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Occurrence of estrogen binding components in mouse thymus
Immunology Letters, 1 (1980) 293-297
© Elsevier/North-HollandBiomedicalPress
OCCURRENCE OF ESTROGEN BINDING COMPONENTS IN MOUSE THYMUS A s t u d y using fluorescence technique Terje KALLAND and John43unnar FORSBERG* Institute of Anatomy, University of Bergen, Norway and *Department of Anatomy, University of Lurid, Sweden
(Accepted 12 March 1980)
The occurrence of estrogen binding components was studied in frozen sections from different organs from adult and neonatal female mice. The sections were incubated with a fluorescein-BSA---estradiol-17/3 complex and studied in a microscope equipped for epi-illumination. A pronounced fluorescence was seen in the reticuloepithelial cells of the thymus while the thymic lymphocytes were negative. Treatment of ovariectomized females with estradiol and progesterone in combination strongly increased fluorescence. The estrogen thymolytic effect is supposed to be due to an interaction between reticuloepithelial cells and thymocytes.
4.5S estrogen binding component which could represent plasma 0t-fetoprotein contamination [ 12]. Studies on estrogen uptake, concentration and retention, were all negative. In spite of this, estrogen treatment of neonatal female mice resulted in pronounced thymic cortical necrosis [ 12]. During recent years, fluorescence methods have been introduced for studies of steroid binding. We have used a fluorescein-albumin--estradiol complex for studying estrogen binding. The purpose of this study was to look for the cellular distribution of estrogen binding components in thymus and possible differences in this aspect between neonatal and adult female mice. Moreover, we were interested in hormonal control of the binding components.
2. Introduction
3. Material and methods
1. Summary
Estrogen effects on the immune system are well documented [1-3]. High doses of estrogens are thymolytic and induce depression of cell-mediated and humeral immunity [4-6]. However, the mechanism of action of estrogens on lymphoid tissue is still unclear. In thymus of adult mice, there is retention of estradiol [7] and estradiol binding components with different characteristics have been described [8,9]. A 7-8S specific estrogen receptor has been localized to the thymic reticuloeplthelial cells while the thymocytes do not contain receptor [10,11 ]. Thymic cytosol from neonatal mice contains a * To whom correspondence should be addressedat: Departmentof Anatomy, Biskopsgatan7, S-223 62 Lund, Sweden.
Female mice, belonging to a dosed stock of the NMRI strain, were used. They were fed a standard pellet diet and given water ad libitum. Six-day-old females were killed after previous treatment with diethylstilbestrol (DES, Sigma; 5 gg in 0.025 ml olive oil subcutaneously for the ftrst 5 days after birth) or estradiol-17fl (E2, Sigma; 5/~g in 0.025 ml olive oil subcutaneously 48, 24 and 3 h before death). Adult females, 8-10-weeks-old, were ovariectomized and starting on the sixth day after the operation, were given daily subcutaneous injections of 0.1/ag E2 for 3 days and/or 0.5 mg progesterone (P, Sigma) for the same time. When combined, E2 and P, each in 0.025 ml olive off, were injected at different sites.The animals were killed 24 h after the last injection. Par293
allel experiments, including both neonatal and adult females, were always done with females injected with olive oil only. The females were killed by cervical dislocation. Parts of thymus, uterine hems, liver and skeletal muscle were rapidly dissected out and immediately frozen onto tissue holders in liquid nitrogen. The frozen tissue was sectioned at 2 tam in a Harris Wide Range Cryostat (Harris, Cambridge, Mass.) kept at --40°C. Sections were air-dried in a refrigerator (4°C) for one hour, rehydrated with a brief rinse in phosphate-buffered saline (PBS) and covered with a 17/~-estradiol(E2) 6-carboxymethyl oxime-bovine serum albumin (BSA)-fluorescein isothiocyanate (FITC) conjugate. The fluorescent estradiol conjugate was obtained from Dr. S. H. Lee, The Hospital of St. Raphael, New Haven, Conn., U.S.A. It preparation and use has been described [13,14]. After incubation with the conjugate in a humid chamber at room temperature for two hours, the sections were carefully washed in PBS for one hour, mounted in buffered glycerin and examined in a Zeiss fluorescence microscope equipped for epiillumination. Adjacent sections were stained with haematoxylin and eosin for comparison. Control sections were incubated in an identical way as test sections, except that the estradiol-conjugate was replaced by: (1)PBS; and (2)a BSA-FITC complex. Competitive experiments with estradiol were not performed for the reasons discussed by Lee [13].
