Enhancing effect of 17β-estradiol on human NK cell activity

bnmunology Letters, 36 (1993) 31 36 0165 2478 / 93 $ 6.00 i: 1993 Elsevier Science Publishers B.V. All rights reserved IMLET 01938

Enhancing effect of 17fi-estradiol on human N K cell activity K e n - i c h i S o r a c h i ~, S h u n i c h i K u m a g a i ~, M a s a h i k o S u g i t a a, Junji Y o d o i b a n d H i r o o I m u r a a ~'Seeond Division ~[' Internal Medicine, Faculty ~f Medicine, and blnstitute Jor Virus Research, Kyoto University, l(voto, Japan (Received 24 April 1992; revision received 8 February 1993; accepted 17 February 1993)

1.

Summary

The in vitro effect of 17fl-estradiol on N K activity was studied. The proliferation and N K activity of Y T - N I 7 (a human NK-like cell line) were enhanced by 17fl-estradiol (E2), and the enhancement was blocked by tamoxifen (Tx), an antagonist of E2. On the contrary, other steroid hormones such as Tx, progesterone, and testosterone had no effect. YT-N17 contained 11.8 fmol/mg protein of estrogen receptor (mean of two independent assays), a value which was 5 10-fold higher than that of other hematopoietic cell lines. An enhancement of NK activity by E2 was also seen in large granular lymphocytes obtained from normal subjects, and it was again suppressed by Tx. These data suggest that E2 is one of the activating factors for N K / L G L cells.

by which E2 acts on immunocompetent cells, however, remains to be elucidated. Since the estrogen receptor was identified in lymphoid cells [5,6], E2 is thought to affect the immune system through its receptor. The NK activity was reported to be decreased by E2 in the mouse in vivo [7], although the suppression may not be caused by direct effects on NK cells [8]. On the other hand, it has been reported that E2 first enhances and then inhibits N K activity [9]. In this study, we investigated the in vitro effects of E2 on a human NK-like cell line, YT-N17, and found that E2 not only enhanced the proliferation but also the N K activity of the cells. Furthermore, the augmentation of NK activity of large granular lymphocyte (LGL) was also observed after 48-h culture with E2.

2.

3.

Introduction

The presence of sex-related differences in the immune system has been indicated [1], and sex hormones, especially 17fl-estradiol (E2), have been thought to be part of the factors responsible for such differences [24]. The exact mechanism Key words: 17/3-Estradiok NK activity; Large granular lymphocyte Correspondence to." Dr. Shunichi Kumagai, The Second Division of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, Japan. Abbreviations: E2,17fl-estradioI; LGL, large granular lymphocyte; DTT, dithiothreitol; Tx, tamoxifen" ER, estrogen receptor.

3. l.

Materials and Methods Cells and cell culture

A human NK-like cell line, YT-N17, and a human erythromyeloid leukemic cell line, K562, were maintained in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% heatinactivated FCS (Commonwealth Serum Laboratory, Melbourne, Australia). YT-NI7, with higher N K activity, was recloned from YT cells [10] by a limiting dilution procedure. YT-N17 phenotypically possesses the same character as the original YT cells: positive for CD56 and negative for CD3, CD4, CD8, CDI6, CD57, TCR-ccfl or TCR-~,3. PBL from three healthy males were freshly separated by Ficoll-Hypaque gradient cen31

trifugation. Subsequently, PBL were depleted of macrophages and B cells by adherence to nylonwool columns [11]. The nonadherent lymphocytes were fractionated by centrifugation on a modified discontinuous Percoll gradient [11]. The L G L fraction consisted of more than 80% LGL, as judged morphologically by Wright staining. More than 95% of the fractionated cells were shown to be viable by trypan blue dye exclusion.

3.2. Proliferation assay Cells (2 x 104) were placed in a 96-well, flatbottomed plate (Coming Glass Works, Coming, NY) in 200 ml of medium with 10 -8 M to 10 - 6 M of E2, progesterone, testosterone (Nakalai Tesque, Tokyo, Japan), and tamoxifen (Tx; Sigma Chemical, St. Louis, MO), and incubated for 48 h. In some experiments (Fig. la), 10 - v M of Tx was added to the culture in addition to E2. D N A synthesis was determined by the uptake of methyl [3H]thymidine (Amersham, Arlington Heights, IL) for the last 4 h of culture.

