Effect of 17β-estradiol on immunoglobulin secretion by human tonsillar lymphocytes In vitro

ft. Steroid Biochem. Molec. Biol. Vol. 48, No. 2/3, pp. 171-177, 1994 Copyright © 1994 Elsevier Science Ltd 0960-0760(93)E0025-3 Printed in Great Britain. All rights reserved 0960-0760/94 $6.00 + 0.00

Pergamon

Effect of 17fl-Estradiol on I m m u n o g l o b u l i n Secretion by H u m a n Tonsillar Lymphocytes In V i t r o Marianthi Evagelatou* and John Farrant Irnrnunodeficiency Diseases Research Group, M R C Clinical Research Centre, Watford Road, Harrow H A 1 3Uff , England

Sex s t e r o i d h o r m o n e s pl ay a r ol e in t h e c o m p l e x n e t w o r k o f i m m u n e r e s p o n s e s b u t t h e m e c h a n i s m o f t h e i r a c t i o n is still u n c l e a r . Effects o f a wide r a n g e o f doses o f 1711-estradiol (E2:0.2-100 n g / m l ) on h u m a n t o n s i l l a r l y m p h o c y t e c u l t u r e s w e r e e x a m i n e d . B a n d T l y m p h o c y t e e n r i c h e d p r e p a r a t i o n s w e r e s t i m u l a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f i n t e r l e u k i n - 2 a n d t he p r o d u c t i o n o f i m m u n o g l ob u lin was m e a s u r e d . A d d i t i o n o f Ez i n c r e a s e d B cell i m m u n o g l o b u l i n p r o d u c t i o n in a T cell d e p e n d e n t way with i n t a c t T cells being o b l i g a t o r y . T h e effects o f E2 w e r e also e x a m i n e d on D N A s y nth es is b y t o n s i l l a r T cells. E 2 alone c a u s e d a significant i n c r e a s e in T cell DNA synthesis. With p h y t o h a e m a g g l u t i n i n - s t i m u l a t e d T cell c u l t u r e s t h e r e was a significant i n c r e a s e in D N A syn th e sis with E 2 at p h a r m a c o l o g i c a l doses. D i f f e r e n t cell s u r f a c e a n d a c t i v a t i o n m a r k e r s ( i n c l u d i n g CD25, p75, H L A - D R , CD28) on t o n s i l l a r l y m p h o c y t e s w e r e also s t u d i e d a f t e r e x p o s u r e to E 2. T h e p r e s e n c e o f E2 m a d e no significant d i f f e r e n c e in the e x p r e s s i o n o f t he m a r k e r s e i t h e r alone or w h e n t h e a c t i v a t i o n a n t i g e n s w e r e i n d u c e d by o t h e r st i m ul i . We have shown t h a t i n t a c t T cells a r e n e e d e d f o r t h e a c t i o n o f E2 on t o n s i l l a r B l y m p h o c y t e d i f f e r e n t i a t i o n a n d have e x c l u d e d s e v e r a l m e c h a n i s m s o f a c t i o n o f E2 since c o m m o n a c t i v a t i o n a nt i gens a r e unaffect ed.

ft. Steroid Biochem. Molec. Biol., Vol. 48, No. 2/3, pp. 171-177, 1994

INTRODUCTION Since the last century, evidence has slowly accumulated that sex steroid hormones affect the immune system [1-3]. For example, a sexual dimorphism exists in the immune response with females having higher immunoglobulin levels than males and raising higher antibody titres to viruses and parasites. By contrast, cell-mediated immunity in females is decreased compared to males. Women are more prone to allergy and to autoimmune diseases. Further evidence is that during pregnancy, gonadectomy and sex steroid hormone replacement, the immune response is altered [1-3]. Receptors for estradiol (E2) have been identified in human peripheral blood mononuclear cells, especially in the CD8 ÷ T cells, thymic cells and splenic lymphocytes [4-6]. There are two hypotheses about the action of E2 on lymphocytes. First, E2 may affect lymphocytes indirectly in vivo through the thymus by a mechanism which may involve thymic factors [1, 7, 8]. Second, there is evidence that E2 has a direct effect on lymphocytes in culture [9-16].