4. Results Control sections, incubated with PBS or a BSAfluorescein complex did not demonstrate any fluorescence, irrespective from which of the different organs sections were obtained. No fluorescence was seen in liver sections when the E2-BSA-FITC conjugate was used. Sections from skeletal muscle contained single scattered cells in the connective tissue, which fluoresced while the muscle fibers were negative. The nature of these fluorescent cells is at present unknown. The uterine stroma was negative for fluorescence while a pronounced fluorescence occurred in the uterine epithelium. In neonatal females, the latter type was restricted to the apical part of the epithelium. Uterine epithelium from adult 294
@
Fig. 1. Sections from thymus of 6-day-oldfemales.A: from a control femaleinjected with olive oil.B: from a female injected with Ea. No difference in pattern or amount of fluorescent material. Magnification: 225 ×.
castrated females was fluorescent apically, but the fluorescence could be followed between the nuclei in the middle part of the epithelium and down to a narrow basal zone of fluorescence. The epithelial fluorescence was more pronounced in the adult compared with the neonatal female. In sections from control neonatal thymus, there was a network of fluorescent cells with non-fluorescent cells in the meshes. The latter cell type represented the lyrnphocytes while the former type had an appearance morphologically identical to the thymic reticuleepithelial cells. The cell body was oval to circular and fluorescent as were the thin cellular processes. The overall picture was the same in sections from adult, ovariectomized females. Injections of neonatal females for 5 days with 5 tag DES or injections with 5 tag E2 at 4 8 , 2 4 and 12 h before killing on day 6 did not
Fig. 2. Sections from thymus from adult females. A: ovariectomized. B: injected with E 2. C: injected with P. D: injected with E 2 plus P. No difference in fluorescence betweenA - C , but a pronounced increase in D. Magnification: 360×. 295
change the fluorescent pattern, nor the amount of fluorescent material. The same result was obtained when thymus from ovariectomized adult animals was compared with that from ovariectomized but not E2or P-injected females. A striking effect on the amount of fluorescent material was obtained when ovariectomized animals were injected with both E2 and P: the fluorescent cells and their processes were overloaded with fluorescent material; they had increased in size strikingly compared with the conditions in females given only E2 or P.
