Dihydrotestosterone regulation of thymocyte function in the rat—mediation by serum factors

J. steroid Biochem. Vol, 19, No. 4, pp. 1459 1467, 1983

0022-4731/83 $3.00+0.00 Copyright x 1983 Pergamon Press ktd

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D I H Y D R O T E S T O S T E R O N E R E G U L A T I O N OF T H Y M O C Y T E F U N C T I O N IN THE R A T - M E D I A T I O N BY S E R U M F A C T O R S CHARLES J. GROSSMAN*t~, LEON J. SHOLITON*§ and GARY A. ROSELLE*H• *Medical Research Service, VA Medical Center, tPhysiology Department, UC College of Medicine, ~Department of Biological Sciences, Xavier University, Cincinnati, Ohio, §Division of Endocrinology and Metabolism, Department of Internal Medicine, UC College of Medicine, Cincinnati, Ohio, ]]Infectious Diseases Section, VA Medical Center, and fDivision of Infectious Diseases, Dept. of Internal Medicine, UC College of Medicine, Cincinnati, Ohio, U.S.A. (Received 27 April 1982)

Summary---We have previously reported that reticuloepithelial cells of rat thymic tissue contain specific, high-attinity estrogen (E) and dihydrotestosterone (DHT) receptors which are not present in thymocytes, and that stimulation of the concanavalin A (Con A) sensitive thymocyte population is by means of a Ihymic serum substance depressed by estradiol. To ascertain if a serum substance modulated by DHT could also effect a thymocyte population, we incubated thymocytes in vitro for 3 days in the presence of the mitogens Con A or phytohaemagglutinin (PHA)+tissue culture media+20~i specific rat serum preparations and pulse labelled with tritiated thymidine. Rat serum samples were prepared from control, castrate, thymectomized (Tx) and castrate-Tx animals as well as from similar groups of animals treated in vitro for 3 days with physiological doses of DHT. It was found that there was a significant enhancement of thymocyte blastogenesis in cultures incubated with castrate rat serum + Con A or PHA vs control serum cultures (P < 0.001). Direct replacement of DHT either to normal or castrate sera in vitro at physiological concentrations did not alter the level of Ihymocyte blastogenesis from normal or castrate controls. Sera orepared from castrate animals treated with DHT at physiological concentrations did not depress the blastogenic response, but sera from animals treated with testosterone were successful in depressing the blastogenic response to noncastrate levels. Sera prepared from Tx animals were stimulatory on Ihymocyte blastogenesis when PHA were utilized (P < 0.02) and this stimulation was depressed in sera from animals treated in vivo with DHT. Sera prepared from Tx or castrate-Tx animals were not able to stimulate thymocyte blastogenesis when Con A was utilized but were stimulatory when prepared from castrate-Tx animals pretreated in rit,o with DHT. It is hypothesized that Con A sensitive thymocytes are depressed by a thymic serum factor under the control of E and that both Con A and PHA sensitive thymocytes are depressed by a serum factor possibly of extrathymic origin that is under the control of DHT. A tentative hypothesis for the regulation of thymocytes by gonadal steroid hormones is presented.

INTRODUCTION Disorders of the cell mediated i m m u n e system frequently result in sex related differences in the expression of the disease process. Examples of such diseases include systemic lupus e r y t h e m a t o s u s in h u m a n s [ l - 4 ] a n d N Z B / N Z W FI mice[5-8], a n d h u m a n r h e u m a t o i d arthritis[9, 10] which are pred o m i n a n t l y expressed in the female. Further, multiple sclerosis in h u m a n s [11] and b o t h h u m a n m u s c u l a r d y s t r o p h y and dystrophic disease in mice[12] are more c o m m o n l y manifested in males t h a n in females. It has also been reported t h a t the function of the cellular i m m u n e system is depressed by estradiol (E) [13 17] a n d d i h y d r o t e s t o s t e r o n e ( D H T ) [5 8] and stimulated by castration [13, 14, 18-20]. We d e m o n s t r a t e d previously that the rat thymus contains specific, high-affinity E and D H T receptors which are present only in the thymic reticuloepithelial matrix a n d not in the thymocyte [21 23]. Recently we s. 19/4

reported that thymocyte function is depressed by serum factors which have their origin in the thymus a n d that these serum factors are m o d u l a t e d by estradiol presumably functioning t h r o u g h the thymic reticuloepithelial E receptors [24]. Since reticulo- epithelial D H T receptors are also present in thymic tissue it appears logical that D H T could also regulate the i m m u n e response. The objective of the present study, therefore, was to ascertain if a n d r o g e n could m o d u l a t e the cell-mediated i m m u n e system a n d if this effect is mediated by thymic serum factors. METHODS Animal mode/

Male H o l t z m a n rats were o b t a i n e d from Harlen Industries of Indiana. The animals were 1-2 m o n t h s old a n d weighed approx. 150 g at the time of use. The rats were m a i n t a i n e d on a s t a n d a r d l a b o r a t o r y diet a n d a 13:11 p h o t o p e r i o d (lights on 1500h to 1800).

