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Effect of chronic or cyclic exposure to estradiol on the humoral immune response and the thymus
lmmunopharmacology, 16 (1988) 45-51 Elsevier
45
IMO00413
Effect of chronic or cyclic exposure to estradiol on the humoral immune response and the thymus G r e g o r y T. E r b a c h I a n d J a n i c e M . B a h r 1'2 1Department of Physiology and ZDepartment of Animal Sciences, University of Illinois, Urbana, IL 61801, U.S.A.
(Received 9 January 1988; accepted 29 April 1988)
Abstract: Estradiol (Ez) is known to enhance humoral immunity. Previous studies examining serum immunoglobulin levels on days within the estrous cycle or in the ovariectomized, chronically E2-implanted rat have determined a positive correlation between serum E2 concentrations and antibody responsiveness. No previous study has investigated the effect of fluctuating E2 concentrations, as during the estrous cycle, throughout the course of a specific humoral immune response. Therefore, in the present study we addressed the influences of cyclic and tonic E2 exposure on the anti-fluorescein response in the rat. Ovariectomized rats were submitted to a 2 x 2 factorial design of chronic E2 implants and 4-day E2 injection (and vehicle control) regimens. Anti-fluorescein antibody production and thymic weights were measured. Animals given a 4-day cycle of E2 showed significantly (p < 0.001) greater anti-fluorescein responses than did animals given tonic or no E2 replacement. Animals which were given implants, with or without cyclic E2 injections, experienced significant (p < 0.001) thymic involution relative to those without implants. The 4-day E2 injections alone did not lead to thymic atrophy. These results indicate that while chronic E2 treatment induced thymic atrophy, cyclic exposure to E2 exerted a greater immunoenhancing influence than did tonic E2 exposure.
Key words: Estradiol; Humoral immunity; Thymic involution
Introduction
E s t r a d i o l has been f o u n d to enhance i m m u n e responsiveness (Stern a n d D a v i d s o h n , 1955; K e n n y et al., 1976; W i r a a n d Sandoe, 1980; P a a v o n e n et al., 1981; M y e r s a n d Petersen, 1985). This observ a t i o n m a y c o n t r i b u t e to an e x p l a n a t i o n o f the m e a s u r e m e n t o f higher a n t i b o d y titres in females t h a n in males (Butcher a n d C h a p m a n , 1965; Terres et al., 1968; Eidinger a n d G a r r e t t , 1972; W e i m e r and Roberts, 1972), and o f greater i m m u n e reCorrespondence." Janice M. Bahr, 102 Animal Genetics Laboratory, University of Illinois, 1301 W. Lorado Taft Drive, Urbana, IL 61801, U.S.A. Abbreviations." BSA, bovine serum albumin; E2, estradiol; FL, fluorescein; FITC, fluorescein isothiocyanate; KLH, keyhole limpet haemocyanin; MONA, multiple of normal activity; OVX, ovariectomy.
sponses in intact females t h a n in o v a r i e c t o m i z e d females ( K e n n y a n d G r a y , 1971). W e previously c o n d u c t e d an e x p e r i m e n t to d e t e r m i n e the quantitative relationship between the c o n c e n t r a t i o n o f E2 in serum a n d the specific in vivo a n t i b o d y p r o d u c tion in o v a r i e c t o m i z e d rats given chronic E2 rep l a c e m e n t by m e a n s o f s u b c u t a n e o u s i m p l a n t s (Trawick a n d Bahr, 1985). W e f o u n d a significant q u a d r a t i c relationship between anti-fluorescein (ant i - F L ) titres a n d serum E2 e x t e n d i n g into the p h a r m a c o l o g i c a l range. W i t h i n the p h y s i o l o g i c a l range, the relationship was linear, p r o v i d i n g for a d o u b l i n g o f a n t i b o d y titre with serum E 2 concentrations between 15 a n d 71 pg/ml. H o w e v e r , extrap o l a t i o n o f the i m m u n e responsiveness o f the ovariectomized, i m p l a n t e d rat to that o f the intact female rat m u s t be m a d e with caution. Physiological h o r m o n e c o n c e n t r a t i o n s a p p r o x i m a t e only one fa-
0162-3109/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
46 cet of physiological hormone replacement. In the normal, intact female, hormone levels fluctuate during the four-day estrous cycle. This condition is different from that of a chronically implanted animal and may impart some other influence on the animal's immunological status. Therefore, we designed the experiment to compare the influences of cyclic and tonic E2 exposure on the anti-FL response and thymic weight of the adult female rat.