5. Discussion
As fluorescence was seen only in sections incubated with the E2-BSA-fluorescein complex and not in sections incubated with PBS or BSA-fluorescein, it is evident that the fluorescence is dependent on the E2part of the complex. Recently, Rao et al. [15] have reported specific binding of the conjugate to a uterine estrogen receptor. Thus the occurrence of fluorescence suggests the presence of E2 binding components in the tissue. Further th~ results from studies of different types of sections indicate some specificity of the E2 binding components: they could not be demonstrated in the liver sections or in muscle fibers, nor in uterine stroma. The uterine epithelium, as expected, was fluorescent. However, there results cannot be taken as definite evidence for or against the presence of estrogen binding components; it could be more a question of limitations in the method to detect below-threshold amounts of estrogen binding components. In thymus, the fluorescence was localized to the reticuloepithelial cells while the thymocytes were negative. The same pattern of distribution has earlier been described for 7 - 8 S estrogen receptors in adult rat thymus [10,11]. The type of fluorescence was the same in neonatal and adult stages. Biochemical studies, using tissue from neonatal females, has demonstrated the occurrence of an 8S estrogen receptor in uterine cytosol but a 4.5S estrogen binding component in thymus cytosol [12]. Other studies argue for the occurrence of a 4S specific estrogen receptor in thymus from adult, ovariectomized females [8]. The 4.5S neonatal thymic component had characteristics similar to c~-fetoprotein [12]. In spite of the thymocytes evidently lacking estro296
gen binding components in the neonatal stage, neonatal estrogen treatment (5 gg DES for the first 5 days after birth) results in pronounced necrosis in thymus cortical area [12]. Our results suggest that this estrogen-induced thymic involution may be mediated through some type of interaction between the reticuloepithelial cells and the thymocytes. The possibility exists that this interaction may influence differentiative processes in the thymus during a critical stage of development, resulting in the decreased and disproportionate T-cell population in the adult animal [ 16,17 ]. Moreover, the estrogen binding components of the thymus may be of importance for the modulatory effects of estrogen on the immune response [18,19]. Neither E2 nor P alone influenced the amount of fluorescent material in the reticuloepithelial cells, but a combined treatment did. The estrogen doses used in neonatal animals were considerably higher than in the adult stage because of estrogen-buffering mechanisms neonatally [20~,1]. The results of the combined E 2 plus P treatment could indicate that the production of the estrogen binding component in thymic reticuloepithelial cells is subject to progesterone control.
Acknowledgement
This investigation was supported by grants from the Norwegian Cancer Society (Landsforeningen mot Kreft).
References
[ 1] Dougherty, T. F. (1952) Physiol. Rev. 32,379. [2] Sljivi~,V. S. and Wart, G. W. (1973) Br. J. Exp. Path. 54, 69. [3] Ahlquist, J. (1976) Acta Endocr. (Kbh) (Suppl.) 206, 3. [4] Luz, N. P., Marques, M., Ayub, A. C. et al. (1969) Am. J. Obstet. Gynec. 105,525. [5] Franks, C. R., Perkins, F. T., Bishop, D. (1975) Br. J.Cancer 31,100. [6] Toivanen, P. (1967) Ann. Meal.Exp. Fenn. 45,152. [7] Seiki, K., Imanishi, Y. and Hamki, Y. (1978) Endocr. Jap. 25,290. [8] Seiki, K., Imanishi, Y., Haruki, Y. and Enomoto, T. (1979) Endocr. Jap, 26, 159. [9] Reichman, M. and ViUee,C. A. (1978) J. Steroid Biochem. 9,637. [10] Grossman,Ch. J., Sholiton, L. J. and Nathan, P. (1979) J. Steroid Biochem. 11, 1233.
[11] Grossman, Ch. J., Sholiton, L. J., Blaha, G. C. and Nathan, P. (1979) J. Steroid Biochem. 11, 1241. [12] Kalland, T., Fossberg, T. M. and Forsherg, J.-G. (1978) Exp. Molec. Path. 28, 76. [13] Lee, S. H. (1978) Am. J. Clin. Path. 70,197. [14] Lee, S. H. (1979) Cancer 44, 1. [15] Rao, B. R., Patrick, T. B., Sweet, F. (1980) Endocrinology 106,356. [16] Kalland, T. (1980) Cell. lmmunol. 50 (in print).
[17] Kalland, T. (1980) J. Immunol. 124,194. [ 18 ] Krzyck, U., Strausser, H. R., Bressler, J. P. and Goldstein, A. L. (1978) J. Immunol. 121, 1603. [ 19 ] Mathur, S., Mathur, R. S., Dowda, H., Williamson, H. D., Faulk, W. P., Fudenberg, H.H. (1978) Clin. Exp. Immunol. 33, 79. [20] Fox, T. O. (1975) Nature 258,441. [21 ] Uriel, J., Bouillon, D. and Dupiers, M. (1975) FEBS Lett. 53,305.
