represent a cell population in which PgRs are constitutively synthesized in an estrogen-independent way. Other examples of human cells expressing PgR without any associated ER include the T47D breast carcinoma cell line3' and the meningiomas32; the stromal cells of late secretory endometrium have been the only normal cells so far reported to share the same phenoa
type.'3 Also our data do not support the alleged expression of sex steroid hormone receptors in normal and neoplastic cells of the exocrine pancreas, except for the single case of papillary and cystic tumor in which PgR immunoreactivity has been detected. The latter tumor type, however,
Figure 3. Double-immunostaining experiments for the simultaneous localization of PgR (KD68 MAb, blue reaction product) and pancreatic hormones (brown reaction product): A: glucagon; B: insulin; C: pancreatic polypeptide in a PP-rich area; and D: somatostatin. (Differential interference contrast optics, X400.)
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Figure 4. A: Almost all the neoplastic cells of this insulin-producing endocrine tumor exhibit nuclear immunoreactivity for PgR, as opposed to the unreactive residual exocrine tissue (top right). In the control section (B), the neoplastic cells are completely unstained. (A: KD68 MAb and hematoxylin counterstain, X250; B: no counterstain, differential interference contrast optics, X250.)
has been shown to have untrastructural and immunocytochemical features indicating an endocrine differentiation.34 Immunoreactivity for PgR of the endocrine pancreatic cells suggests that progesterone, acting through its specific receptorial protein, exerts some still poorly defined regulatory function on the secretory activity of these cells. Progesterone might play a major role in the occurrence of hyperinsulinemia in pregnant women and in women ingesting oral contraceptives,'14 as well as in the derangements of glucose homeostasis, which may complicate pregnancy.3 Indeed experimental investigations have documented that the level of progesterone binding to rat Langerhans islets, unlike estrogen binding, correlates with the rate of insulin secretion, suggesting that progesterone actually may regulate insulin release and insulinemia in pregnancy, as well as during sex steroid administration.9 Our findings, however, indicate that progesterone would preferentially bind to glucagon-secreting A cells, which exhibit the highest content of PgR. The final effects on insulin secretion, therefore, could be mediated by a paracrine regulation of the A cells on the insulin-producing B cells. Finally PgR immunoreactivity has been demonstrated in almost 40% of the endocrine tumors investigated. An immunocytochemical study on a much larger series of cases is in progress to determine any possible correlation of PgR immunoreactivity with the morphofunctional and clinical features of these tumors, with particular reference to their degree of malignancy.
References 1. Tyson JE, Felig P: Medical aspects of diabetes in pregnancy and the diabetogenic effects of oral contraceptives. Med Clin North Am 1971, 55:947-959 2. Spellacy WN, Buhi WC, Birk SA: Effects of estrogens on carbohydrate metabolism: Glucose, insulin, and growth hormone studies on 171 women ingesting premarin, mestranol, and ethinyl estradiol for six months. Am J Obstet Gynecol
1972, 114:378-392 3. Posner NA, Silverstone FA, Tobin EH: Changes in carbohydrate tolerance during long-term oral contraception. Am J Obstet Gynecol 1975, 123:119-127 4. Goldman JA: Effect of ethynodiol diacetate and a combination-type oral contraceptive compound on carbohydrate metabolism. Diabetologia 1977, 13:89-91 5. Costrini NV, Kalkhoff RK: Relative effects of pregnancy, estradiol and progesterone on plasma insulin and pancreatic insulin secretion. J Clin Invest 1971, 50:992-999 6. Howell SL, Tyhurst M, Green IC: Direct effects of progesterone on rat islets of Langerhans in vivo and in tissue culture. Diabetologia 1977, 13:579-583 7. Lenzen S: Effects of ovariectomy and treatment with progesterone or oestradiol 17B on the secretion of insulin by the perfused rat pancreas. J Endocrinol 1978, 78:153-154 8. Sufter-Dub M-Th: Effects of pregnancy and progesterone and/or oestradiol on the insulin secretion and pancreatic insulin content in the perfused rat pancreas. Diab Metab 1979, 5:47-56 9. El Seifi S, Green IC, Perrin D: Insulin release and steroidhormone binding in isolated islets of Langerhans in the rat: Effects of ovariectomy. J Endocrinol 1981, 90:59-67