Methods
Animals Lewis inbred female rats, aged 70 days, were purchased from Harlan-Sprague Dawley, Indianapolis, IN. They were housed in NIH approved facilities in 14L:10D and were provided Purina Rat Chow and water ad libitum. Implants Steroid implants were made from Silastic tubing, 0.95 mm i.d., 1.25 mm o.d. (Storz Instruments, St. Louis, MO). The tubing was cut into 0.5 cm lengths, filled with crystalline E2 (Sigma Chemical, St. Louis, MO) and sealed at both ends. The implants released E2 to maintain serum concentrations of 60 pg/ml (Trawick and Bahr, 1985). Surgeries Animals were ovariectomized through bilateral incisions under ketamine/acepromazine anaesthesia three weeks prior to immunization. At the time of surgery, E2 implants or sham (empty) implants were placed subcutaneously between the scapulae.
In/ections Subcutaneous injections of 0.25 ml of a 1 /~g/ml solution of E2 in sesame oil, or sesame oil alone, were administered beginning the day before immunization and every fourth day. In a preliminary study to characterize the E2 injection, ten ovariectomized rats were anaesthetized and bled for basal serum E2 assessment, then injected with the dose described above. Two rats each were bled at either 0.5, 1.0, 2.0, 3.0, 4.0, 6.0 or 8.0
h after injection. E 2 w a s measured in these samples so that a s e r u m E 2 profile could be drawn. The injection produced a n E2 surge approximately 4 11 in duration, reaching peak concentrations of 40 pg/ml above basal levels approximately 1.75 h after injection, and returning to basal levels 6 h after injection. This level and profile of Ez are similar to the endogenous E2 surge of the estrous cycle (Butcher et al., 1974). At the end of the present experiment, terminal trunk blood was collected from all animals by decapitation 1.75 h after E2 or oil injections for measurement of E2 surges, which confirmed expected levels of 40 pg/ml above basal.
Immunizations and measurements of E2 and antibody titres Fluorescein (FL) as fluorescein isothiocyanate (FITC) was covalently conjugated to keyhole limpet haemocyanin (KLH) (Sigma, St. Louis, MO) as previously described (Trawick and Bahr, 1985). Rats were immunized against F L - K L H by injecting subcutaneously 0.25 ml of a 1 mg/ml solution in water. Blood samples were obtained from the tail. Anti-FL activity in serum was determined by a specific, solid-phase enzyme-linked immunosorbent assay (Trawick et al., in preparation). Prior to the assay, serial dilutions were carried out on representative antiserum samples from each experimental group to determine the dilution at which the assay endpoint (optical density) fell on the linear portion of all the titration curves. In the assay, this dilution (1:400) of sample antiserum was added to microtitre wells previously coated with FL-conjugated bovine serum albumin. After unbound antibodies had been removed, a 1:1000 dilution of a second antiserum (horseradish peroxidase-conjugated rabbit anti-rat gamma globulin) was added. After unbound second antibodies had been removed, hydrogen peroxide was added with 2,2'-azinobis(3ethylbenzthiazoline sulfonic acid) (ABTS), which develops colour in the presence of free oxygen. The optical density of the 1:400 sample was compared to the optical densities of a complete titration of a pooled sample. Relative titres of sample antisera were determined by conversion to multiples of nor-
47 real activity (MONA) by the method of Greenfield et al. (1983). Hormone concentrations were measured by radioimmunoassay as previously described (Bahr and Ben-Jonathan, 1981). The E 2 antiserum No. 244 was obtained from G.D. Niswender, Colorado State University, Fort Collins, CO. It was prepared against 6-hemisuccinate-17#-estradiol and crossreacts 0.44% with estrone.
Experimental design Animals were randomly assigned to four treatment groups: (1) ovariectomy (OVX), empty implants and oil injections; (2) OVX, empty implants and E2 injections; (3) OVX, E2 implants and oil injections; and (4) OVX, E2 implants and E2 injections. Surgeries (OVXes and insertion of implants) were performed when rats were 70 days old (Fig. 1). Injections of E2, designed to mimic the E2 surge of proestrus, were begun three weeks after surgeries and were administered every fourth day until the conclusion of the experiment. The day following the first injection of E2, all animals were immunized against FL-KLH. Blood samples (20-40 /A) were taken 3, 6, 12, 15 and 21 days after immunization and were assayed for anti-FL activity. On day 23, tail blood samples for measurement of tonic E2 levels were taken from groups 2 and 4. One week later, terminal trunk blood was collected from all animals by decapitation 1.75 h after E2 or oil injections for measurement of E 2 surges. Also, thymi and uteri were excised and weighed.
with the use of the SAS programme (SAS Institute, 1985).
Results
A diagrammatic representation of levels and profiles of E2 replacement for each group through the course of the experiment, extrapolated from measurements made during preliminary studies and in terminal blood samples, is provided in Fig. 2. Rats given empty implants and oil injections had low serum E2 (mean 23.6 4- 1.0 pg/ml). The group having only implants had a serum E2 concentration of 56.5 + 1.1 pg/ml. The group receiving empty implants and injections of E2 had an E2 surge for 3 h every fourth day with peak levels of 60.6 + 2.6 pg/ml. The group of rats receiving both E2 implants and E2 injections experienced the same four-day hormone cycle with serum E2 concentrations between 64.6:1:3.4 and 96.8 + 4.9 pg/ml. With this model, we were able to compare the relative effects of the level and pattern of E2 exposure on the humoral immune response.