3.4.

Estrogen receptor assay

Cells (3~4 x 107) were suspended in H E D buffer (10 mM HEPES, 1.5 m M E D T A , 0.5 mM D T T pH 7.4) containing 0.5 m M phenylmethylsulfonyl fluoride (Sigma Chemical, St. Louis, MO) and 20 m M sodium molybdate (Nakalai) [12]. Cells were homogenized by 6 x 10-s bursts in a Teflon-glass homogenizer on ice, and the homogenates were centrifuged at 105000 x g for I h at 4°C. The protein concentrations of the supernarant were determined by Coomassie brilliant blue staining. The amount of ER in each cell lysate was determined by an ELISA using an ABBOTT ER EIA monoclonal (Dainabot, Abbot Park, IL). The manufacturer's instructions were strictly followed.

3.5. Statistical analysis The statistical significance was determined by Student's t-test. P < 0.05 was considered to be significant.

3.3. Cytotoxicity assay 4.

The N K activity of YT-N17 and L G L against K562 target cells was studied using 51Cr (Amersham) releasing assay. Effector cells were cultured with E2 (concentrations of l0 8 and 10 6 M), or other hormones, such as progesterone, testosterone, and Tx, for 48 h and washed three times with R P M I before using in the killing assay. In some experiments, cells were precultured with or without 10 7 M of Tx for 48 h and then cultured for another 48 h in the presence of the indicated concentrations of E2. Target cells (5 x 103) were cocultured with effector cells at various E:T ratios for 4 h at 37°C. The entire supernatant was collected using a Titertek Supernatant Collection System (Flow Laboratories, McLean, VA), and the radioactivity was counted. Maxim u m 51Cr release was determined by the addition of 0.1% Nonidet P-40 (Nakalai) to the target cells. The spontaneous release (SR) without effector cells was also determined. The percent (%) cytotoxicity was calculated as follows: % cytotoxicity - 100 x (cpm test sample - SR)/(maximum release - SR). 32

4.1.

Results

Effect of E2 on proliferation

Fig. la shows two independent trials of the proliferation assay of YT- NI7. Proliferation was significantly increased in the presence of E2 in a dose-dependent manner. The cell number was also increased by E2 from 1.2 x 105 to 1.3 x 10 5 and 1.6 x 105 (10 -8 and 10 -6 M of E2, respectively). To determine whether E2 affects the cells through a specific receptor, the effect of Tx pretreatment was examined. Tx competes with E2 for estrogen receptor (ER) binding sites, and ER bound with Tx does not have trans-activating ability [13]. When cells were pretreated with 10 7 M Tx, the enhancing effect of E2 was completely blocked. The viability of the cells was unaffected. Effects of other steroid hormones such as Tx, progesterone, and testosterone on the proliferation of YT-N17 were also studied. As shown in Fig. lb, none of them had any effect on YT-NI7.

a)

b)

cpm 6000

cpm 6000

5000

5000 •

4000

----

Exo-1 Exp-1 Tx(+) Exp-2 Exp-2 Tx(+)

3000

3000'

;

10 -8

U

2000

i

2000

3-amoxiten Progesterone Testosterone

4000

10 "7

10-6

E2 (M)

10 -8

10 .7

10.6

M

Fig. 1. (a) Augmentation of proliferation of YT-N17 by E2 and its inhibition by tamoxifen. Two independent experiments (circles for experiment 1, squares for experiment 2) were shown. YT-NI7 were cultured in the presence of the indicated concentrations of E2 for 48 h after preincubation for 48 h with (O and II) or without (C) and [2]) 10 7 M Tx. (b) The effects ofTx (C)), progesterone ([~), and testosterone (A) were also studied. The [3H]thymidine uptake during the last 4 h was estimated. Asterisks indicate a significant difference (*P<0.05 and **P<0.01, respectively) compared with E2-free controls. The bars indicate SD of triplicate samples.

4.2.