In the present study, we have quantified the direct effect of E 2 on human tonsillar lymphocytes in vitro principally by measuring immunoglobulin production. T o help to elucidate the mechanisms of the action of Ez, we have also studied its effect on T cell proliferation and the expression of lymphocyte activation markers.

*Correspondence to M. Evagelatou. Received 19 June 1993; accepted 27 Oct. 1993. 171

MATERIALS AND METHODS

Cell preparations Human tonsils were obtained after routine operations for chronic tonsillitis and used as sources of B and T lymphocytes [17]. In brief, after the tonsils were teased into medium [RPMI-1640 + 10% foetal calf serum (FCS)], the cell suspension was passed through a fine cell strainer to remove remnants of tissue and then layered onto Ficoll-Paque (Pharmacia, UK). T he mononuclear cells obtained from the interface of the Ficoll-Paque were rosetted overnight with neuraminidase-treated sheep erythrocytes (n-SRBC) to separate T cells from a n o n - T preparation. T he T ceils were recovered by lysing the treated sheep erythrocytes with ammonium chloride lysis fluid. By flow cytometry, the T cell preparation contained more than 90%

172

Marianthi Evagelatou and John Farrant

C D 3 ÷ cells and less than 3 % CD19 ÷ cells. T h e n o n - T preparation was layered onto a Percoll (Sigma, U K ) gradient and only the band of high-density cells (45 to 55% Percoll) was used for the B cell preparations. By flow cytometry, there were about 9 0 - 9 5 % CD19 ÷ cells and no more than 2% of C D 3 ÷ cells.

Cell cultures B cells (40,000/well) were cultured with or without T cells (80,000/well) in 20/~1 hanging drops in 60-well Terasaki plates (Cel-Cult, Sterilin, U K ) in 5% CO2 in air [18]. T h e m e d i u m was R P M I - 1 6 4 0 , with penicillin/streptomycin (100 I U / m l ) and L-glutamine ( 2 m M ) with heat-inactivated F C S (10%). T h e F C S used had been screened for E 2 levels which were very low (max. 0.02 ng/ml) compared to the levels added later in cultures (2 to 100 ng/ml). N o attempt was made to strip the steroid hormones from the F C S as cultures with and without added E2 would both be exposed to the same m e d i u m and the effects of added E2 that we report would be unaffected by any background levels of E 2. In pilot experiments we found that the effects of extrinsic E2 in the dose range that we used was unchanged whether or not the F C S had been stripped of steroid hormones. T h e m i n i m u m n u m b e r of replicate wells for each set of conditions was six. Initial cell viability was checked with T r y p a n blue dye exclusion and always exceeded 85%. Cultures were done from 4 different tonsils.

Stimulants Recombinant interleukin-2 ( r I L - 2 , British Biotechnology Products Ltd, U K ) was reconstituted according to the manufacturer's instructions, aliquoted and stored at - 7 0 ° C . Dilutions in m e d i u m were made immediately before use at a range of final concentrations of 1 0 - 2 0 0 U / m l . E2 (Sigma) supplied as a lyophilized and g a m m a - r a y irradiated powder, was reconstituted in 1 ml of sterile absolute alcohol followed by 4 9 m l of R P M I making a stock solution of 20/~g/ml and aliquoted before storage at - 7 0 ° C . Dilutions of E 2 w e r e made with m e d i u m immediately before use at a final concentration ranging from 0.2-100ng/ml. Phytohaemagglutinin (PHA, Wellcome, U K ) was obtained in freeze-dried form, reconstituted in sterile water, stored at - 7 0 ° C and used at final concentrations of 0.5 and 1/~g/ml.

Immunoglobulin (IgM and IgG) assay B cell preparations were cultured with or without T cells in the presence of r I L - 2 and with or without E2 for 8 days. In some instances, T cell supernatants replaced intact T cells. T o prepare these supernatants, T cells were incubated overnight with I L - 2 . T h e supernatant was then added to B cells and the cultures stimulated in the presence of I L - 2 and E 2 a t various doses. T h e amounts of I g M and I g G secreted by B cells were assessed by E L I S A as described previously [19].