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Fig. 1. Scheduleof treatments. Arrows indicate the days E2 injections were given. Boxes indicate the days tail blood samples were taken. Immunizations were done on day 0.
Fig. 2. A diagrammatic representationof serum E2 profilesduring the course of the experimentfor each treatment group, from measurements made during preliminary studies and at the end of the experiment.The duration of the Ez surge in groups given E2 injectionsis approximately3 h. Borders of shaded areas represent means :E SEM, n = 12-14.
48 The anti-FL response for each of the treatment groups is shown in Fig. 3. The profiles of the responses of all the groups were identical. Peak MONA values were achieved on day 12, and plateau values which were not different from peak values occurred on days 15 and 21. The log phase of the response was achieved earlier by the Ez-injected groups with or without E2 implants, so that on day 6 their titres were significantly (p < 0.001) greater than those of the oil-injected groups. A significant difference (p < 0.02) between E2- and oil-injected groups was extended to day 12, when there was also a trend (p < 0.14) toward a difference between the Ez-implanted, oil-injected group and the sham-implanted, oil-injected group. Although E2 concentrations at their peaks were equivalent between the E2-implanted, oil-injected group and the sham-im-
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Fig. 4. A comparison of terminal thymic and uterine weights lbr treatment groups. Bars represent the means ± SEM. Numbers within bars indicate the numbers of rats per group. Different letters indicate significant differences for tissues within experimental groups (p < 0.001).
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Fig. 3. Anti-FL titres (MONA values) versus time after immunization. Each point represents the mean :t: SEM of 12 14 rats. p < 0.00t for comparisons made between treatments by repeated measures analysis, denoted by different letters.
planted, E2-injected group, the group exposed to a cyclic regimen of E2 showed a higher anti-FL titre (p < 0.02). Throughout the course of the humoral response, the cyclic E2 injection treatment exerted a greater (p < 0.001) immunoenhancing influence than the chronic E2 implant treatment. Terminal thymic and uterine weights are compared among treatments in Fig. 4. Injections of Ez which produced cyclic increases of 40 pg/ml above basal serum concentrations once every four days did not cause involution of thymic tissue (compared to no E 2 replacement), whereas both groups given E2 implants had marked (p < 0.001) and equivalent thymic involution. Uteri, which respond to prolonged E2 exposure with growth and proliferation, weighed more (p < 0.001) in the implanted groups.
Discussion
The results of the present experiment demonstrate that chronic and cyclic regimens of E 2 administration lead to different immunological responses. We conclude that, first, in vivo E2 replacement after OVX in rats enhances the humoral immune re-
49 sponse against FL. This immunoenhancement is not merely an effect of serum E2 concentrations, but of the pattern of E2 administration in surges which mimic those of the estrous cycle. Second, tonic Ez exposure in ovariectomized rats causes thymic involution, whereas intermittent exposure by injection of physiological levels of E2 once every four days does not. The novel findings of this study lie in the comparison of antibody titres of groups which were exposed to cyclic or tonic regimens of E 2. The groups which received E2 replacement either by implant alone or by injection alone, both had serum concentrations of E2 of approximately 60 pg/ml. Yet, of these two groups, the one exposed to a four-day cyclic regimen of E 2 displayed a significantly greater immune response against FL than did the other, which was exposed to the same serum level of E2 but in a tonic fashion. A comparison of the two groups exposed to four-day cycles of E2 surges shows that although one group had significantly higher basal and peak E2 levels than the other, both groups displayed equivalent anti-FL responses. If the strength of the immune response were dependent on E2 concentration alone, we would expect, according to our previous publication (Trawick and Bahr, 1985), the group with the higher serum E2 concentration to exhibit the stronger anti-FL titre. Rather, these results demonstrate that in this model of measuring in vivo antibody production, cyclic exposure to E2 had a stronger immunoenhancing influence than had the absolute level of exposure to the hormone. Our experiment did not specifically address the question of when during the course of the immune response the E 2 signal (surge) was necessary to produce the immunoenhancement observed in rats given E2 injections every four days. Kenny et al. (1976) showed that a single injection of E2 early in the proliferative phase of the humoral response, one day before or two days after immunization, was adequate to increase significantly the number of cells secreting antibody against heat-killed E. coli antigen in mice. In our study, rats were given surge injections the day before and three days after immunization. Though not apparent in Fig. 3, a sig-