1459

1460

CHARIA'SJ. GROSSMANel al.

Surgical procedures

All surgical procedures were carried out under ether anesthesia. Groups consisting of 20 animals were either castrated, adrenalectomized or thymectomized 3 weeks prior to their use [24]. If more than one surgical treatment was used, 2 weeks were allowed between procedures, For all procedures adequate sham-operated controls were employed. Steroid treatment

For certain studies experimental animals (20 per group) were surgically prepared and these animals, along with intact controls, were treated daily with 151~g of DHT or T prepared by dissolving 50#g of the steroid in 1 ml of corn oil. This dosage was chosen because from previous studies it was shown that this concentration would produce an immunological effect in vivo [24, 26] and also was adequate to translocate thymic reticuloepithelial steroid receptor from the cytoplasm into the nucleus [22, 27]. Preparation ~/ thynw~3'tes

Thymi were removed from intact male rats under sterile conditions and immediately placed in Eagle's minimal essential media with Earles salts (MEM) in Petri dishes. Thymocytes were purified as described previously [24] and after washing, the thymocytes were diluted in MEM containing 1 mM L glutamine, 100 units/ml penicillin, 100pg/ml streptomycin and 20,~; rat serum (as specified below) to a final concentration of 12.5 x 10~ cells/ml. Serum selection and cell viability

From previous studies [24] it was determined that incubation of rat thymocytes in rat serum allowed for greater than 95"~i of the cells originally placed in the culture to remain viable after 3 days' incubation as measured by Tryptan Blue dye exclusion. Preparation o[ rat serum

Sera were prepared from the various groups of rats by decapitating the animals, collecting the blood and allowing it to clot at room temperature as described previously [24]. The sera was stored at - 3 C and before use was sterilized by filtration (0.45 p filter, Millipore Corp., Bedford, MA) and heat inactivated at 56"C for 30 min in a constant temperature water bath. Blastogenic assays

Blastogenic assays were carried out in triplicate as described previously [24] in 96 well pt plates using 0.2 ml of cell suspension per well (2.5 x 105 cells). Ten id of varying concentrations of the mitogens phytohaemagglutinin-M (PHA, 1:5 and 1:2 dilutions) and concanavalin A (Con A, 2 and 5 pg) were added to the appropriate cultures. These dilutions were chosen because they consistently yielded a strong response in this assay system. After 3 days of

incubation at 37~C in a 5!!ii CO2 atmosphere, the cultures were pulse-labelled for 2h with I i~Ci (methyl-3H) thymidine (sp. act., 6 Ci/mmo[, SchwarzMann, Orangeburg, NY), The cells were then collected on glass-fiber filters using a multiple automated sample harvester (MASH II, Microbiological Products, Walkersville, MD). The amount of incorporated radioactivity was determined by scintillation counting and the results expressed as disintegrations per minute (dpm). In certain experiments, steroids were added directly to the cell cultures so that the final concentration was in the physiologic range for rat serum. These concentrations were as follows: T 2 Ng/ml [28, 29] and DItT 1 Ng/ml [30, 31] of culture medium. Controls consisted of cell cultures with all tested serum in the absence of mitogen. Statistical analysis

Blastogenic assay results were computed from three assay wells containing thymocytes from a single animal along with one dosage of a particular mitogen. The mean of the results from the three wells were then calculated to produce the actual dpm in all cases, maximum dpm/serum sample/mitogen dosage was used for statistical analysis. These maximum dpm were then combined to produce a final mean, SD and SE of the mean. Data were analyzed using the Welch test for two samples with unequal variance [32] with 0.05 as the upper level of significance. This method is utilized widely in immunological assay to eliminate biological variability between preparations. In these studies n = separate animal preparations utilized. RESULTS