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10. Lenzen S, Bailey CJ: Thyroid hormones, gonadal and adrenocortical steroids and the function of the islets of Langerhans. Endocrinol Rev 1984, 5:411-434 11. Nielsen JH: Growth and function of the pancreatic B-cell in vitro: Effects of glucose, hormones and serum factors in mouse, rat and human pancreatic islet in organ culture. Acta Endocrinol 1985, S266:1-39 12. Greenway B, lqbal MJ, Johnson PJ, Williams R: Oestrogen receptor proteins in malignant and fetal pancreas. Br Med J 1981, 283:751-753 13. Corbishley TP, lqbal MJ, Johnson PJ, Williams R: Progesterone receptors in malignant and foetal pancreas tissue. IRCS Med Sci 1984,12:575-576 14. Corbishley TP, lqbal MJ, Wilkinson ML, Williams R: Androgen receptor in human normal and malignant pancreatic tissue and cell lines. Cancer 1986, 57:1992-1995 15. Winborn WB, Sheridan PJ, McGill HC: Estrogen receptor in the islet of Langerhans of baboons. Cell Tissue Res 1983, 230:219-223 16. Winborn WB, Sheridan PJ, McGill HC: Localization of progestin receptors in the islets of Langerhans. Pancreas 1987, 2:289-294 17. Greene GL, Nolan C, Engler GP, Jensen EV: Monoclonal antibodies to human estrogen receptor. Proc Natl Acad Sci USA 1980, 77:5115-5119 18. Perrot-Applanat M, Logeat F, Groyer-Picard MT, Milgrom E: Immunocytochemical study of mammalian progesterone receptor using monoclonal antibodies. Endocrinology 1985, 116:1473-1483 19. Press MF, Greene GL: Localization of progesterone receptor with monoclonal antibodies to the human progestin receptor. Endocrinology 1988, 123:1165-1175 20. Wilson BS, Lloyd RV: Detection of chromogranin in neuroendocrine cells with a monoclonal antibody. Am J Pathol
1984,115:458-468 21. Wiedenmann B, Franke WW: Identification and localization of synaptophysin, integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell 1985, 41: 1017-1028 22. Sternberger LA, Hardy PM Jr, Cuculis JJ, Meyer MG: The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970, 18:315-333 23. Hsu S-M, Raine L, Fanger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. A com-
24.
25. 26. 27.
28.
29.
30. 31.
32. 33.
34.
35.
parison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981, 29:577-580 Doglioni C, Dell'Orto P, Coggi G, luzzolino P, Bontempini L, Viale G: Choroid plexus tumors. An immunocytochemical study with particular reference to the coexpression of intermediate filament proteins. Am J Pathol 1987,127:519-529 Hiort 0, Kwan PWL, DeLellis RA: Immunohistochemistry of estrogen receptor protein in paraffin sections. Am J Clin Pathol 1988, 90:559-563 Unger ER, Budgeon LR, Myerson D, Bngati DJ: Viral diagnosis by in situ hybridization. Description of a rapid simplified colorimetric method. Am J Surg Pathol 1986,10:1-8 Greene GL, Press MF: Immunochemical evaluation of estrogen receptor and progesterone receptor in breast cancer. In Ceriani RL, ed. Immunological Approaches to the Diagnosis and Therapy of Breast Cancer. New York, Plenum Press, 1987, pp 119-136 Vu Hai MT, Jolivet A, Ravet V, Lorenzo F, Perrot-Applanat M, Citerine M, Milgrom E: Novel monoclonal antibodies against human uterine progesterone receptor. Mapping of receptor immunogenic domains. Biochem J 1989,260:371376 Pertschuk L, Money S, Thelmo W, Carter A, Kim D, Gamaris Z, Jaffe B, Greene G: Immunocytochemical localization of estrogen and progesterone receptors in the endocrine and exocrine pancreas of man and monkey. Lab Invest 1990, 62:78 (Abstr) Leavitt WW, Chen TJ, Allen TC: Regulation of progesterone receptor formation by estrogen action. Ann NY Acad Sci 1977, 286:210-225 Horwitz KB, Mockus MB, Lessey BA: Variant T47D human breast cancer cells with high progesterone-receptor levels despite estrogen and antiestrogen resistance. Cell 1982,28: 633-642 Tilzer LL, Plapp FV, Evans JP, Stone D, Alward K: Steroid receptor proteins in human meningiomas. Cancer 1982, 49: 633-636 Bergeron C, Ferenczy A, Toft DO, Schneider W, Shyamala G: Immunocytochemical study of progesterone receptors in the human endometrium during the menstrual cycle. Lab Invest 1988, 59:862-869 Miettinen M, Partanen S, Fraki 0, Kivilaakso E: Papillary cystic tumor of the pancreas. An analysis of cellular differentiation by electron microscopy and immunohistochemistry. Am J Surg Pathol 1987, 11:855-865 Freinkel N: Of pregnancy and progeny. Diabetes 1980, 29: 1023-1035