"297
© Elsevier/North-HollandBiomedicalPress
OCCURRENCE OF ESTROGEN BINDING COMPONENTS IN MOUSE THYMUS A s t u d y using fluorescence technique Terje KALLAND and John43unnar FORSBERG* Institute of Anatomy, University of Bergen, Norway and *Department of Anatomy, University of Lurid, Sweden
(Accepted 12 March 1980)
The occurrence of estrogen binding components was studied in frozen sections from different organs from adult and neonatal female mice. The sections were incubated with a fluorescein-BSA---estradiol-17/3 complex and studied in a microscope equipped for epi-illumination. A pronounced fluorescence was seen in the reticuloepithelial cells of the thymus while the thymic lymphocytes were negative. Treatment of ovariectomized females with estradiol and progesterone in combination strongly increased fluorescence. The estrogen thymolytic effect is supposed to be due to an interaction between reticuloepithelial cells and thymocytes.
4.5S estrogen binding component which could represent plasma 0t-fetoprotein contamination [ 12]. Studies on estrogen uptake, concentration and retention, were all negative. In spite of this, estrogen treatment of neonatal female mice resulted in pronounced thymic cortical necrosis [ 12]. During recent years, fluorescence methods have been introduced for studies of steroid binding. We have used a fluorescein-albumin--estradiol complex for studying estrogen binding. The purpose of this study was to look for the cellular distribution of estrogen binding components in thymus and possible differences in this aspect between neonatal and adult female mice. Moreover, we were interested in hormonal control of the binding components.
2. Introduction
3. Material and methods
1. Summary
Estrogen effects on the immune system are well documented [1-3]. High doses of estrogens are thymolytic and induce depression of cell-mediated and humeral immunity [4-6]. However, the mechanism of action of estrogens on lymphoid tissue is still unclear. In thymus of adult mice, there is retention of estradiol [7] and estradiol binding components with different characteristics have been described [8,9]. A 7-8S specific estrogen receptor has been localized to the thymic reticuloeplthelial cells while the thymocytes do not contain receptor [10,11 ]. Thymic cytosol from neonatal mice contains a * To whom correspondence should be addressedat: Departmentof Anatomy, Biskopsgatan7, S-223 62 Lund, Sweden.
Female mice, belonging to a dosed stock of the NMRI strain, were used. They were fed a standard pellet diet and given water ad libitum. Six-day-old females were killed after previous treatment with diethylstilbestrol (DES, Sigma; 5 gg in 0.025 ml olive oil subcutaneously for the ftrst 5 days after birth) or estradiol-17fl (E2, Sigma; 5/~g in 0.025 ml olive oil subcutaneously 48, 24 and 3 h before death). Adult females, 8-10-weeks-old, were ovariectomized and starting on the sixth day after the operation, were given daily subcutaneous injections of 0.1/ag E2 for 3 days and/or 0.5 mg progesterone (P, Sigma) for the same time. When combined, E2 and P, each in 0.025 ml olive off, were injected at different sites.The animals were killed 24 h after the last injection. Par293
allel experiments, including both neonatal and adult females, were always done with females injected with olive oil only. The females were killed by cervical dislocation. Parts of thymus, uterine hems, liver and skeletal muscle were rapidly dissected out and immediately frozen onto tissue holders in liquid nitrogen. The frozen tissue was sectioned at 2 tam in a Harris Wide Range Cryostat (Harris, Cambridge, Mass.) kept at --40°C. Sections were air-dried in a refrigerator (4°C) for one hour, rehydrated with a brief rinse in phosphate-buffered saline (PBS) and covered with a 17/~-estradiol(E2) 6-carboxymethyl oxime-bovine serum albumin (BSA)-fluorescein isothiocyanate (FITC) conjugate. The fluorescent estradiol conjugate was obtained from Dr. S. H. Lee, The Hospital of St. Raphael, New Haven, Conn., U.S.A. It preparation and use has been described [13,14]. After incubation with the conjugate in a humid chamber at room temperature for two hours, the sections were carefully washed in PBS for one hour, mounted in buffered glycerin and examined in a Zeiss fluorescence microscope equipped for epiillumination. Adjacent sections were stained with haematoxylin and eosin for comparison. Control sections were incubated in an identical way as test sections, except that the estradiol-conjugate was replaced by: (1)PBS; and (2)a BSA-FITC complex. Competitive experiments with estradiol were not performed for the reasons discussed by Lee [13].