D H T treatment

In order to determine if the treatment regime of the animals utilizing DHT injection in vitro resulted in systemic physiological levels, male rats were castrated. After 3 weeks different groups were injected with oil as controls, 15/~g DHT/day in oil or 301tg DHT/day in oil for 2 weeks after which time the groups were killed and prostate weights measured. As can be seen in Table 1, castrate animals injected with oil only possessed a prostate weight of 0.044 mg which was significantly less (P <0.001) than the weight of the prostate in normal (non-castrate) male rats (0.285 rag). Treatment of castrate rats with 15 ~Lg DHT resulted in a significant elevation of prostate weight (P < 0.001 I) to 0.189 mg and treatment with 30/~g DHT produced a prostate weight of 0.195 mg which was significantly greater than untreated castrates (P < 0.001) but was not significantly different from the 15/~g-treated group. While the increase in prostate weight in the DHT-treated groups was not as great as that observed in the intact control rats, it was significantly greater than the untreated castrate group and was 68°/~ of normal levels. Further, treatment of castrate male rats with 15/tg DHT for 3 days

Dihydrotestosterone regulation of thymocyte function in the rat

g r o u p 3), it did depress the in vitro blastogenic response when the mitogen P H A was utilized (Table 5; g r o u p 2 vs g r o u p 3). T r e a t m e n t of castrate rats with s u b c u t a n e o u s injections of T resulted in a loss of the ability o f the serum to stimulate thymocyte activity in assays where P H A or C o n A was added (Tables 4 and 5; group 2 vs group 4). Also, serum prepared from animals which were thymectomized was not able to stimulate thymocyte blastogenesis utilizing Con A as the mitogen (Table 4; g r o u p 1 vs group 5) but was able to produce a significant thymocyte stimulation above n o r m a l levels when the mitogen P H A was employed (Table 5; group 1 vs group 5). While this stimulus of the PHA-sensitive thymocytes in thymectomized serum was significantly a b o v e n o r m a l levels (Table 5; group 1 vs g r o u p 5) it was not as great as the stimulus o b t a i n e d in PHA-sensitive thymocytes incubated in castrate serum (Table 5: g r o u p 2 vs g r o u p 5). This stimulatory effect of thymectomized serum on P H A sensitive cells was reversed when thymectomized rats were pretreated in vivo with D H T , (Table 5: group 2 vs group 6). F o r both Con A a n d P H A sensitive cells there was no effect of castrate-thymectomized serum on blast t r a n s f o r m a t i o n a b o v e n o r m a l levels (Tables 4 and 5: g r o u p 1 vs group 7). However, for the Con A sensitive cells t r e a t m e n t o f castrate-thymectomized rats in vivo with D H T resulted in a stimulation above n o r m a l levels (Table 4; group 1 vs group 8) which was not significantly different from the stimulus obtained with castrate serum (Table 4; group 2 vs group 8).

Table 1. The effect of/n vivo treatment of long-term castrate male rats on maintenance of prostate weight Concentration DHT injected (/~g)

Prostate weight (mg) Castrate

0

0.044 + 0.0078 n=10 0.189 + 0.0067 n = 10 0.195 _+0.0067 n=10

15 30 Normal None

0.285 _-4-0.0145 n=10

Male rats were castrated and after 3 weeks were treated with various concentrations of DHT in oil or oil alone for 2 weeks after which the animals were killed and prostate weights measured. There was a significant increase in prostate weights between treated groups and castrate controls (P < 0.001). There was a significant difference in prostate weight between treated groups and noncastrate controls (P < 0.001). There was no significant difference between prostate weights of the treated groups. Significance was measured using the Student's t-test, n = separate animal preparations. X _+ SEM. results in t r a n s l o c a t i o n of D H T receptor into the nucleus o f the reticuloepithelial cells of the thymus [27]. These findings suggest that the levels of D H T utilized in this study for the in vivo t r e a t m e n t regime are within the physiological range. Thymocyte responses As has been reported previously [24] castrate serum in the presence of the mitogens C o n A or P H A produced a greater stimulation of thymocytes than did n o r m a l serum with mitogens present (Table 2). In the present study, in vitro replacement of T or D H T to castrate serum did not significantly alter the stimulatory effects o f castrate serum on thymocytes in blastogenic assay. F u r t h e r m o r e , in vitro addition o f D H T to n o r m a l serum did not result in stimulation of thymocyte blastogenesis (Table 3). However, while t r e a t m e n t of castrate animals with D H T in vivo did not significantly decrease the ability o f the serum to stimulate thymocyte blastogenesis in vitro when the mitogen C o n A was employed (Table 4; g r o u p 2 vs