nificant effect of the injection was detected by statistical analysis as early as day 3 post-immunization. It is possible that the very first surge of Ez was sufficient to effect the responses observed. It is also conceivable that any subsequent E2 surge influenced a later phase of the response, such as cell differentiation or antibody synthesis. Several targets for E2 within the immune system are known. E 2 receptors have been characterized and measured in T-cells of the suppressor/cytotoxic phenotype in humans (Cohen et al., 1983), and, in rats and other animals, in spleen cells (Myers et al., 1986) and cells of the epithelial matrix of the thymus (Reichman and Villee, 1978; Grossman et al., 1979). The function of E2 in the thymus is inferred to promote the release of factors which affect local or peripheral immune functions (Stimson and Crilly, 1981; Dardenne et al., 1986; Savino et al., 1988). Although the exact mechanism is not known, prolonged exposure to E2 also causes thymic involution (Chiodi, 1940; Dougherty, 1952; Sobhon and Jirasarrham, 1974). For this reason we measured thymic weights as a function of our E2 treatments. Only the animals given chronic E 2 replacement by implant displayed involution of the thymus. In contrast, animals given E 2 replacement only by injection once every four days displayed no thymic atrophy. However, it was the cyclic regimen of E2 replacement which had the greatest effect on the humoral endpoint of the immune network. We are currently conducting experiments to determine the role of the thymus in the Ez-enhancement of humoral immunity. There are conflicting data regarding the responsiveness of lymphocytes to E2. Prior studies report that addition of E2 tO cultures of T cells did not change their responsiveness to mitogens (Holdstock et al., 1982; Grossman et al., 1982; Myers et al., 1986), while serum from Ez-treated, thymus-intact animals altered the mitogenic responses of T cell cultures (Grossman et al., 1982). However, in a study of humoral immunity, Paavonen et al. (1981) observed an increase in the number of IgM-secreting cells in vitro by the addition of physiological concentrations of E2 to cultures of human peripheral blood lymphocytes. They found the primary
50 effect on a subpopulation of T cells, rather than a direct action on B cells; yet Myers and Petersen (1985) measured in Ee-treated rats an increase in antibody titres against Type llI-pneumococci polysaccharide, a T-independent antigen. Therefore, a direct effect of E2 on B cells cannot be ruled out. We conclude from the results of the present experiment that the administration of E2 in cyclic surges exerts a greater enhancing influence on the anti-FL response than does the absolute level of E2 replacement. The results show also that tonic E 2 exposure in ovariectomized rats leads to involution of the thymus, while cyclic exposure does not. Further investigation into the E2 responsiveness of the thymus and its role in humoral immupity is being undertaken currently.
Acknowledgements This work was supported by a grant from Sigma Xi. The authors thank Ms. Cheryl Green, Ms. Jennifer Marcinkiewicz and Mr. Robert Dickerman for technical assistance, Mr. Stephen Palmer for the statistical analyses, and Dr. David Trawick and Dr. Patricia Johnson for helpful criticism.
References Bahr JM, Ben-Jonathan N. Preovulatory depletion of ovarian catecholamines in the rat. Endocrinol 1981 ; 108:1815. Batchelor JR, C h a p m a n BA. The influence of sex upon the antibody response to an incompatible tumor. Immunology 1965; 9:553. Butcher RL, Collins WE, Fugo NW. Plasma concentrations of LH, FSH, prolactin, progesterone, and estradiol-17beta throughout the 4-day estrous cycle of the rat. Endocrinology 1974; 94:1704. Chiodi H. The relationship between the thymus and the sexual organs. Endocrinology 1940; 26:107. Cohen JHM, Danel L. Cordier G, Saez S, Revillard J-P. Sex steroid receptors in peripheral T cells: absence of androgen receptors and restriction of estrogen receptors to OKT8-positive cells. J Immunol I983; 131:2767. Dardenne M, Savino W, Duval D, Kaiserlian D, Hassid J, Bach FJ. Thymic hormone-containing cells. VII. Adrenals and gonads control the in vivo secretion of thymulin and its plasmatic inhibitor, ,1 lmmunol 1986:136:I 303.