Proliferation assay T cell cultures were stimulated with P H A , with or without E~ and incubated for 3 days. [3H]Thymidine ([3H]Tdr, 0.2/~Ci, 2 Ci/mmol, Amersham, U K ) in 1/~1 aliquots was added to each 20 #1 well of the inverted Terasaki plates 2 h prior to harvesting and counting in a liquid scintillation counter [18]. Counts (dpm per well) from these microcultures are m u c h lower than those of standard 200/~1 cultures, but the ratio of response to background is superior.

Activation markers Directly conjugated rnonoclonal antibodies used to assess lymphocyte surface antigens including activation markers by flow cytometry (Becton Dickinson F A C S C A N ) were: I L - 2 R g chain (CD25) F I T C , Becton Dickinson (BD, UK); I L - 2 R fl chain (p75) F I T C , Endogen, USA; H L A - D R F I T C , (BD) CD28 F I T C , I m m u n o t e c h , France; C D 4 5 R O F I T C , Dakopatts, Denmark; C D 4 5 R A F I T C , Dakopatts, D e n mark. Cells were assessed by flow cytometry after 1 or 2 days of culture in the presence or absence of E 2 (0.2-100 ng/ml). Various cytokines and mitogens were used for the stimulation of the cell cultures (see Results). All the above markers were tested on C D 3 ÷ (CD4 ÷ and C D 8 + subsets also), C D 1 9 ÷ and CD14 ÷ cells. Antibodies for these cell subsets were direct conjugated with PE or P e r C P (BD). T h e standard procedure of staining was carried out on ice. Data was assessed both by the percentage of cells positive for the particular antigen and by the level of expression per cell (mean channel).

Statistical analysis Analysis of variance (ANOVA) of the data on immunoglobulin production and D N A synthesis was p e r f o r m e d using a computerized statistical p r o g r a m (Genstat) to assess the treatment effects within tonsils. Other data were analysed by Student's t test. RESULTS

Effects of E2 on IgM secretion by human normal tonsillar H lymphocytes in the absence of other stimuli T h e effect of E 2 w a s studied on B cell preparations with and without the addition of irradiated T cells, and the production of I g M and I g G compared. E2 caused an increase in I g M production at all of its doses in the cultures when T cells were present (Fig. 1). In B cell cultures without added T cells the addition of most doses of E2 had no effect. Only at a high dose of E2 (20 ng/ml) was there a significant increase in I g M production in the absence of T cells.

Effects of E2 on I L - 2 driven IgM secretion by human normal tonsillar B lymphocytes I L - 2 was used to drive I g M secretion in a range of doses from 10-200 U / m l and E 2 was added at the same time as I L - 2 at the start of the 8 day cultures. T a b l e 1

173

Estradiol on Human Tonsillar Lymphocytes 6-n

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Ea (ng/rnl) Fig. 1. M e a n p r o d u c t i o n o f I g M ( / t g / m l ) b y h i g h d e n s i t y t o n s i l l a r B c e l l s in t h e p r e s e n c e ( m ) a n d a b s e n c e ([-1) o f i r r a d i a t e d T cells. (I) a n d (II) r e p r e s e n t t h e s t a n d a r d error of the differences between the treatment means with or w i t h o u t T cells. T h e s e w e r e c a l c u l a t e d w i t h i n t o n s i l s using analysis of variance (Genstat). Significant differences b e t w e e n t h e t r e a t m e n t m e a n s w i t h E 2 a n d t h e m e a n v a l u e s in t h e a b s e n c e o f E 2 w e r e t e s t e d b y S t u d e n t ' s t t e s t ( * P < 0.05, * * P < 0.01, * * * P < 0.001, at d e g r e e s o f f r e e d o m of t h e e r r o r v a r i a n c e = 43 a n d 30 in t h e p r e s e n c e or a b s e n c e o f i r r a d i a t e d T cells, r e s p e c t i v e l y ) . In t h e a b s e n c e of T cells t h e d o s e o f E 2 = 100 n g / m l w a s n o t t e s t e d .