In vivo dose dependem3' In an effort to ascertain if these effects observed with in vivo D H T t r e a t m e n t might be dose d e p e n d e n t we treated castrate male rats with various levels of D H T in oil for 1 week and prepared serum samples. These samples were then run in blastogenic assay with n o r m a l thymocytes. As can be seen in Fig. 1 in the presence of C o n A as mitogen increasing the D H T dosage from 15 to 1 0 0 p g did not result in any significant increase in blastogenic response that was different from untreated castrate serum. While the trend appears to suggest that 3 0 p g produced a maximum stimulation, this proved to be

Table 2. The effects of normal vs castrate serum on normal thymocytes with or without the addition of the mitogens concanavalin A (Con A) or phytohaemagglutinin (PHA) Control

?/

x SEM

PHA

Normal serum

Castrate serum

Normal serum

Castrate serum

77 383 dpm _+40

56 439 dpm _+49

77 823 dpm _+73 P* < 0.001

56 1814dpm _+ 159

NS

1461

Con A Normal serum

Castrate serum

77 56 1269dpm 5646 dpm _+ 184 + 769 P* < 0.001

*Significant as measured between normal and castrate serum using the Welch test for two samples with unequal variances, n = number of separate animal preparations utilized to generate thymocytes for blastogenic assays. X _+ SEM.

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CHARLES J. GROSSMAN et al.

Table 3. The effect of in vitro addition of testosterone (T) or dihydrotestosterone (DHT) to cultures of normal thymocytes Castrate Serum n X SEM

Phytohaemagglutinin Castrate serum Castrate serum + 2 ng/ml T + I ng/ml DHT

56 1814dpm +159

15 161l dpm +208 P-NS I

Normal serum 10 302 dpm + 62 P-NS 2

n X SEM

Concanavalin A Castrate serum Castrate serum + 2 ng/ml T + 1 ng/ml DHT

Castrate serum

15 1598 dpm +_251

56 5646 dpm +769

15 7026 dpm +_1573 P-NS j

Normal serum + 1 ng/ml DHT 10 440 dpm + 123

15 5931 dpm +974

Normal serum 10 2182 dpm +-481 P-NS 2

Normal serum + 1 ng/ml DHT 10 2744 dpm _+827

(1) There was no significant difference in T-cell stimulation in vitro utilizing castrate serum alone or with the addition of 2 ng/ml DHT within the group. (2) There was no significant difference in T-cell stimulation in vitro utilizing normal serum alone or with the addition of 1 ng/ml DHT within the group, n = Number of separate animal preparations utilized to generate thymocytes for blastogenic assay. nonsignificant when measured by the Welch test for samples with unequal variance as well as by Student's t-test. W h e n the mitogen P H A is employed, however, a different picture emerges (Fig. 2). Here, as the levels

o f D H T are increased from 15 to 100/tg, there is a significant depression o f thymocyte function below untreated castrate serum. These results suggest that the thymocyte population responding to the mitogen

Table 4. Pooled sera were prepared from groups of normal or surgically manipulated rats. After clotting, centrifugation and millipore filtering, the sera were heat inactivated at 56°C for 30 min. Thymocytes were extracted from normal rat thymi and incubated in blastogenic assay at a final concentration of 2.5 x 102 cells per assay. Each assay well also contained 20°~, rat serum plus the mitogen Con A, minimal essential media with Earles salts, 1 mM L-glutamine, penicillin and streptomycin. Incubation was for 3 days at 37' C after which time the culture were pulse labelled for 2 h with tritiated thymidine, collected on glass fiber filters and counted for incorporated radioactivity. In this table an "X" drawn through a symbol for an organ indicates organ ablation and a double line drawn around the symbol for an organ indicates stimulation. Significance between groups was measured using the Welch test for two samples with unequal variance. X -+ SEM, n = number of separate animal preparations utilized to generate thymocytes for blastogenic assays Concanavalin A Gonads T cell Thymus 1 Normal

~

2 Castrate

3

a

t

a

~

o+O.