Dougherty EF. Eff,ect of hormones oil lymphatic tissue. Physiol Rev 1952; 32:379. Eidinger D, Garrett TJ. Studies of the regulatory eff,ects of the sex hormones on antibody formation and stem cell differentiation. J Exp Med 1972: 136:1098. Greenfield RA, Stephens JL, Bussey M J, Jones JM. Quantitation of antibody to Candida m a n n a n by enzyme-linked irnmunosorbent assay. J Lab Clin Med 1983', 10I:758. Grossman C J, Sholiton L J. Nathan P. Rat thymic estrogen receptor: I. Preparation, location and physicochcmical properties. J Steroid Biochem 1979; 11:1233. G r o s s m a n C J, Sholiton LJ, Roselle GA. Estradiol regulation of thymic lymphocyte function in the rat: mediation by serum thymic factors. J Steroid Biochcm 1982: 16:683. Holdstock G, Chasternay BF, Krawitt EL. Effects of testosterone, oestradiol and progesterone on immune regulation. Clin Exp l m m u n o l 1982: 47:449. Kenny JF, Gray JA. Sex differences in immunological response. Studies of antibody production by individual spleen cells after stimulation with Escheriehia coli antigen. Pediatr Res 197I; 5:246. Kenny JF, Pangburn PC, Trail G. Effect ofestradiol on immune competence: In vivo and in vitro studies. Infect h n m u n 1976: 13:448. Myers M J, Butler LD, Petersen BH. Estradiol-induced alteration in the immune system, lI. Suppression ot" cellular immunity in the rat is not the result of direct estrogenic action. I m m u n o p h a r m a c o l o g y 1986; 11:47. Myers M J, Helm MC, Hirsch KS, Quenner SF. Petersen BH. Translocatable estrogen receptors in rat splenocytes. Life Sci 1986; 39:313. Myers M J, Petersen BH. Estradiol induced alterations of the immune system. I. Enhancement of lgM production, lnt J Immunopharmacol I985; 7:207. Paavonen T, Andersson LC, Adlcrcreutz H. Sex hormone regulation of the in vitro immune response. Estradiol enhances h u m a n B cell maturation via inhibition of suppressor T cells in PWM-stimulated cultures. J Exp Med 1981: 154:1935. Reichman ME, Villec CA. Estradiol binding by rat thymus cytosol. J Steroid Biochem 1978; 9:637. SAS Institute Inc. SAS User's Guide: Statistics, Version 5 Edition. Cary, NC: SAS Institute, Inc., 1985. Savino W, Bartoccioni E, Homo-Delarche F, ltoh T, Dardennc M. In vitro modulation of thymic endocrine function by steroid hormones. J Steroid Biochem 1988: in press. Sobhon P, Jirasarrham C. Effcct of sex hormones on the thymus and lymphoid tissue of ovariectomized rats. Acta Anat I974: 89:21 I. Stern K, Davidsohn I. Effect of estrogen and cortisone on immune hemoantibodies in mice of inbred strains. J lmmunol 1955: 74:479. Stilnson WH, Crilly PJ. Effects of steroids on the secretion of immunoregulatory factors by lhymic epithelial cells. Immunology 1981; 44:401. Terres G. Morrison SL, Habecht GS. A quantitative diff'erence
51 in the immune response between male and female mice. Proc Soc Exp Biol Med 1968; 127:664. Trawick DR, Bahr JM, Modulation of the primary and secondary antifluorescyl antibody response in rats by 17beta-estradiol. Endocrinology 1985; 118:2324. Voss EW. Fluorescein hapten: an immunologic probe. Boca Raton, FL: CRC Press, 1984.
Weimer H, Roberts DM. Sex differences in the immunologic and phlogistic responses to Salmonella typhosa H antigen in the albino rat. Comp Biochem Physiol [B] 1972; 47:713. Wira CR, Sandoe CP. Hormonal regulation of immunoglobins: influence of estradiol on immunoglobulins A and G in the rat uterus. Endocrinology 1980; 106:1020.
45
IMO00413
Effect of chronic or cyclic exposure to estradiol on the humoral immune response and the thymus G r e g o r y T. E r b a c h I a n d J a n i c e M . B a h r 1'2 1Department of Physiology and ZDepartment of Animal Sciences, University of Illinois, Urbana, IL 61801, U.S.A.
(Received 9 January 1988; accepted 29 April 1988)
Abstract: Estradiol (Ez) is known to enhance humoral immunity. Previous studies examining serum immunoglobulin levels on days within the estrous cycle or in the ovariectomized, chronically E2-implanted rat have determined a positive correlation between serum E2 concentrations and antibody responsiveness. No previous study has investigated the effect of fluctuating E2 concentrations, as during the estrous cycle, throughout the course of a specific humoral immune response. Therefore, in the present study we addressed the influences of cyclic and tonic E2 exposure on the anti-fluorescein response in the rat. Ovariectomized rats were submitted to a 2 x 2 factorial design of chronic E2 implants and 4-day E2 injection (and vehicle control) regimens. Anti-fluorescein antibody production and thymic weights were measured. Animals given a 4-day cycle of E2 showed significantly (p < 0.001) greater anti-fluorescein responses than did animals given tonic or no E2 replacement. Animals which were given implants, with or without cyclic E2 injections, experienced significant (p < 0.001) thymic involution relative to those without implants. The 4-day E2 injections alone did not lead to thymic atrophy. These results indicate that while chronic E2 treatment induced thymic atrophy, cyclic exposure to E2 exerted a greater immunoenhancing influence than did tonic E2 exposure.