shows the effect of E 2 on I L - 2 stimulated cultures of B cells without the addition of T cells [Table I(A)] and with irradiated T cells present [Table I(B)]. When T cells were not added, E2 had little effect on IgM production of B cell cultures: however, a low dose of E2 (2 ng/ml) caused a decrease in IgM production that was significant [Table 1(A)]. T h e significant effect of E2 (at 10 ng/ml) in increasing IgM production induced by 1 0 U / m l of I L - 2 was due to data from 1 out of the 4 tonsils. On the contrary, in the presence of T cells, E2 significantly increased the I L - 2 driven production of IgM [Table I(B)].

Table 1. Effect of different doses of E2 on the mean production of I g M (pg/ml) in tonsillar B cell cultures in the absence ( A ) or presence (B) of irradiated T cells in response to different concentrations of I L - 2 E2(ng/ml) IL-2 (U/ml)

0

10 50 100 200

4.7 11.8 13.0 14.0

10 50 100 200

4.9 10.9 10.9 11.4

2

10

6.5 17.0*(a) 19.9"**(a) 19.4"**(a)

20

100

(ng/ml]

Effects of E 2 on IgG secretion by human normal tonsillar B lymphocytes in the absence of other stimuli Figure 2 illustrates the effect of E 2 on I g G production from B cell preparations with and without irradiated T cells in the absence of any added IL-2. T h e presence of irradiated T cells allowed E2 to increase significantly the IgG production by these cultures. Effects of E 2 on I L - 2 driven IgG secretion by human normal tonsillar B lymphocytes In the presence of IL-2, E2 did not increase the I L - 2 driven IgG production in the B cell cultures without T cells [Table 2(A)]. In some instances, E2 caused a significant decrease in the IgG production from these cultures. However, when irradiated T cells were present and the cultures were stimulated with I L - 2 at 100 or 200 U/ml, E 2 at some doses increased IgG production significantly [Table 2(B)].

Table 2. Effect of different doses of E 2 on the mean production of IgG (#g/ml) in tonsillar B cell cultures in the absence ( A ) or presence (B) of irradiated T cells in response to different concentrations of I L - 2

20

100

SED of means

6.9 8.9 11.2 10.5

ND ND ND ND

1.1 1.6 1.6 2.1

10 50 100 200

2.3 4.1 4.7 4.5

6.1 12.7 13.8 14.5

7.6"(a) 16.0"(a) 17.4"*(a) 18.5"**(a)

1.1 2.6 2.2 1.9

10 50 100 200

8.8 17.6 14.2 12.8

E2 (ng/ml) IL-2 (U/ml)

0

2 2.6 2.8"*(b) 3.3 2.7"(b)

10

20

100

SED of means

2.7 2.7"*(b) 3.5 3.2

ND ND ND ND

0.4 0.4 0.7 0.9

(A) Absence

(B) Presence 7.7"(a) 10.1 10.9 12.3

10

Fig. 2. M e a n p r o d u c t i o n o f I g G ( ~ g / m l ) b y h i g h d e n s i t y t o n s i l l a r B cells in t h e p r e s e n c e ( . ) a n d a b s e n c e ([3) o f i r r a d i a t e d T cells. O t h e r d e t a i l s as in Fig. 1.

(A ) Absence 3.9 7.7"(a) 7.2"(b) 9.1 6.8"**(b) 11.9 6.5"**(b) 11.2

2

2.6 2.9"*(b) 3.1*(b) 4.4

(B) Presence

(a) =increased and (b) = decreased at *P <0.05, **P <0.01, ***P < 0.001; N D = not done, SED of means = standard error of the differences between the means.

10.3 13.2"(b) 14.4 6.2"(a)

11.4 9.3 10.4 15.8 11.0**(b) 19.9 17.5"(a) 12.2 16.6 21.0***(a) 13.8 17.5"*(a)

1.4 2.0 1,7 1.6

(a) = increased and (b) = decreased at * P < 0 . 0 5 , * * P < 0 . 0 1 , ***P < 0.001; N D = not done, SED of means = standard error of the differences between the means.