ITI

[~

1269 + 184 n - 77

--

(~)

5646+769 n - 56

P<0.00I

M.Y

x

Castrate + T Rx

6 Thymectomy + DHT Rx

~

14

-

[--"1 t.._.J

~

753 _+ 216 n 7

NS

P < 0.001

O

~

O

1081+143 n - 26

NS

P < 0.001

O

]~

O

2493 + 587

NS

P < 0.002

NS

P < 0.001

P<0.002

NS

DHT

n

7 Castrate thymectomy

~

]~[

8

~

~[

Castratethymectomy+DHTRx

P < 0.001

<000

n

r 5 Thymectomy

Significance compared to Normal Castrate

~

'X"'X" k " - ' ~ -~L~--J ~DHT 4

_ Dpm (X _+SEM)

D H T

O

(~

9

-

2014+397 n-18 7112+1232 n

--

9

1463

Dihydrotestosterone regulation of thymocyte function in the rat Table 5. Pooled sera and thymocytes were prepared as described in the text and Table 4. Each assay well also contained 20% rat serum plus the mitogen PHA, minimal essential media with Earles salts, 1 mM L-glutamine, penicillin and streptomycin. Incubation methods and symbol explanation is described in Table 4. Statistical significance between groups was measured using the Welch test for two samples with unequal variance. X _+ SEM, n = number of separate animal preparations utilized to generate thymocytes for blastogenic assay Phytohaemagglutinin Gonads T-cell _ Dpm Thymus (X + SEM) 1 Normal

Q~}

2

~

~

(~

~

[~

(~)1234_+175n_14

Castrate

3 Castrate+DHTRx

[]

C)

Significance compared to Normal Castrate

823 + 73 n -77

P < 0.001

1814-I-159n_56 P<0.001

--

P<0.05

P<0.02

NS

P < 0.002

DHT 4

Castrate + T Rx

[---.-]

826 _+ 211 n 7

L.-_J

T 5 Thymectomy

O

~

6

f"'l t.,./, DHT

~

7 Castrate thymectomy

~

8

"~

Thymectomy + DHT Rx

Castrate thymectomy + DHT Rx

~

1239_+157n_26 P < 0 . 0 2

P<0.02

#'~ k.J

972 _+ 272 n-9

NS

P < 0.02

~[

Q~)

743_+7n_18

NS

P<0.001

~f

('-'3

1031 _+ 277 n-9

NS

P < 0.02

DI..tT

Con A, CASTRATE SERUM

DPM 20,000

PHA, CASTRATE SERUM

DPM 15,000

--

10,000

--

5,000

--

2,000

1,000

0

500 0

15

30

60

100

pg DHT (IN VIVO)

Fig. I. Castrate male rats were treated with various concentrations of DHT in oil or oil alone for 5 days after which time the animals were killed and the serum prepared as described in the text. Serum fractions were run in blastogenie assay along with thymocytes prepared from normal male rats and the mitogen Con A. After 3 days of incubation the cultures were pulse labelled with tritiated thymidine and counted for radioactivity. Statistically, there was no significant difference in thymocyte stimulation between control serum and serum prepared from DHT-treated animals. Significance was measured by the Welch test for two samples with unequal variance, X + SEM, n = number of separate animal preparations utilized to generate thymocytes for blastogenic assay. In Fig. 1 for all groups n=10.

0 0

15

30

60

100

~Jg D H T (IN V I V O )

Fig. 2. Castrate male rats were treated with various concentrations of DHT in oil or oil alone as described in Fig. 1 and the test. Thymocytes and serum preparations were incubated along with the mitogen PHA and tritiated thymidine uptake measured. Statistically, there was a significant decrease in thymocyte stimulation in serum prepared from DHT-treated animals vs control serum (see text for details). Significance was measured by the Welch test for two samples with unequal variance. X _+ SEM, n = number of separate animal preparations utilized to generate thymocytes for blastogenic assay. In Fig. 2 for all groups n-10.

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CHARLES J. GROSSMAN et al.