4. Results Control sections, incubated with PBS or a BSAfluorescein complex did not demonstrate any fluorescence, irrespective from which of the different organs sections were obtained. No fluorescence was seen in liver sections when the E2-BSA-FITC conjugate was used. Sections from skeletal muscle contained single scattered cells in the connective tissue, which fluoresced while the muscle fibers were negative. The nature of these fluorescent cells is at present unknown. The uterine stroma was negative for fluorescence while a pronounced fluorescence occurred in the uterine epithelium. In neonatal females, the latter type was restricted to the apical part of the epithelium. Uterine epithelium from adult 294
@
Fig. 1. Sections from thymus of 6-day-oldfemales.A: from a control femaleinjected with olive oil.B: from a female injected with Ea. No difference in pattern or amount of fluorescent material. Magnification: 225 ×.
castrated females was fluorescent apically, but the fluorescence could be followed between the nuclei in the middle part of the epithelium and down to a narrow basal zone of fluorescence. The epithelial fluorescence was more pronounced in the adult compared with the neonatal female. In sections from control neonatal thymus, there was a network of fluorescent cells with non-fluorescent cells in the meshes. The latter cell type represented the lyrnphocytes while the former type had an appearance morphologically identical to the thymic reticuleepithelial cells. The cell body was oval to circular and fluorescent as were the thin cellular processes. The overall picture was the same in sections from adult, ovariectomized females. Injections of neonatal females for 5 days with 5 tag DES or injections with 5 tag E2 at 4 8 , 2 4 and 12 h before killing on day 6 did not
Fig. 2. Sections from thymus from adult females. A: ovariectomized. B: injected with E 2. C: injected with P. D: injected with E 2 plus P. No difference in fluorescence betweenA - C , but a pronounced increase in D. Magnification: 360×. 295
change the fluorescent pattern, nor the amount of fluorescent material. The same result was obtained when thymus from ovariectomized adult animals was compared with that from ovariectomized but not E2or P-injected females. A striking effect on the amount of fluorescent material was obtained when ovariectomized animals were injected with both E2 and P: the fluorescent cells and their processes were overloaded with fluorescent material; they had increased in size strikingly compared with the conditions in females given only E2 or P.