Rapid Communication Immunocytochemical Localization of Progesterone Receptors in Endocrine Cells of the Human Pancreas Claudio Doglioni,* Marcello Gambacorta,t Giuseppe Zamboni,t Guido Coggi, and Giuseppe Vialet From the Second Department of Pathology, University of Milan School of Medicine, Milan, and the Departments of Surgical Pathology, Ospedale Civile,* Feltre, Ospedale Borgo Roma,t Verona, and Ospedale Ca' Granda,j Milan, Italy
ing assays on tissue homogenates, in pancreatic adenocarcinomas, and in fetal pancreas, whereas the existence of such receptors in normal adult pancreas has long been controversial.12-14 These studies, based on the use of receptor-binding assays, could not assess any possible restriction of steroid hormone receptor expression to specific cell types. Using autoradiographic techniques, however, selective estrogen and progesterone binding to pancreatic islet cells recently was documented in ba-
boons.15'16 Progesterone receptors (PgR) have been immunocytochemically localized in the nuclei of several (40% to 75%) endocrine cells of the human pancreas and in a more variable number of neoplastic cells of 7 of 18 endocrine pancreatic tumors. Conversely the exocrine epithelial cells of the pancreas did not exhibit any PgR immunoreactivity in normal as well as in different pathologic conditions, including pancreatic adenocarcinomas. Estrogen receptors were not detected in any of the pancreatic samples investigated. Double immunocytochemical experiments have documented that PgR immunoreactivity in normal Langerhans islets is a consistentfeature of most (75%) glucagon-producing A cells, of approximately 5% to 20% of insulin-producing B cells, and of a variable percentage ofpancreatic polypeptide (PP)-producing cells, ranging from 5% to 70%. Thesefigures were not affected by the sex, age, or underlying disease of the patients. The reportedfindings corroborateprevious clinical and experimental evidence indicating that sex steroid hormones may have some regulatory effects on thefunctional activity of the endocrinepancreas. (Am J Pathol 1990, 13 7:999-1005)
The pancreas is not usually included among the sex steroid hormone target organs, although existing clinical'-4 and experimental5-11 data indicate that steroid hormones exert some effects at least on its endocrine functions. In humans, receptors for estrogen, progesterone, and androgen hormones have been detected by ligand-bind-
Because of the availability of specific monoclonal antibodies (MAbs), it is now possible to immunocytochemically localize estrogen (ER) and progesterone receptors (PgR) in hormone-responsive cells.17-19 This prompted us to investigate the possible expression of these receptors in the human pancreatic cells, both in normal and diverse pathologic conditions. The present report documents the immunocytochemical localization of PgR in a major subpopulation of normal islet cells as well as in some insular neoplasms. Double immunocytochemical staining techniques for the simultaneous localization of PgR and islet cell hormones, chromogranin, and synaptophysin also have been performed to precisely define the nature and the functional activity of the PgR-immunoreactive cells.
Materials and Methods Tissues Pancreatic tissue samples were obtained from 95 patients undergoing surgery for different pathologic conditions, as detailed in Table 1. Fifty-eight patients were men, aged 10 to 82 years, and 37 women, aged 24 to 79 years; none of the patients was known as diabetic. Menstrual data from the 10 premenopausal women were not recorded. Accepted for publication August 22, 1990. Address for reprint requests to G. Viale, MD, Department of Pathology, Via Di Rudini 8, 20142 Milano, Italy.