Key words: Estradiol; Humoral immunity; Thymic involution
Introduction
E s t r a d i o l has been f o u n d to enhance i m m u n e responsiveness (Stern a n d D a v i d s o h n , 1955; K e n n y et al., 1976; W i r a a n d Sandoe, 1980; P a a v o n e n et al., 1981; M y e r s a n d Petersen, 1985). This observ a t i o n m a y c o n t r i b u t e to an e x p l a n a t i o n o f the m e a s u r e m e n t o f higher a n t i b o d y titres in females t h a n in males (Butcher a n d C h a p m a n , 1965; Terres et al., 1968; Eidinger a n d G a r r e t t , 1972; W e i m e r and Roberts, 1972), and o f greater i m m u n e reCorrespondence." Janice M. Bahr, 102 Animal Genetics Laboratory, University of Illinois, 1301 W. Lorado Taft Drive, Urbana, IL 61801, U.S.A. Abbreviations." BSA, bovine serum albumin; E2, estradiol; FL, fluorescein; FITC, fluorescein isothiocyanate; KLH, keyhole limpet haemocyanin; MONA, multiple of normal activity; OVX, ovariectomy.
sponses in intact females t h a n in o v a r i e c t o m i z e d females ( K e n n y a n d G r a y , 1971). W e previously c o n d u c t e d an e x p e r i m e n t to d e t e r m i n e the quantitative relationship between the c o n c e n t r a t i o n o f E2 in serum a n d the specific in vivo a n t i b o d y p r o d u c tion in o v a r i e c t o m i z e d rats given chronic E2 rep l a c e m e n t by m e a n s o f s u b c u t a n e o u s i m p l a n t s (Trawick a n d Bahr, 1985). W e f o u n d a significant q u a d r a t i c relationship between anti-fluorescein (ant i - F L ) titres a n d serum E2 e x t e n d i n g into the p h a r m a c o l o g i c a l range. W i t h i n the p h y s i o l o g i c a l range, the relationship was linear, p r o v i d i n g for a d o u b l i n g o f a n t i b o d y titre with serum E 2 concentrations between 15 a n d 71 pg/ml. H o w e v e r , extrap o l a t i o n o f the i m m u n e responsiveness o f the ovariectomized, i m p l a n t e d rat to that o f the intact female rat m u s t be m a d e with caution. Physiological h o r m o n e c o n c e n t r a t i o n s a p p r o x i m a t e only one fa-
0162-3109/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
46 cet of physiological hormone replacement. In the normal, intact female, hormone levels fluctuate during the four-day estrous cycle. This condition is different from that of a chronically implanted animal and may impart some other influence on the animal's immunological status. Therefore, we designed the experiment to compare the influences of cyclic and tonic E2 exposure on the anti-FL response and thymic weight of the adult female rat.
Methods
Animals Lewis inbred female rats, aged 70 days, were purchased from Harlan-Sprague Dawley, Indianapolis, IN. They were housed in NIH approved facilities in 14L:10D and were provided Purina Rat Chow and water ad libitum. Implants Steroid implants were made from Silastic tubing, 0.95 mm i.d., 1.25 mm o.d. (Storz Instruments, St. Louis, MO). The tubing was cut into 0.5 cm lengths, filled with crystalline E2 (Sigma Chemical, St. Louis, MO) and sealed at both ends. The implants released E2 to maintain serum concentrations of 60 pg/ml (Trawick and Bahr, 1985). Surgeries Animals were ovariectomized through bilateral incisions under ketamine/acepromazine anaesthesia three weeks prior to immunization. At the time of surgery, E2 implants or sham (empty) implants were placed subcutaneously between the scapulae.
In/ections Subcutaneous injections of 0.25 ml of a 1 /~g/ml solution of E2 in sesame oil, or sesame oil alone, were administered beginning the day before immunization and every fourth day. In a preliminary study to characterize the E2 injection, ten ovariectomized rats were anaesthetized and bled for basal serum E2 assessment, then injected with the dose described above. Two rats each were bled at either 0.5, 1.0, 2.0, 3.0, 4.0, 6.0 or 8.0
h after injection. E 2 w a s measured in these samples so that a s e r u m E 2 profile could be drawn. The injection produced a n E2 surge approximately 4 11 in duration, reaching peak concentrations of 40 pg/ml above basal levels approximately 1.75 h after injection, and returning to basal levels 6 h after injection. This level and profile of Ez are similar to the endogenous E2 surge of the estrous cycle (Butcher et al., 1974). At the end of the present experiment, terminal trunk blood was collected from all animals by decapitation 1.75 h after E2 or oil injections for measurement of E2 surges, which confirmed expected levels of 40 pg/ml above basal.