PHA is depressed by a serum factor under the control of DHT. Since direct addition of DHT to castrate serum was ineffective in altering the blastogenic response of thymocytes from control values (Table 3) these findings along with the results presented in Tables 4 and 5 could be interpreted to mean that serum factors under the control of DHT can depress the PHA-sensitive thymocyte response. DISCUSSION

We have reported previously that estradiol was able to depress the cell-mediated immune response in vitro as regulated through a thymic serum factor. The present study describes the effects of the androgens, testosterone and dihydrotestosterone on in vitro immune function. As can be seen (Tables 4 and 5) variations in the androgen effect on immune response depend on whether PHA or Con A is employed as the mitogen. Since these differences may be due to a differential effect of these mitogens on thymocyte subpopulations, the results will be evaluated separately. Con A ~ c t s

Removal of the sex steroids by castration results in a significant increase in thymic size [19,20] along with the production of a thymic stimulatory factor which activates a Con A sensitive thymocyte population (Table 4). Replacement by a subcutaneous injection of DHT fails to reverse this effect but injection with T reverses this stimulation. This depression of the serum factor by T treatment is similar to the depression observed by in viro E treatment [24]. The immunologically suppressive effect of T may possibly be due to its peripheral aromatization into E [33, 34]. In addition, conversion of T into DHT has been shown to take place in androgen-sensitive tissues containing steroidal 5~-reductase[35, 37] and, according to recent information, this enzyme is present within thymic tissue [38]. The E formed from aromatization of T may possibly be responsible for the effect noted on the thymus after T injection. This tentative hypothesis is supported by the observation that sera prepared from castrate animals pretreated in vivo with T produces the same effect on blastogenesis as does sera prepared from castrate animals pretreated in vivo with E [24]. Further support for this hypothesis comes from the observation that thymic reticuloepithelial cells contain E receptors[21,22] which could mediate these effects. Another possible explanation for the apparent lack of effect of DHT in vivo could be its conversion to androstanediols which circulate in the plasm. Since these substances must be reconverted to DHT at the target organ to enter the nucleus, a lack of reconversion to DHT would result in the depressed response to DHT treatment [39,40]. Although we do not at present know if such conversion of the androstanediols to DHT takes place in thymus, we do

know that DHT is able to translocate receptor from the cytoplasm to the nucleus in both rat and human thymus tissue and in castrate rats pretreated with 15#g DHT/day for 3 days [27]. While serum prepared from thymectomized, castrate-thymectomized and DHT-treated thymectomized rats was not stimulatory, DHT replacement into castrate-thymectomized animals produced a significant blastogenic response. It therefore follows that the stimulatory effect elicited with DHT may be mediated through in vivo interactions at some nonthymic site and expressed in the absence of gonadal factor interactions. P H A ~[J?cts

PHA (Table 5) stimulation of thymocytes is significantly enhanced by serum factors produced in castrate rats compared with serum from normal animals. In vivo treatment with DHT does not depress this stimulation; however, injection with T reverses this effect. The depression of the serum factor by T treatment is similar to that observed for E treatment in vivo [24] and has been previously mentioned could be a result of peripheral aromatization of T into E [33, 34]. Serum prepared from thymectomized animals is significantly stimulatory on T-lymphocyte blastogenesis which is different from the effects observed in vitro with thymectomized serum in the presence of Con A. Serum prepared from either thymectomized DHT-treated animals, castrate-thymectomized DHT-treated animals or castrate-thymectomized serum from untreated groups is also unable to produce T-cell blastogenesis in assay. Also, DHT treatment appears to depress castrate serum stimulation of blastogenesis as well as thymectomized serum stimulation of thymocytes. Taken together these findings suggest that PHAsensitive thymocytes are regulated by a thymic inhibitory factor and stimulated by a gonadal factor. These findings are different from the effects of serum from DHT treated rats in the presence of Con A in vitro. Overall consideration

It is clear from the preceding discussion that the effects of androgens on T-lymphocyte function depends on the mitogen employed in vitro. This suggests that the T-lymphocytes responding are from different subpopulations. Our observations that DHT depresses the PHA-sensitive thymocyte population suggests an explanation for the findings reported by Roubinian et al.[5 8] working with the FI NZB/NZW lupus mouse model. They showed that the female normally dies of this disease but not if treated with DHT, while prepubertal castration of the male results in enhanced mortality which is promoted by estradiol treatment. According to reports, the mitogen PHA is stimulatory on a large variety of thymocytes subclasses [41]. Depression of the PHAsensitive thymocyte class would probably include