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Table 1. Tissues Enclosed in the Current Investigation
Samples Normal pancreas* Heterotopic pancreast Chronic pancreatitis Pancreatic adenocarcinoma Ampullary adenocarcinoma Microcystic cystadenoma Papillary-cystic neoplasm Endocrine tumorst
Number of cases (male/female)
21 (12/9) 5 (3/2) 11 (8/3) 22 (17/5) 15 (8/7) 2 (1/1) 1 (0/1) 18 (9/9)
* Normal pancreatic tissue removed at surgery for technical reasons during gastrectomy or colectomy. Residual normal pancreas also was present in all the remaining cases under study. t Located in the stomach (two cases) or in Meckel's diverticulum (three cases). t Predominant hormone content (as revealed by immunocytochemical staining): insulin (3 cases), glucagon (3 cases), somatostatin (3 cases), gastrin (3 cases), and pancreatic polypeptide (3 cases). No pancreatic hormones were detected in the remaining three cases. Two tumors had metastasized at the time of surgery (one producing somatostatin and one lacking immunoreactivity for the different hormones).
In all the cases, formalin-fixed, paraffin-embedded samples including normal pancreatic tissue were available for immunocytochemical studies. In 25 cases (10 normal pancreas, 12 pancreatic adenocarcinomas, and 3 ampullary adenocarcinomas extending into the pancreas) frozen material also was investigated. Frozen and formalinfixed, paraffin-embedded sections of 10 breast carcinomas (with a content of ER and PgR more than 10 fmol/ mg of cytosol protein as evaluated by dextran-coated charcoal ligand-binding assays) and of a late proliferative endometrium of a 28-year-old woman served as positive controls for the immunocytochemical localization of both ER and PgR.
Immunocytochemical Staining The specificity, working dilution, and source of the antibodies used in the current investigation are detailed in Table 2.
Frozen tissue sections were immunostained for ER and PgR only, using the peroxidase-anti-peroxidase (PAP) complex staining technique.22 With the rat MAbs, the reaction was performed using all the reagents in kit form (ER-ICA and PR-ICA, Abbott Laboratories, North Chicago, IL) and following the manufacturers' instructions. Formalin-fixed, paraffin-embedded tissue sections were immunostained for ER and PgR, pancreatic hormones, chromogranin, and synaptophysin using the avidin-biotinylated peroxidase complex (ABC) method,' as previously reported.24 Double enzymatic pretreatment with trypsin and DNAse was performed before immunostaining for ER, according to Hiort et al.25 Control sections for specificity were incubated with the immunoglobulin fraction of nonimmune rat, mouse, or rabbit sera, and were constantly unstained. Double immunocytochemical staining experiments were performed on 20 formalin-fixed, paraffin-embedded tissue samples containing normal islets from both the pancreatic polypeptide (PP)-rich and the PP-poor pancreatic areas. Briefly, the immunoreactions for insulin, glucagon, PP, somatostatin, chromogranin A, and synaptophysin were performed first, on consecutive serial sections, according to the PAP staining technique and using diaminobenzidine as a chromogenic substrate to give a brown end product. On the very same sections, PgR then were immunolocalized using rat KD68 MAb, 1/200 dilutions of species-specific biotinylated anti-rat IgG antiserum (Amersham, Buckinghamshire, UK) and 1/200 dilution of alkaline phosphatase-labeled streptavidin (Dakopatts, Glostrup, Denmark). Alkaline phosphatase activity was developed with the McGadey reagent, as reported by Unger et al,26 to produce a blue end product. The results of the immunoreactions for ER and PgR were expressed as the percentage of the cells showing nuclear immunostaining in at least 10 normal Langerhans islets, or more that 2000 neoplastic cells from different areas of the endocrine neoplasms. Similarly, in double immunocytochemical experiments, the percentage of cells showing simultaneous immunoreactivity for the endocrine
Table 2. Specificity, Working Dilution, and Source of the Antibodies Used in the Study Clone or Working Reagent code number dilution Source Anti-ER (MAb) H222 Undiluted Abbott Lab. Anti-PgR (MAb)* KD68 1/5 Abbott Lab. Anti-PgR (MAb) LET126 1/50 Transbio Anti-PgR (MAb) Li417 1/25 Transbio Anti-chromogranin A (MAb) LK2H10 1/5000 Hybritech Anti-synaptophysin (MAb) Sy38 1/20 Boehringer Anti-glucagon (poly) L1813 1/10 Dakopatts Anti-insulin (poly) K512 1/5 Dakopatts Anti-somatostatin (poly) L1840 1/20 Dakopatts Anti-PP (poly) K532 1/10 Dakopatts
Ref. 17 19 18 18 20 21
ER, estrogen receptors; PgR, progesterone receptors; PP, pancreatic polypeptide; MAb, monoclonal antibody; poly, polyclonal antiserum. *
Used undiluted on frozen tissue sections.
