Modulation of nonspecific cell-mediated growth inhibition by estrogen metabolites

Immunopharmacology, 10 (1985) 127-135 Elsevier

127

IMO00296

Modulation of Nonspecific Cell-Mediated Growth Inhibition by Estrogen Metabolites Richard W. Pfeifer* and Rachel M. Patterson Systemic Toxicology Branch~National Toxicology Program, P.O. Box 12233, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, U.S.A. (Received 15 August 1984; accepted 16 July 1985)

Al~traet: Chronic exposure of mice to estrogens such as 17-fl estradiol and diethylstilbestrol inhibits natural killer cell-mediated cytotoxicity in vivo. In this report, we investigated the direct in vitro effects of 17-fl estradiol and its major metabolites on nonspecific effector cell function measured as the ability of naive lymphocytes to inhibit the growth of the YAC-1 lymphoma, a classical natural killer-sensitive target cell. Without exception, the effects of individual estrogen metabolites on the growth inhibitory properties of these cells were accompanied, at every concentration of compound, by identical effects on the blastogenic response of lymphocytes to the T cell lectin phytohemagglutinin. These observations suggested membrane-mediated immunomodulation of lymphocyte function by estrogen metabolltes. As suggested by previous studies with quinone metabolites of benzene, the catechol estrogen metabolite 2-OH estrone was significantly more potent than the parent compound at suppressing lymphocyte functions in vitro; however, dosing regimens of 2-OH estrone that suppressed blastogenic response in vivo failed to inhibit nonspecific cell-mediated growth inhibition. Key words:

Estrogen metabolites; Nonspecific growth inhibition; Natural killing; Membrane effects

Introduction

Nonspecific cell-mediated cytotoxicity refers to the observation that naive rodents, as well as human donors, demonstrate significant lymphocyte-mediated cytotoxicity against various syngeneic, allogeneic or xenogeneic tumor cells and some normal cells (Kiessling et al., 1975a,b; Herberman and Holden, 1978; Riccardi et al., 1979). The failure to find increased frequencies of polyclonal tumors in nude mice and neonatally thymectomized animals, and in immunosuppressed human subjects, has discredited the T cell as a primary effector of tumor immunosurveillance (Mtller and MSller, 1976). Therefore, increased interest has been expressed in nonspecific host defense mechanisms such as natural killer (NK) activity as mediators of immunosurveillance. In toxicology, there may be a significant relationship between suppression of natural

host defenses and the enhanced expression of chemically initiated neoplastic lesions. Suspected carcinogens often suppress NK activity, along with other immune parameters, at tumorigenic doses (Keller, 1983). Female mice treated neonatally with diethylstilbestrol (Kalland and Forsberg, 1981) and adult animals chronically administered 17-fl estradiol (Seaman et al., 1979a) demonstrate suppression of NK activity. In adult animals, the loss of NK function was not thymus-dependent and appeared linked to suppression of interferon-induced maturation; in

* Present address: Department of Pharmacology and Toxicology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907, U.S.A. Abbreviations: NK, natural killer; PHA, phytohemagghitinin; EC/TC, effector/target cell; ACT, Tris-buffered ammonium chloride.

0162-3109/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

128 vitro incubation with 17-fl estradiol did not affect NK activity (Seaman et al., 1978; 1979b). The objectives of this report were two-fold. First, we aimed to standardize conditions for the measurement of nonspecific effector cell function in terms of growth inhibition of the YAC-1 lymphoma target cell; relative to the Slchromium release methodology traditionally used to measure nonspecific cell-mediated cytotoxicity, this approach may offer greater convenience and sensitivity. The second objective was to evaluate the role of estrogen metabolites in modulation of nonspecific growth inhibition via direct membrane effects. We previously suggested that estrogen metabolites modulated lymphocyte activation at the cell surface because of identical effects, for every in vitro concentration of compound, on phytohemagglutinin (PHA)-induced lymphocyte agglutination, an early event of cell activation, and blastogenesis (Pfeifer and Patterson, 1985); herein, we suggest that a similar in vitro modulation of nonspecific effector cell function occurs. As suggested by previous studies with quinone metabolites of benzene, 2-OH estrone was the most potent estrogen at inhibiting lymphocyte functions in vitro.