174

Marianthi Evagelatou and John Farrant 20

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I L - 2 (U/ml) Fig. 3. P r o d u c t i o n of IgM ( ~ g / m l ) by high density tonsillar B cells w h e n i n t a c t T cells w e r e p r e s e n t (solid symbols) or w h e n T cell s u p e r n a t a n t s w e r e a d d e d (open symbols). The cells were s t i m u l a t e d with various doses of IL-2 (abscissa) and doses o f E 2 ( 0 , O 0 n g / m l ; m, [] 2 ng/ml; V, ~ 2 0 n g / m l ; ~ , r ~ 100 ug/ml). The above g r a p h s r e p r e s e n t data f r o m two different e x p e r i m e n t s with 6 r e p l i c a t e s for each t r e a t m e n t condition.

11" 177

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I

I

i

I

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2

10

20

100

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(ng/ml)

Fig. 5. M e a n levels of the uptake o f [3H]thymidine by tonsillar T cells s t i m u l a t e d with P H A ([7, 0 p g / m l ; . , 0 . 5 p g / m l ; [], 1 p g / m l ) and various doses of E 2 (abscissa). The degrees o f f r e e d o m of the e r r o r v a r i a n c e were 100. The s t a n d a r d e r r o r of the differences between the t r e a t m e n t m e a n s were (II) 46, (I) 177 a n d (II) 284 d p m for the three doses of PHA; r e s p e c t ively. These were calculated within tonsils using analysis of v a r i a n c e (Genstat), (**P < 0.01; ***P < 0.001).

T cell supernatants. A similar pattern was followed for I g G production in the same cultures (Fig. 4). With 50 U / m l of I L - 2 there was a significant decrease in the production of I g G after addition of E 2 at two doses.

Effect of E2 on IgM production by B lymphocytes in the presence of irradiated T cells or T cell supernatants W h e t h e r intact T cells are required for E 2 t o act was tested by comparing the effects of adding T cells or T cell supernatants. I L - 2 driven B cell cultures produced higher levels of I g M when intact irradiated T cells were present compared to when T cell supernatants were substituted for T cells (Fig. 3). In addition, the effect of E2 was only seen when irradiated T cells were present but not when T cells were substituted with

Role of E 2 on P H A -driven D N A synthesis by tonsillar T lymphocytes T o see whether the effects of E 2 on T - d e p e n d e n t I g M secretion could be reflected by an effect on T cell proliferation, doses of E 2 w e r e added to T cell cultures. E 2 alone (at all doses tested) significantly increased the D N A synthesis by the T cells (Fig. 5). However, when the T cells were stimulated with P H A (0.5 or 1 #g/ml), the lower doses of E 2 had no effect on the D N A synthesis. Only the addition of high doses of E 2 (20 and 100 ng/ml) increased D N A synthesis significantly.

Effects of E2 on surface antigens of tonsillar lymphocytes from normal donors 30

:20

o ~o~10

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0

I

I

I

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0

10

50

100

200

I L - 2 (g/rnl) Fig. 4. P r o d u c t i o n o f IgG ( p g / m l ) by high density tonsillar B cells w h e n i n t a c t T cells were p r e s e n t (solid s y m b o l s ) or w h e n T cell s u p e r n a t a n t s were a d d e d (open symbols). O t h e r details as in Fig. 3.

I,n order to explore the mechanism of the action of E 2 o n lymphocyte function, the expression of a n u m b e r of different lymphocyte surface antigens and activation markers were tested for changes after addition of E 2 t o the lymphocyte cultures. These antigens were examined by flow cytometry on CD3 + (CD4 + and C D 8 + subgroups), C D 1 9 + and CD14 + cells. In general, no effect of E2 was discerned on these antigens either when E2 was used alone or when the activation marker expression was increased by stimulation. CD25 (IL-2 receptor, ~-chain). T h i s was assessed on CD3 +, CD19 + and CD14 + cells after they had been cultured for 2 days with different stimulants in the presence or absence of E2 = 20 ng/ml. T h e stimulants used were: pokeweed mitogen ( P W M , 1 #g/ml), I L - 2 (100U/ml), I L - 4 ( 1 0 U / m l ) , I L - 5 ( 1 0 U / m l ) , I L - 6 (100U/ml), 7 - I F N ( 1 0 0 U / m l ) and combinations including I L - 2 and 6, I L - 4 and 5, I L - 6 and 7 - I F N .