5. Discussion
As fluorescence was seen only in sections incubated with the E2-BSA-fluorescein complex and not in sections incubated with PBS or BSA-fluorescein, it is evident that the fluorescence is dependent on the E2part of the complex. Recently, Rao et al. [15] have reported specific binding of the conjugate to a uterine estrogen receptor. Thus the occurrence of fluorescence suggests the presence of E2 binding components in the tissue. Further th~ results from studies of different types of sections indicate some specificity of the E2 binding components: they could not be demonstrated in the liver sections or in muscle fibers, nor in uterine stroma. The uterine epithelium, as expected, was fluorescent. However, there results cannot be taken as definite evidence for or against the presence of estrogen binding components; it could be more a question of limitations in the method to detect below-threshold amounts of estrogen binding components. In thymus, the fluorescence was localized to the reticuloepithelial cells while the thymocytes were negative. The same pattern of distribution has earlier been described for 7 - 8 S estrogen receptors in adult rat thymus [10,11]. The type of fluorescence was the same in neonatal and adult stages. Biochemical studies, using tissue from neonatal females, has demonstrated the occurrence of an 8S estrogen receptor in uterine cytosol but a 4.5S estrogen binding component in thymus cytosol [12]. Other studies argue for the occurrence of a 4S specific estrogen receptor in thymus from adult, ovariectomized females [8]. The 4.5S neonatal thymic component had characteristics similar to c~-fetoprotein [12]. In spite of the thymocytes evidently lacking estro296
gen binding components in the neonatal stage, neonatal estrogen treatment (5 gg DES for the first 5 days after birth) results in pronounced necrosis in thymus cortical area [12]. Our results suggest that this estrogen-induced thymic involution may be mediated through some type of interaction between the reticuloepithelial cells and the thymocytes. The possibility exists that this interaction may influence differentiative processes in the thymus during a critical stage of development, resulting in the decreased and disproportionate T-cell population in the adult animal [ 16,17 ]. Moreover, the estrogen binding components of the thymus may be of importance for the modulatory effects of estrogen on the immune response [18,19]. Neither E2 nor P alone influenced the amount of fluorescent material in the reticuloepithelial cells, but a combined treatment did. The estrogen doses used in neonatal animals were considerably higher than in the adult stage because of estrogen-buffering mechanisms neonatally [20~,1]. The results of the combined E 2 plus P treatment could indicate that the production of the estrogen binding component in thymic reticuloepithelial cells is subject to progesterone control.
Acknowledgement
This investigation was supported by grants from the Norwegian Cancer Society (Landsforeningen mot Kreft).
References
[ 1] Dougherty, T. F. (1952) Physiol. Rev. 32,379. [2] Sljivi~,V. S. and Wart, G. W. (1973) Br. J. Exp. Path. 54, 69. [3] Ahlquist, J. (1976) Acta Endocr. (Kbh) (Suppl.) 206, 3. [4] Luz, N. P., Marques, M., Ayub, A. C. et al. (1969) Am. J. Obstet. Gynec. 105,525. [5] Franks, C. R., Perkins, F. T., Bishop, D. (1975) Br. J.Cancer 31,100. [6] Toivanen, P. (1967) Ann. Meal.Exp. Fenn. 45,152. [7] Seiki, K., Imanishi, Y. and Hamki, Y. (1978) Endocr. Jap. 25,290. [8] Seiki, K., Imanishi, Y., Haruki, Y. and Enomoto, T. (1979) Endocr. Jap, 26, 159. [9] Reichman, M. and ViUee,C. A. (1978) J. Steroid Biochem. 9,637. [10] Grossman,Ch. J., Sholiton, L. J. and Nathan, P. (1979) J. Steroid Biochem. 11, 1233.
[11] Grossman, Ch. J., Sholiton, L. J., Blaha, G. C. and Nathan, P. (1979) J. Steroid Biochem. 11, 1241. [12] Kalland, T., Fossberg, T. M. and Forsherg, J.-G. (1978) Exp. Molec. Path. 28, 76. [13] Lee, S. H. (1978) Am. J. Clin. Path. 70,197. [14] Lee, S. H. (1979) Cancer 44, 1. [15] Rao, B. R., Patrick, T. B., Sweet, F. (1980) Endocrinology 106,356. [16] Kalland, T. (1980) Cell. lmmunol. 50 (in print).
[17] Kalland, T. (1980) J. Immunol. 124,194. [ 18 ] Krzyck, U., Strausser, H. R., Bressler, J. P. and Goldstein, A. L. (1978) J. Immunol. 121, 1603. [ 19 ] Mathur, S., Mathur, R. S., Dowda, H., Williamson, H. D., Faulk, W. P., Fudenberg, H.H. (1978) Clin. Exp. Immunol. 33, 79. [20] Fox, T. O. (1975) Nature 258,441. [21 ] Uriel, J., Bouillon, D. and Dupiers, M. (1975) FEBS Lett. 53,305.
"297