Materials and Methods

Preparation of cell suspensions After sacrifice by CO 2 asphyxia, spleens and thymuses were removed aseptically from 6-8-week-old female CD (Sprague-Dawley) rats (Charles River, Portage, MI). Two organs were pooled per 10 ml RPMI-1640 tissue culture medium (Gibco; Flow Laboratories) supplemented with 10% heat-inactivated fetal calf serum (Gibco) and Hepes buffer. Tissue was teased apart, disaggregated by aspiration in a 10-ml syringe and the debris allowed to settle out for 5 rain on ice. 5-ml aliquots of cell suspension were washed once, centrifuged at 300 x g for 5 min, and resuspended in Tris-buffered ammonium chloride (ACT) solution to lyse contaminating erythrocytes; spleen and thymus cell suspensions were diluted 1:6 (v/v) and kept for 5 min

on ice. The leukocytes were centrifuged, the supernatant (containing erythrocyte stroma) decanted and the cells washed before chemical exposure. The nucleated cell count per rat spleen was 5-6 x 108 cells; the thymuses contained approximately 7.5-9 x 108 cells. Organs were removed from 6-8-week-old female B6C3F1 mice (Charles River), pooled at four organs per 10 ml of medium and cell suspensions treated with ACT solution. The nucleated cell count per mouse spleen was 108 cells. During the preparation of lymphoid cell suspensions, the cells were kept on ice to maintain viability.

Chemical exposure 17-fl estradiol and three of the major metabolites, 2-OH estrone, 2-OCH3 estrone and estrone (Fig. 1), were preincubated, at various concentrations, with lymphoid cells to determine the effects on nonspecific cell-mediated growth inhibition of the YAC-1 lymphoma cell line; all estrogens were obtained from Sigma (St. Louis, MO). After exposure to ACT solution, cells were resuspended in serumfree RPMI-1640 medium. Estrogens were prepared a s 10 - 2 M stock solutions in ethanol and the highest concentration of solvent to which the cells were exposed was 5%. This maximum concentration caused no toxicity to lymphocyte functions. The chemical incubation step was for 30 min at 370C in a humidified, 5% CO2/air incubator at a cell concentration of 2 x 106 cells/ml. Although chemical preincubations were routinely performed for 30 min at 370C, a 5 min incubation at either 37°C or 0°C was sufficient to induce the response. After chemical exposure, the cells were washed and resuspended in 0.9 ml tissue culture medium (RPMI-1640 medium supplemented with 10% fetal calf serum), 1% (2 mM) glutamine and 0.1% (0.05 mg/ml) gentamycin at appropriate cell concentrations to yield a series of effector cell/target cell (EC/TC) ratios of 10, 50 and 100:1.

Nonspecific cell-mediated growth inhibition To these mixing tubes, 0.1-ml aliquots (5 x 104

129 OH

-

4- H YDROXYE STRADIOL"--~ 4-HYDROXYESTRONE

ESTETROL

ESTRADIOL

HO ~'~'~-j~',,-f

HO

2-METHOXYESTRONE

Jr

HO

2- HYDROXYESTRONE

2-METHOXYESTRADIOL

2- HYDROXYESTRADIOL

HO ESTRONE

2-HYDROXYESTRIOL

16-~ HYDROXYESTRONE

',

ESTRIOL

Fig. 1. Molecular structures of major metabolitesof 17-/3estradiol: the catechol estrogen metabolite, 2-OH estrone, its modifiedderivative, 2-OCH3 estrone, estrone and 16 ~t-OHestrone.

cells) of washed, log-phase YAC-1 lymphoma cells, routinely passaged in vitro in this laboratory, were added. The tubes were triturated gently and 0.2-ml aliquots dispensed in triplicate wells in fiat-bottom microtiter plates. The cell suspensions were centrifuged at 300 x g for 1 rain to promote cellular interaction and then incubated for 4 h at 37°C in a humidified, 5% CO2/air incubator. At the end of this time, cultures were pulsed with [3H]thymidine (6.7 Ci/mmol; New England Nuclear) at 1.0 #Ci/ well. After an additional overnight incubation (18 h total), the cultures were harvested onto fiberglass strips with a multiple automatic sample harvester. The filters were dried, placed in minivials and counted by scintillation spectrophotometry. The nonspecific growth inhibition (/) was calculated, relative to thymus cell suspensions which demonstrated no growth inhibition against YAC-1 lymphoma ceils, using the equation:

I=

1-

cpm(spleen ceils

+ YAC-1)

x 100

c p m ( t h y m u s cells + YAC-1)

Chemical modulation was represented as a percent of the control response (Fig. 2). In general, one standard deviation did not vary more than 5%

from the mean of triplicate samples; statistically significant treatment effects were indicated at the p < 0.05 level by Student's t-test.

PHA-induced lymphocyte blastogenesis Mouse splenic lymphocytes were prepared and exposed to estrogens in a similar manner to that described for the growth inhibition assay. The cells were washed and resuspended in RPMI-1640 medium supplemented with 5% heat-inactivated fresh-frozen human AB serum (Irvine Scientific), 1% (2 mM) glutamine and 0.1% (0.05 mg/ml) gentamycin at a cell concentration of 106 cells/ml. PHA was added to obtain a final concentration of 1.0 #g/ml and the mixing tube rack gently shaken. The suspended cells were dispensed in triplicate wells (2 x 10 s cells/0.2-ml aliquot) in flat-bottom microtiter plates and cultured in a humidified, 5% CO2/air incubator at 37°C for 72 h (the optimal time point for blastogenic response). Plates were pulsed with [3H]thymidine 6 h before harvesting onto fiberglass strips with a multiple automatic sample harvester. The filters were dried, placed in minivials and counted by scintillation spectrophotometry. Chemical modulation was represented as a percent of the control response (Fig. 2). In general, for cpm

130 TABLE 1 Rat nonspecific cell-mediated growth inhibition Target"

EC/TC ratio

Growth inhibitionc (%)

Incorporation (cpm) b

Thymus YAC-1

MBL-I

0:I 10:1 50:1 100:1

118885 116635 109046 106058

0:1 10:1 50:1 I00:1

77207 87135 95398 91 113

Spleen 4+ 44-

6831 3913 2335 3043

122362 75415 25950 3908

4444-

3557 7413 1914 369

35 76 96

4- 2149 + 4612 4- 1836 + 3024

88944 75929 20815 4020

+ 444-

7858 3041 962 994

13 78 96

YAC-I lymphoma or MBL-2 leukemia target cells were added to yield a series of EC/TC ratios and the growth inhibition measured after an 18 h incubation period. b Erythrocytes were removed from cell suspensions by incubating with ACT. Values given represent mean 4- S.D. c The growth inhibition was calculated relative to thymocyte-tumor cell suspensions as described in Materials and Methods.

a

greater t h a n 1000, one s t a n d a r d d e v i a t i o n did n o t v a r y m o r e t h a n 10% f r o m the m e a n o f triplicate samples; statistical significance was i n d i c a t e d a t the p < 0.05 level b y S t u d e n t ' s t-test. Blastogenic response r a n g e d b e t w e e n 13 186 a n d 33449 c p m for lectin c o n c e n t r a t i o n s b e t w e e n 0.5 a n d 2.0 #g/ml. Results

Nonspecific cell-mediated growth inhibition: characteristics of the effector cell

cell growth; these cells were used as a c o n t r o l for cell c r o w d i n g effects (Tables I a n d III). A t all E C / T C ratios, t h y m u s a n d spleen cell i n c o r p o r a t i o n o f label was insignificant relative to t u m o r cell inc o r p o r a t i o n . Effector cells m e d i a t i n g nonspecific g r o w t h i n h i b i t i o n were enriched in plastic n o n a d h e r e n t cell p o p u l a t i o n s ( T a b l e II), thus i n d i c a t i n g t h a t a d h e r e n t cells ( m a c r o p h a g e s ) were n o t signific a n t p a r t i c i p a n t s in the cytostasis. TABLE II Rat nonspecific cell-mediated growth inhibition: plastic adher-