Effect o/' E2 and other hormones on N K activity in YT-N17

I n c u b a t i o n w i t h E2 f o r 48 h m a r k e d l y enh a n c e d N K activity, a n d the m a x i m u m effect was o b s e r v e d at 10 . 6 M o f E 2 (Fig. 2a). I n c u b a t i o n

~

b) Tamoxifen (+)

a) Tamoxifen (-) 30-

.ca

20-

10"

,

,

2.5:1

5:1 E:T

,

10:1

i *

30-

o o o

w i t h E2 for 24 h did n o t affect N K a c t i v i t y ( d a t a n o t s h o w n ) . T h e specificity o f this e n h a n c i n g effect was also c o n f i r m e d by u s i n g Tx. T h e E2 enh a n c e m e n t was c o m p l e t e l y b l o c k e d by 10 - 7 M T x p r e t r e a t m e n t (Fig. 2b). O t h e r s t e r o i d h o r m o n e s , s u c h as p r o g e s t e r o n e , t e s t o s t e r o n e , a n d Tx, w e r e also i n v e s t i g a t e d f o r t h e i r effects o n N K a c t i v i t y o f Y T - N I 7 . Y T - N 1 7 w e r e i n c u b a t e d w i t h several c o n c e n t r a t i o n s o f these a g e n t s for 48 h a n d w a s h e d t h r e e times, a n d the N K a c t i v i t y was a s s a y e d . N e i t h e r p r o g e s t e r o n e n o r t e s t o s t e r o n e i n f l u e n c e d the N K a c t i v i t y o f Y T - N 1 7 , a l t h o u g h T x slightly e n h a n c e d it ( T a ble 1).

10

.'"

0

20:1

2.5:1

ratio

&

5:1 10:1 E:T ratio

20:1

E20 E210 "8M E210 "6M

Fig. 2. Enhancing effects of E2 on NK activity ofYT-NI7. YTNI7 were cultured with l0 6 M ( L ~ ) and 10 - 6 M ( ~ 1 ) of E2 for 48 h, and NK activity was examined at various E:T ratios (a). YT-N17 precultured with 10 7 M tamoxifen for 48 h were cultured for a further 48 h in the presence of E2, and NK activity was assayed (b). The vertical bars indicate SD of quadruplicate samples. Asterisks indicate a significant difference (*P<0.05 and **P<0.01, respectively) compared with E2-free controls ( ~ ) . The bars indicate SD of triplicate samples.

TABLE 1 Effect of other hormones on NK activity of YT-N17. Agent

Concentration (M)

Percentage"

Testosterone

10 10 10 10 10 10

106.6 109.0 91.9 111.2 142.1 121.3

Progesterone Tamoxifen

7 6 7 6 7 6

~'The values indicate % increase or decrease against agent-free control at E:T ratio of 20:1. 33

cell lines examined. MCF-7 (a human m a m m a carcinoma cell line) was used as a positive control.

TABLE 2 Number of estrogen receptors. Cell line

4.4.

ER"

Monocyte Myelomonocyte T cell B cell NK cell M a m m a r y cell

U-937 HL-60 Jurkat MSB-3 YT N17 MCF 7

Exp-1

Exp-2

2.2 3.0 < 2b 2.2 8.9 270.0

< 2b 2.4 NT" NT ~ 14.6 251.9

~'Each experiment was done in duplicate, and the results were shown as fmol/mg protein, bThese values were less than 2 fmol/ mg protein, the detection sensitivity of" the assay kit. CNot tested.

4.3.

Concentration of estrogen receptors

Blockade of the E2 enhancement of Y T - N I 7 by Tx pretreatment suggests that estrogen affects YT-N17 through its own receptor. The amount of the receptor in the cells might be related to the sensitivity to E2. We therefore tried to determine the amount of ER, using the A B B O T T ER EIA kit. Table 2 shows the results of two assays. Each assay was done in duplicate. YT-N17 contained 11.8 fmol/mg protein (mean value of two independent assays), a value which was about five- to tenfold higher than that of the other hematopoietic

Subject 1

Subject 2

•

i"

70-

i,o

~ ..................... E:T ralio

Subject 3

t o

5~i: lt0° : . . . . . . . . . . . . . . . . . . . . . T r E : T ratio

E20 E2,OS ",,07M

Fig. 3. Effect of E2 on NK activity in LGL. LGL were cultured withl0 6M(~)andl0 S M ( [ D U]) of E2 or l0 7 M T x ( t ~ - D ) for 48 h, and N K activity was assayed. In subject l, Tx treatment was not done. The vertical bars indicate SD of triplicate s a m p l e s . A s t e r i s k s i n d i c a t e a s i g n i f i c a n t d i f f e r e n c e (*P<0.05 and **P<0.01, respectively) compared with E2-free controls (C>-O). The bars indicate SD of triplicate samples.