Estradiol on Human Tonsillar Lymphocytes T h e presence of E 2 made no difference in the expression of CD25 on the above cell subsets. Moreover, T cell preparations cultured with O K T 3 (1 #g/ml) showed no change in CD25 expression on C D 4 ÷ and C D 8 ÷ cells when 2 or 20 ng/ml of E2 were added to the culture for 1 or 2 days. With P H A (0.5 or 1 pg/ml) there was a small increase in CD25 expression due to E2 after 2 days of culture [Fig. 6(b)]. p75 (IL-2 receptor, fl-chain). T cell preparations were cultured for 2 days with P H A (0.5 or 1/~g/ml) or with O K T 3 (1/~g/ml) and the p75 antigen was assessed on C D 4 ÷ and C D 8 ÷ cells after the addition of E2 = 20 ng/ml. T h e r e was no effect of E2 on p75 expression. Additionally, B cells alone or B cells with irradiated T cells cultured with I L - 2 (100 U/ml) and E2 = 20 ng/ml, showed no change of p75. H L A - D R (class I1). B cells alone or cultures of B cells with T cells were stimulated with I L - 2 (100 U/ml)

for 2 days and tested for any changes in H L A - D R expression on the C D 4 +, C D 8 + and CD19 + subsets after addition of E z = 20 ng/ml. Again E z made no difference [Fig. 6(d)]. Also, tonsillar Ficoll-separated mononuclear cells cultured for 2 days with various doses of E2 (0.2-100 ng/ml) alone, or with different cytokines including 7 - I F N (100 or 1000U/ml), I L - 6 (100 U/ml) or combinations of y - I F N and I L - 6 ( 1 0 0 U / m l each), showed no changes of H L A - D R expression on C D 4 +, C D 8 +, CD19 + and C D 1 4 + subsets after addition of E2. CD28. Tonsillar T cells were cultured with P H A (1/~g/ml) or P W M (1/~g/ml) for 1 or 2 days. Addition of E 2 (0.2-100 ng/ml) had no effect on C D 2 8 expression on C D 4 + or C D 8 ÷ subsets. CD45RO/RA. T h e same cultures as for C D 2 8 were tested for C D 4 5 R O and C D 4 5 R A expression after addition of E 2. N o change was observed. E z had no effect on the T cell D N A synthesis or B cell

(a) 20 -I

175

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101

102

103

104

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102

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0

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Fig. 6. Flow c y t o m e t r y h i s t o g r a m s s h o w i n g the effect o f E 2 (20 n g / m l ) on the e x p r e s s i o n o f IL-2R = c h a i n (CD25) on CD3 + cells w i t h o u t (a) a n d w i t h (b) s t i m u l a t i o n by P H A (0.5/~g/ml) a n d on the e x p r e s s i o n of H L A - D R on CD4 + cells w i t h o u t (c) and with (d) s t i m u l a t i o n by IL-2 (100 U / m l ) . O n P H A s t i m u l a t i o n there w a s a s m a l l i n c r e a s e in the e x p r e s s i o n o f CD25 p e r cell in the p r e s e n c e o f E 2 [see (b)].