C o n s o n a n t with studies o f N K c y t o t o x i c i t y as m e a s u r e d b y s l C r release, m o u s e splenic l y m p h o cytes d e r i v e d f r o m naive a n i m a l s d e m o n s t r a t e d g r o w t h i n h i b i t i o n o f the Y A C - 1 l y m p h o m a , the classical N K - s e n s i t i v e t a r g e t cell, b u t d i d n o t d e m o n s t r a t e activity a g a i n s t the N K - r e s i s t a n t M B L - 2 l e u k e m i a (Table III). A l t e r n a t i v e l y , r a t splenic lymp h o c y t e s d e m o n s t r a t e d significant g r o w t h inhibition a g a i n s t b o t h t a r g e t cells ( T a b l e I), suggesting a g r e a t e r N K p o t e n c y ; after an 18 h i n c u b a t i o n , a 100:1 E C / T C r a t i o resulted in n e a r l y c o m p l e t e s u p p r e s s i o n o f t u m o r cell p r o l i f e r a t i o n . A l s o c o n s o n a n t with studies o f N K c y t o t o x i c i t y , thymocytes demonstrated no inhibition of tumor

eno~

a

Target

Growth inhibition Unpurified

Nonadherent

YAC-1

62

75*

MBL-2

76

89*

" Data collection and definition of terms as described for Table I. Incubation was for 1 h at 370C on a plastic tissue culture flask (Falcon) lying flat: recovery of splenic adherent cells was 5%; nonadherent cells, 78%. The EC/TC ratio was 50:1. The experiment was repeated and similar results obtained. * Significantly different from unpurified cells.

131

Effects of estrogen metabolites on nonspecific cellmediated growth inhibition

~J 100 g c

For every in vitro concentration of compound, estrogen metabolites modulated nonspecific cell-mediated growth inhibition, and PHA-induced lymphocyte blastogenesis, in an identical manner (Fig. 2). At pharmacologically relevant concentrations (less than 100 #M in vitro), the catechol estrogen metabolite, 2-OH estrone, demonstrated significant suppression of nonspecific growth inhibition and blastogenesis relative to the parent compound, 17fl estradiol, and other metabolites (Fig. 2). However, at dosing regimens of 2-OH estrone that significantly inhibited splenic lymphocyte blastogenesis in viva (36% of the control response), nonspecific effector cell function remained unimpaired (Table IV). Preincubation with in vitro pharmacological concentrations of the metabolites estrone and 2-OCH3 estrone enhanced both PHA-induced lymphocyte blastogenesis and nonspecific growth inhibition, whereas 17-fl estradiol had negligible effects (Fig. 2). At 100 #M concentrations (not pharmacologically relevant), all the estrogens were suppressive to cell function.

Discussion

Growth inhibition versus cytotoxicity Lymphocyte-mediated growth inhibition does not distinguish between cytostatic and lytic effector cell interactions with the tumor cell targets. The results reported in the literature for the measurement of N K cell-mediated cytotoxicity do not address the possible importance of growth inhibition (Cerottini and Brunner, 1974; Herberman and Holden, 1978). Although there have been previous reports of assays measuring T cell killing in a growth inhibition protocol (Grant et al., 1973), measurement of N K activity by this methodology has only recently been reported (Hagner, 1984) despite the possibility that suppression of tumor growth in viva may be more a function of contact inhibition than lysis. Presumably, one reason why T cell-mediated