Immunizations and measurements of E2 and antibody titres Fluorescein (FL) as fluorescein isothiocyanate (FITC) was covalently conjugated to keyhole limpet haemocyanin (KLH) (Sigma, St. Louis, MO) as previously described (Trawick and Bahr, 1985). Rats were immunized against F L - K L H by injecting subcutaneously 0.25 ml of a 1 mg/ml solution in water. Blood samples were obtained from the tail. Anti-FL activity in serum was determined by a specific, solid-phase enzyme-linked immunosorbent assay (Trawick et al., in preparation). Prior to the assay, serial dilutions were carried out on representative antiserum samples from each experimental group to determine the dilution at which the assay endpoint (optical density) fell on the linear portion of all the titration curves. In the assay, this dilution (1:400) of sample antiserum was added to microtitre wells previously coated with FL-conjugated bovine serum albumin. After unbound antibodies had been removed, a 1:1000 dilution of a second antiserum (horseradish peroxidase-conjugated rabbit anti-rat gamma globulin) was added. After unbound second antibodies had been removed, hydrogen peroxide was added with 2,2'-azinobis(3ethylbenzthiazoline sulfonic acid) (ABTS), which develops colour in the presence of free oxygen. The optical density of the 1:400 sample was compared to the optical densities of a complete titration of a pooled sample. Relative titres of sample antisera were determined by conversion to multiples of nor-
47 real activity (MONA) by the method of Greenfield et al. (1983). Hormone concentrations were measured by radioimmunoassay as previously described (Bahr and Ben-Jonathan, 1981). The E 2 antiserum No. 244 was obtained from G.D. Niswender, Colorado State University, Fort Collins, CO. It was prepared against 6-hemisuccinate-17#-estradiol and crossreacts 0.44% with estrone.
Experimental design Animals were randomly assigned to four treatment groups: (1) ovariectomy (OVX), empty implants and oil injections; (2) OVX, empty implants and E2 injections; (3) OVX, E2 implants and oil injections; and (4) OVX, E2 implants and E2 injections. Surgeries (OVXes and insertion of implants) were performed when rats were 70 days old (Fig. 1). Injections of E2, designed to mimic the E2 surge of proestrus, were begun three weeks after surgeries and were administered every fourth day until the conclusion of the experiment. The day following the first injection of E2, all animals were immunized against FL-KLH. Blood samples (20-40 /A) were taken 3, 6, 12, 15 and 21 days after immunization and were assayed for anti-FL activity. On day 23, tail blood samples for measurement of tonic E2 levels were taken from groups 2 and 4. One week later, terminal trunk blood was collected from all animals by decapitation 1.75 h after E2 or oil injections for measurement of E 2 surges. Also, thymi and uteri were excised and weighed.
with the use of the SAS programme (SAS Institute, 1985).
Results
A diagrammatic representation of levels and profiles of E2 replacement for each group through the course of the experiment, extrapolated from measurements made during preliminary studies and in terminal blood samples, is provided in Fig. 2. Rats given empty implants and oil injections had low serum E2 (mean 23.6 4- 1.0 pg/ml). The group having only implants had a serum E2 concentration of 56.5 + 1.1 pg/ml. The group receiving empty implants and injections of E2 had an E2 surge for 3 h every fourth day with peak levels of 60.6 + 2.6 pg/ml. The group of rats receiving both E2 implants and E2 injections experienced the same four-day hormone cycle with serum E2 concentrations between 64.6:1:3.4 and 96.8 + 4.9 pg/ml. With this model, we were able to compare the relative effects of the level and pattern of E2 exposure on the humoral immune response.
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Fig. 2. A diagrammatic representationof serum E2 profilesduring the course of the experimentfor each treatment group, from measurements made during preliminary studies and at the end of the experiment.The duration of the Ez surge in groups given E2 injectionsis approximately3 h. Borders of shaded areas represent means :E SEM, n = 12-14.
48 The anti-FL response for each of the treatment groups is shown in Fig. 3. The profiles of the responses of all the groups were identical. Peak MONA values were achieved on day 12, and plateau values which were not different from peak values occurred on days 15 and 21. The log phase of the response was achieved earlier by the Ez-injected groups with or without E2 implants, so that on day 6 their titres were significantly (p < 0.001) greater than those of the oil-injected groups. A significant difference (p < 0.02) between E2- and oil-injected groups was extended to day 12, when there was also a trend (p < 0.14) toward a difference between the Ez-implanted, oil-injected group and the sham-implanted, oil-injected group. Although E2 concentrations at their peaks were equivalent between the E2-implanted, oil-injected group and the sham-im-
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Fig. 4. A comparison of terminal thymic and uterine weights lbr treatment groups. Bars represent the means ± SEM. Numbers within bars indicate the numbers of rats per group. Different letters indicate significant differences for tissues within experimental groups (p < 0.001).
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Fig. 3. Anti-FL titres (MONA values) versus time after immunization. Each point represents the mean :t: SEM of 12 14 rats. p < 0.00t for comparisons made between treatments by repeated measures analysis, denoted by different letters.
planted, E2-injected group, the group exposed to a cyclic regimen of E2 showed a higher anti-FL titre (p < 0.02). Throughout the course of the humoral response, the cyclic E2 injection treatment exerted a greater (p < 0.001) immunoenhancing influence than the chronic E2 implant treatment. Terminal thymic and uterine weights are compared among treatments in Fig. 4. Injections of Ez which produced cyclic increases of 40 pg/ml above basal serum concentrations once every four days did not cause involution of thymic tissue (compared to no E 2 replacement), whereas both groups given E2 implants had marked (p < 0.001) and equivalent thymic involution. Uteri, which respond to prolonged E2 exposure with growth and proliferation, weighed more (p < 0.001) in the implanted groups.