Dihydrotestosterone regulation of thymocyte function in the rat both helper and suppressor cells and this depression may be under the control of thymic inhibitory factors. In the absence of helper cell function autoantibody synthesis by B-lymphocytes would be inhibited and the disease would not be expressed. Further support for this hypothesis comes from our previous studies on the effects of estradiol in regulation of the cell-mediated immune response and also from the observations that the cell population stimulated by the mitogen Con A is a suppressor Tlymphocyte class [42, 43]. Our results indicate that estradiol depresses the function of the Con A sensitive T-lymphocyte and that this effect is mediated by inhibition of a stimulatory factor of thymic origin released into the serum. A similar finding has been reported by Stimson and Hunter[44] who showed that estradiol was able to enhance humoral immunity and depress cellular immunity as mediated by a low molecular weight thymic-derived factor. It follows from these findings that if the cell population depressed by estradiol through absence of thymic stimulatory factor interaction is a suppressor T-lymphocyte, then in the absence of suppressor cell function autoantibody synthesis would be stimulated and the disease would be expressed. Thus, in the female or castrated male treated with estradiol, autoimmune disorders would be evident. In a recent study it was shown that some patients with SLE had alterations in their estrogenic metabolites resulting in an increase in estrogen activity [1]. It has also been noted that patients with SLE lack some suppressor cell function [2]. Such suppressor cell activity could be induced if the cells from many of these patients were incubated with thymosin, possibly acting as the thymic stimulatory factor or cultured thymic epithelium [2] which might act to produce such a stimulatory factor, although genetic factors have also been implicated [3, 4]. These studies suggest that T-cell function can be mediated by thymic hormones and modulated by sex steroids in humans as well as the rat. We suggest, therefore, that there is a balance between the effects produced by E and the effects produced by DHT such that E can depress suppressor T-lymphocyte function while DHT can depress a diverse population of thymocytes. We further hypothesize that this response to E is mediated through thymic stimulatory factors released into the serum and that these factors may have their origin in the thymic reticuloepithelial cells. These thymic reticuloepithelial cells have also been shown to contain receptors for both E and DHT[21-33], thus these hormones could conceivably regulate the release of thymic serum factors through receptor-mediated events. In the male, E may be supplied by peripheral conversion of T. Thus, in the absence of E (such as in castration) the inhibition is removed and stimulation of T-lymphocytes by thymic serum factors takes place. Further support for our findings is suppled by the recent study of Stimson and Crilly[45]

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who showed that thymic epithelial culture supernatants pepared from testosterone-conditioned cultures enhanced PHA and Con A stimulated thymocyte proliferation. Thymic epithelial culture supernatants prepared from estradiol-treated cultures reduced the thymocyte mitogenic response. Finally, as has been mentioned previously, thymectomy alone produces an increase in the concentration of a serum factor that is stimulatory on T-lymphocytes incubated in the presence of the mitogen PHA but not Con A. This response is not present in thymectomized animals treated in ~,it,o with DHT. This suggests that thymectomy removes the source of a thymic-derived serum factor which is inhibitory on PHA-sensitive T-lymphocyte blastogenesis and in the absence of this factor T-lymphocyte stimulation occurs. This stimulation is reversed in thymectomized animals receiving DHT but because DHT is ineffective when added directly to in t,itro blastogenic assays this suggests that the DHT functions via some in t~it,o mechanism. Such a mechanism may involve a gonadal stimulatory factor because in the castrated thymectomized model no stimulation occurs. Since this effect is observed with the mitogen PHA but not with Con A we suggest that the responding Tlymphocytes are probably composed of more than one class of thymocytes including suppressor and helper cells. In summary our findings suggest that: (1) Con A sensitive thymocytes are activated by a thymicderived serum stimulatory factor. (2) The thymicderived serum stimulatory factor appears to be inhibited by a gonadal factor, T and E. (3) PHA-sensitive thymocytes are activated by the removal of a thymic-derived serum inhibitory factor. (4) PHA-sensitive thymocytes are activated by the presence of a gonadal stimulatory factor. (5) The gonadal stimulatory factor is inhibited by DHT and E. (6) The production of thymic-derived serum inhibitory factor depends on the presence of a gonadal factor. (7) The thymic derived serum inhibitory factor is stimulated by T, E and possible DHT. (8) Because the gonads interact with, regulate, and are regulated by the hypothalmic-pituitary axis, it is conceivable that the gonadal factors responsible for thymocyte effects may release pituitary substances which affect immune response. This hypothesis is presently under investigation. The clinical significance of these results remains to be elucidated. Acknowledgements The authors wish to express their appreciation to Mrs Patricia Uebel, Ms Susan Madaris and Mrs Peggy Warth for their expert technical assistance, to Mr Richard L. Clark for his preparation of the figures and to Ms Joan Mason for typing the manuscript. This work was supported by funds supplied by the Veterans Administration. REFERENCES

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