34

Ej.Ject (?f E2 on NK activity in LGL

We further investigated whether or not N K activity in L G L was also enhanced by E2 in vitro (Fig. 3). Male subjects were selected in order to exclude differences of E2 concentration in serum, which may influence N K cells. The N K activity in L G L from all three subjects was significantly enhanced after 48 h culture with E2. The enhancing effect of E2 was again blocked by Tx. 5.

Discussion

It has long been thought that E2 decreases NK activity in vivo [7]. However, this might result from an indirect effect of E2. E2 injection causes bone marrow cell depletion and results in a decrease of N K activity [8]. Recently, Screpanti et al. reported that E2 exhibits a biphasic effect on N K activity, first enhanced and then inhibited [9]. In the present study, we demonstrated that E2 enhanced both the proliferation and cytotoxic activity of YT-N17 in vitro. The effects of E2 on YT-N17 were almost completely blocked by Tx, a competitor of E2 on ER. Moreover, the number of ER was greater in Y T - N I 7 than in other hematopoietic cell lines. These data suggest that E2 was one of the activating factors of Y T - N I 7 and that the activation was mediated by ER. Interestingly, NK cells accumulate in decidua during the first trimester of pregnancy [14]. These cells are phenotypically the same as YT-NI7, which is positive for CD56 and negative for CD3, CD16, CD57. Although 10 6 M E2, in which a marked enhancement of N K activity was observed, is higher than the serum concentration of E2, the E2 concentration in the pregnant uterus could reach as high as 10 -6 M [15]. These facts suggest that E2 might play an important role in the accumulation and proliferation of decidual N K cells and in part in the regulation of their N K activity. The augmentation of N K activity by E2 and its blockage by Tx were also observed in LGL. CD16 CD56 ~ CD57 N K cells, although a minor subset, exist in PBL, through which E2

could affect N K activity [16]. Or it might be possible that the enhancement of N K activity seen in E2-treated L G L is brought about by the activation of not only the CD16 CD56 + C D 5 7 - subset but also other N K subsets. Our data suggest that E2 is one of the activating factors of N K / L G L cells. Although the precise mechanism has not yet been clarified, E2 might in part affect the immune system through the activation of N K / L G L cells.

Acknowledgements This work was supported in part by a research grant for autoimmune diseases from the Japanese Ministry of Health and Welfare.

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(1989) J. Immunol. 142, 3378. [4] Ahmed, S.A., Dauphinee, M.J., Montoya, A.I. and Talal, N. (1989) J. Immunol. 142, 2647. [5] Stimson, W.H. (1988) Scand. J. Immunol. 28, 345. [6] Cohen, J.H.M., Danel, L., Cordier, G., Saez, S. and Revillard, J.-P. (1983) J. Immunol. 131, 2767. [7] Seaman, W.E., Blackman, M.A., Gindhart, T.D., Roubinian, J.R., Loeb, J.M. and Talal, N. (1978) J. lmmunol. 121, 2193. [8] Seaman, W.E., Gindhart, T,D., Greenspan, J.S., Blackman, M.A. and Talal, N. (1979) J. Immunol. 122, 2541. [9] Screpanti, I., Santoni, A., Gulino, A., Herberman, R.B. and Frati, L. (1987) Cell. Immunol. 106, 191. [10] Yodoi, J., Teshigawara, K., Nikaido, T., Fukui, K., Noma, T., Takigawa, M., Sasaki, M., Minato, N., Tsudo, M., Uchiyama, T. and Maeda, M. (1985) J. Immunol. 134, 1623. [11] Phillips, J.H., Warner, N.L. and Lanier, L.L. (1983/84) Natl. Immun. Cell Growth Reg. 3, 73. [12] Leake, R.E. and Habib, F. (1987) in: Steroid Hormones (B. Green and R.E. Leake, Eds.) pp. 67 98, 1RL Press, Washington, DC. [13] Webster, N.J.G., Green, S., Jin, J.R. and Chambon, P. (1988) Cell 54, 199. [14] Ritson, A. and Bulmer, J.N. (1987) Immunology 62, 329. [15] Diczfalusy, E. and Troen, P. (1961) Vitam. Horm. 19, 229. [16] Manaseki, S. and Searle, R.F. (1989) Cell. Immunol. 121, 166.

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