Discussion
The results of the present experiment demonstrate that chronic and cyclic regimens of E 2 administration lead to different immunological responses. We conclude that, first, in vivo E2 replacement after OVX in rats enhances the humoral immune re-
49 sponse against FL. This immunoenhancement is not merely an effect of serum E2 concentrations, but of the pattern of E2 administration in surges which mimic those of the estrous cycle. Second, tonic Ez exposure in ovariectomized rats causes thymic involution, whereas intermittent exposure by injection of physiological levels of E2 once every four days does not. The novel findings of this study lie in the comparison of antibody titres of groups which were exposed to cyclic or tonic regimens of E 2. The groups which received E2 replacement either by implant alone or by injection alone, both had serum concentrations of E2 of approximately 60 pg/ml. Yet, of these two groups, the one exposed to a four-day cyclic regimen of E 2 displayed a significantly greater immune response against FL than did the other, which was exposed to the same serum level of E2 but in a tonic fashion. A comparison of the two groups exposed to four-day cycles of E2 surges shows that although one group had significantly higher basal and peak E2 levels than the other, both groups displayed equivalent anti-FL responses. If the strength of the immune response were dependent on E2 concentration alone, we would expect, according to our previous publication (Trawick and Bahr, 1985), the group with the higher serum E2 concentration to exhibit the stronger anti-FL titre. Rather, these results demonstrate that in this model of measuring in vivo antibody production, cyclic exposure to E2 had a stronger immunoenhancing influence than had the absolute level of exposure to the hormone. Our experiment did not specifically address the question of when during the course of the immune response the E 2 signal (surge) was necessary to produce the immunoenhancement observed in rats given E2 injections every four days. Kenny et al. (1976) showed that a single injection of E2 early in the proliferative phase of the humoral response, one day before or two days after immunization, was adequate to increase significantly the number of cells secreting antibody against heat-killed E. coli antigen in mice. In our study, rats were given surge injections the day before and three days after immunization. Though not apparent in Fig. 3, a sig-
nificant effect of the injection was detected by statistical analysis as early as day 3 post-immunization. It is possible that the very first surge of Ez was sufficient to effect the responses observed. It is also conceivable that any subsequent E2 surge influenced a later phase of the response, such as cell differentiation or antibody synthesis. Several targets for E2 within the immune system are known. E 2 receptors have been characterized and measured in T-cells of the suppressor/cytotoxic phenotype in humans (Cohen et al., 1983), and, in rats and other animals, in spleen cells (Myers et al., 1986) and cells of the epithelial matrix of the thymus (Reichman and Villee, 1978; Grossman et al., 1979). The function of E2 in the thymus is inferred to promote the release of factors which affect local or peripheral immune functions (Stimson and Crilly, 1981; Dardenne et al., 1986; Savino et al., 1988). Although the exact mechanism is not known, prolonged exposure to E2 also causes thymic involution (Chiodi, 1940; Dougherty, 1952; Sobhon and Jirasarrham, 1974). For this reason we measured thymic weights as a function of our E2 treatments. Only the animals given chronic E 2 replacement by implant displayed involution of the thymus. In contrast, animals given E 2 replacement only by injection once every four days displayed no thymic atrophy. However, it was the cyclic regimen of E2 replacement which had the greatest effect on the humoral endpoint of the immune network. We are currently conducting experiments to determine the role of the thymus in the Ez-enhancement of humoral immunity. There are conflicting data regarding the responsiveness of lymphocytes to E2. Prior studies report that addition of E2 tO cultures of T cells did not change their responsiveness to mitogens (Holdstock et al., 1982; Grossman et al., 1982; Myers et al., 1986), while serum from Ez-treated, thymus-intact animals altered the mitogenic responses of T cell cultures (Grossman et al., 1982). However, in a study of humoral immunity, Paavonen et al. (1981) observed an increase in the number of IgM-secreting cells in vitro by the addition of physiological concentrations of E2 to cultures of human peripheral blood lymphocytes. They found the primary
50 effect on a subpopulation of T cells, rather than a direct action on B cells; yet Myers and Petersen (1985) measured in Ee-treated rats an increase in antibody titres against Type llI-pneumococci polysaccharide, a T-independent antigen. Therefore, a direct effect of E2 on B cells cannot be ruled out. We conclude from the results of the present experiment that the administration of E2 in cyclic surges exerts a greater enhancing influence on the anti-FL response than does the absolute level of E2 replacement. The results show also that tonic E 2 exposure in ovariectomized rats leads to involution of the thymus, while cyclic exposure does not. Further investigation into the E2 responsiveness of the thymus and its role in humoral immupity is being undertaken currently.
Acknowledgements This work was supported by a grant from Sigma Xi. The authors thank Ms. Cheryl Green, Ms. Jennifer Marcinkiewicz and Mr. Robert Dickerman for technical assistance, Mr. Stephen Palmer for the statistical analyses, and Dr. David Trawick and Dr. Patricia Johnson for helpful criticism.
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