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Figure 1. Almost all the) eoplastic cells of this control breast carcinoma show immunoreactitvity forER(A) and PgR (B, KD68AMAb). (Formalin-fixed, paraffin-embedded sections with no couniterstain, X400.)
markers and for PgR over the number of cells immunoreactive for the endocrine marker was recorded.
Results In frozen and paraffin sections of the control breast carcinomas, ER and PgR were detected in the nuclei of a variable percentage (from 40% to 100%) of the neoplastic cells (Figure 1), while in the endometrium nuclear immunostaining for ER and PgR was observed in more than 75% of the epithelial cells and in more than 60% of the stromal cells. These results were not affected by the use of the different MAbs to PgR or by the staining of frozen or paraffin sections. In all the cases under study, ER immunoreactivity was never identified on either frozen and paraffin-embedded sections. On the contrary, a definite nuclear immunoreactivity for PgR was a consistent feature of the pancreatic endocrine cells. The number of immunostained cells did not significantly differ in frozen and fixed sections of the same specimens or when different MAbs to PgR were used, although KD68 rat MAb usually resulted in more intense nuclear staining and cleaner background than obtained with the remaining MAbs. PgR immunoreactivity was confined strictly to endocrine cell subpopulations of the normal pancreas and to a variable percentage of the neoplastic cells of some endocrine pancreatic tumors. Indeed in normal pancreatic islets, including those of heterotopic pancreas, 40% to 75% of the endocrine cells showed nuclear PgR immu-
noreactivity. This was particularly intense in the peripheral islet cells, while it was weaker in the more centrally located cells (Figure 2). Double immunocytochemical stainings demonstrated that PgR-immunoreactive cells simultaneously expressed chromogranin A and synaptophysin. Furthermore these experiments allowed us to ascertain that almost 75% of the glucagon-containing A cells showed intense PgR immunoreactivity (Figure 3A), while only a minor percentage (varying from approximately 5% in most cases to almost 20% in very few islets) of the insulin-producing B cells displayed a weaker PgR immunoreactivity (Figure 3B). In the PP-poor regions, approximately 5% of the PP-immunoreactive cells showed intense nuclear immunostaining for PgR, but in the PP-rich regions this percentage increased to 70% (Figure 3C). Finally somatostatin-containing D cells lacked any PgR immunoreactivity (Figure 3D). Progesterone-receptor-immunoreactive cells were not identified outside the Langerhans islets, except for scattered extrainsular endocrine cells (Figure 2A) and occasional basal cells of larger ducts, which were simultaneously immunoreactive for PgR, chromogranin A, synaptophysin, and in the vast majority of the cases, for PP. The distribution and staining intensity of PgR-immunoreactive cells were not affected by the sex, age, preor post-menopausal status of the female patients, or by the underlying disease. Seven (2 insulinomas, 3 glucagonomas, and 2 PPomas) of the 18 endocrine pancreatic tumors showed a variable percentage (from 5% to 80% of the neoplastic cells) of PgR-immunoreactive cells (Figure 4). Weak immunoreactivity also was observed in most neoplastic cells
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Figure 2. Progesterone-receptor immunoreactivity in several islet cells and in a few extrainsular endocrine cells in a normal pancreas (A). All the immunoreactive cells showed endocrine markers in double labeling experiments. In the control section for specificity (B), no staining of the endocrine and exocrine pancreatic cells is apparent. (A: KD68 MAb, differential interference contrast optics, X250; B: differential interference contrast optics, X600.)
in the single case of papillary-cystic tumor. Progesteronereceptor-immunoreactive neoplastic cells were never identified in the pancreatic and ampullary adenocarcinomas, although scattered chromogranin- and synaptophysin-immunoreactive cells were present in three cases.