176

Marianthi Evagelatou and John Farrant

immunoglobulin responses of any of the above stimuli used to modulate activation antigens other than the effects already reported. DISCUSSION T o our knowledge, the present study is the first to examine the effects of E2 on tonsillar lymphocytes. Our observations on tonsillar cells suggest that E2 has an enhancing effect on lymphocyte differentiation and proliferation in vitro. E2 at various doses significantly increased the background and I L - 2 driven I g M and I g G production in vitro in a T - d e p e n d e n t way, observations which extend Paavonen's previous findings [2, 20]. H e demonstrated that addition of E 2 to P W M - s t i m u l a t e d cultures of h u m a n P B L enhanced the I g M levels although there was no effect on B cell proliferation. However, when B cells were stimulated with Staphylococcus aureus there was no significant effect of E2 which strongly suggests that the enhancing mechanism acts through T cells. Moreover, from our experiments the presence of intact T cells seems to be essential. It is not clear why E 2 induced a significant reduction in I g M secretion in our B cell cultures under some conditions. Perhaps this effect is due to a small n u m b e r of residual T cells in the n o n - T p r e p aration. W e e t m a n ' s group [11] also examined the effect of E 2 on I g G synthesis by P W M - s t i m u l a t e d h u m a n P B L and found a significant increase in I g G plaqueforming cells in the presence of E2 but only at physiological concentrations. Additionally, it has been reported that physiological concentrations of E 2 added to h u m a n P B M C significantly augment the Ag-specific i m m u n e response and enhance antibody production [21, 22]. Our present findings that E2 alone at all doses tested exerts a significant enhancing effect on B lymphocyte I g M production seem to contradict partially with K a l m a n ' s data [10]. Although the cell preparations were different, his experiments with h u m a n P B L from normal male donors showed that E 2 alone had no effect but at physiological levels increased the numbers of I g M producing cells in P W M - s t i m u l a t e d cultures. Sthoeger [12] described a significant enhancement of P W M - i n d u c e d generation of plaque formation cells by addition of physiological concentrations of E2 in vitro. Bellini [13] also demonstrated a direct effect of E2 on P H A - s t i m u l a t e d h u m a n PBL. However, preincubation of these cells with E2 decreased significantly the cytosolic free Ca ++ normally induced by P H A stimulation. Accepting the different cellular preparations used, our observations on tonsillar cells agree with the above groups' work on h u m a n PBL, that E2 has an enhancing effect on B cell differentiation through a T-cell dependent pathway. A possible mechanism for E 2 is through C D 8 ÷ T cells (suppressor T cells) which express estrogen receptors in their cytoplasm [4, 5]. Inhibition of C D 8 + cells by E2 m a y improve T helper cell activity which would enhance B cell maturation and

subsequently increase immunoglobulin production [14]. We have clear evidence that E2 added alone significantly increases D N A synthesis by tonsillar T cells at all doses tested. However, when the above cultures were stimulated also with P H A , only the higher concentrations of E2 (20 and 100ng/ml) induced a significant increase in D N A synthesis, data which coincide with Paavonen's [2] observations in h u m a n PBL. By contrast, various studies have reported that h u m a n P B M C stimulated with P H A or Con A showed no effect on D N A synthesis after addition of E2 [15, 23, 24]. Other studies describe the inhibitory effect of supraphysiological doses of E2 on P H A - s t i m u l a t e d lymphocyte proliferation [3,11,25,26]. Our data cannot be compared directly with the above studies on h u m a n P B L since we used tonsillar T cells with distinctly different ratios of their subpopulations compared to blood. Similar experiments with blood, splenic and thymic lymphocytes from mice and rats showed no response to E2 [1, 14] or an enhancing dose related effect of E2 [27]. T h e mechanism of action of E 2 w a s also studied by assessing whether it upregulates different activation markers and surface molecules on the tonsillar lymphocytes. Several possible mechanisms were unlikely since there was little effect of E2 on any of these markers after 1 or 2 days culture, data which agrees with literature reports [15, 16]. Clearly, the mechanism by which Ez modulates T and B lymphocyte function remains to be elucidated in more detail. It is clear that tonsillar lymphocytes are not as sensitive to low doses of E 2 as other systems e.g. breast cells. E 2 is known to affect the transcription of several genes in various cell p o p u lations after binding to its intracellular receptors. N o w that we have established the action of E 2 in the tonsillar system, it will be useful to study the molecular mechanism of the action of E 2 o n lymphocyte function in this system including its effects on cytokine gene transcription. Acknowledgements--M. Evagelatou is currently holding an MRC-

PhD studentship. We thank Ms A. E. Bryant for her helpful advice on many technical points and Dr A. D. B. Webster for his useful comments.

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