Discussion The current investigation demonstrates the occurrence of nuclear PgR immunoreactivity in a major subpopulation of normal human pancreatic endocrine cells and in a certain percentage of endocrine tumors of the pancreas as well. Because very similar results have been obtained with three different MAbs recognizing diverse epitopes on the receptor molecule,18'27'28 false-positive staining due to unwanted cross-reactivity with nuclear protein(s) unrelated to PgR is very unlikely. The endocrine nature of the immunostained cells also has been documented by their simultaneous immunoreactivity, in double-labeling experiments, for general neuroendocrine markers, as synaptophysin and chromogranin. The immunocytochemical localization of PgR in pancreatic endocrine cells has not been previously documented. After completion of this investigation, however,
report on ER and PgR immunoreactivity in both endocrine and exocrine pancreatic cells of humans and monkies appeared in abstract form only.29 At variance with the results of the latter study and of previous investigations using ligand-binding assays, we could not immunocytochemically localize ER in normal pancreatic cells. The reasons for these discrepancies may be many and possibly include the use of different MAbs to ER or the synthesis by pancreatic cells of an estrogen-binding protein in which the epitope recognized by H222 MAb is lacking or inaccessible. Although in sex steroid hormone target tissues the synthesis of PgR is considered to be induced by estradiol,V' the PgR-immunoreactive endocrine cells of the pancreas could well represent a cell population in which PgRs are constitutively synthesized in an estrogen-independent way. Other examples of human cells expressing PgR without any associated ER include the T47D breast carcinoma cell line3' and the meningiomas32; the stromal cells of late secretory endometrium have been the only normal cells so far reported to share the same phenoa
type.'3 Also our data do not support the alleged expression of sex steroid hormone receptors in normal and neoplastic cells of the exocrine pancreas, except for the single case of papillary and cystic tumor in which PgR immunoreactivity has been detected. The latter tumor type, however,
Figure 3. Double-immunostaining experiments for the simultaneous localization of PgR (KD68 MAb, blue reaction product) and pancreatic hormones (brown reaction product): A: glucagon; B: insulin; C: pancreatic polypeptide in a PP-rich area; and D: somatostatin. (Differential interference contrast optics, X400.)
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Figure 4. A: Almost all the neoplastic cells of this insulin-producing endocrine tumor exhibit nuclear immunoreactivity for PgR, as opposed to the unreactive residual exocrine tissue (top right). In the control section (B), the neoplastic cells are completely unstained. (A: KD68 MAb and hematoxylin counterstain, X250; B: no counterstain, differential interference contrast optics, X250.)
has been shown to have untrastructural and immunocytochemical features indicating an endocrine differentiation.34 Immunoreactivity for PgR of the endocrine pancreatic cells suggests that progesterone, acting through its specific receptorial protein, exerts some still poorly defined regulatory function on the secretory activity of these cells. Progesterone might play a major role in the occurrence of hyperinsulinemia in pregnant women and in women ingesting oral contraceptives,'14 as well as in the derangements of glucose homeostasis, which may complicate pregnancy.3 Indeed experimental investigations have documented that the level of progesterone binding to rat Langerhans islets, unlike estrogen binding, correlates with the rate of insulin secretion, suggesting that progesterone actually may regulate insulin release and insulinemia in pregnancy, as well as during sex steroid administration.9 Our findings, however, indicate that progesterone would preferentially bind to glucagon-secreting A cells, which exhibit the highest content of PgR. The final effects on insulin secretion, therefore, could be mediated by a paracrine regulation of the A cells on the insulin-producing B cells. Finally PgR immunoreactivity has been demonstrated in almost 40% of the endocrine tumors investigated. An immunocytochemical study on a much larger series of cases is in progress to determine any possible correlation of PgR immunoreactivity with the morphofunctional and clinical features of these tumors, with particular reference to their degree of malignancy.
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