Estrone

2-OCH3 Estrone

17-# Estrodiol

2-OH Estrone

o o c~

i

0

-100 '1' ~ '1o'~ ,go ', '5 '1o'501o'o '1' 5 '1o'~'1oo '1 '~ ',o'~o'1oo

Concentration(JJm) Fig. 2. Illustration of the identical effects, relative to in vitro concentration, of individual estrogen metabolites (estrone, 2OCH3 estrone, 17flestradiol and 2-OH estrone) on rat nonspecificcell-mediatedgrowthinhibition(Fq)and PHA-inducedblastogenesis (11). The effectsof chemicalpreincubationare indicated as a percentage of control response. For the growth inhibition, the EC/TC ratio was 40:I, for the blastogenicresponse, the final concentration of PHA was 1 /~g/ml. Murine nonspecific cell-mediatedgrowthinhibitionwas modulatedin a similarmanner in vitro. growth inhibition is not significantly employed is the problem encountered with proliferation of effector cells. We found, for example, that the increased incorporation of [3H]thymidine by rapidly proliferating spleen cells taken from DBA/2 (H-2 d) mice alloimmunized 9-13 days earlier with 107 MBL-2 leukemia cells (H-2 b) (Schultz et al., 1976a,b), was 3-6 times that of unsensitized control cells. At higher EC/TC ratios, e.g. greater than 40:1, this label incorporation by proliferating effector cells becomes significant relative to tumor cell incorporation. However, although it is possible that induction of nonspecific effector cell activity in viva with biologicals such as interferon or poly I:C may result in significant spleen cell proliferation, a simple analysis of in viva or in vitro chemical effects on growth inhibition mediated by lymphoid cells derived from naive animals should present no problem. Furthermore, in situations in which significant effector cell proliferation exists, enrichment for cytotoxic T cells

132 or nonspecific effector cells on nylon wool columns (Berke et al., 1972; Pfeifer and Bosmann, 1982) and the use of lower EC/TC ratios or shorter incubation times for assays may represent potential solutions.

Characteristics of the effector cell As reported for NK cell-mediated cytotoxicity (Herberman and Holden, 1978), both rat and mouse splenic lymphocytes derived from naive animals demonstrated growth inhibition of the YAC1 lymphoma, the classical NK-sensitive target cell (Tables I and III). After an 18 h incubation (Altman and Rapp, 1978), complete suppression of tumor cell proliferation was observed for rat lymphocytes at an EC/TC ratio of 100:1. Therefore, rat lymphocytes demonstrated somewhat greater potency (Tables I and III). This observation is in accord with studies demonstrating species differences for NK cell and associated K cell activity. Although K cell activity is negligible in the spleens of most mouse strains (Berger and Amos, 1977), rat spleens demonstrate significantly greater activity (Pfeifer and Bosmann, 1982). The demonstration of growth inhibition against a NK-resistant tumor line, MBL-2 (Table I), by rat lymphocytes is consistent with observations of rat NK cytotoxicity against

other NK-resistant mouse tumor cell lines such as L1210 leukemia (Pfeifer and Bosmann, 1982) and P815 mastocytoma (Britten et al., 1984). It has been suggested recently that this represents an activation of the resting NK cell (Britten et al., 1984). Mouse lymphocytes failed to demonstrate significant growth inhibition against MBL-2 tumor cells (Table III). In further support of species differences in NK activity, Reynolds et al (1981) report difficulties in detecting large granular lymphocytes in mouse blood or spleens. In the mouse and rat, NK cells mediating cytotoxicity are prevalent in the spleen, lung and peripheral blood but are absent from the thymus (Herberman and Holden, 1978; Reynolds et al., 1981). In this report, a similar anatomical distribution for nonspecific cell-mediated growth inhibition was found for spleen and thymus, and thymocytes were therefore used to control for cell crowding effects in culture (Herberman and Holden, 1978). Also, in agreement with demonstrations that the effector cell mediating NK and K cell cytotoxicity in naive rodents is nonadherent to nylon wool and plastic (Herberman and Holden, 1978; Pfeifer and Bosmann, 1982), effector cells mediating NK growth inhibition were enriched for in plastic nonadherent spleen cell populations (Table II).

TABLE III Murine nonspecific cell-mediated growth inhibition a

Target

EC/TC ratio

Growth inhibition

Incorporation (cpm)

(%) Spleen

Thymus YAC-1

MBL-1

0:1 10:1 50:1 100:I

58176 59357 51923 53642

+ 444-

2660 4474 3041 4090

57790 50527 30162 19726

• 2506 • 6604 ± 1301 • 655

NS b 42 63

0:1 10:1 50:1 100:1

30128 31806 30502 28433

4-4± 4-

712 1815 2369 1393

24177 30244 24153 23231

4444-

NS NS NS

a Data collection and definition of terms as described for Table I. b Nonsignificant difference.

2336 2796 6404 2657

133

Modulation of nonspecific cell-media[ed growth inhibition by estrogen metabolites: similarity to effects on lymphocyte activation For every in vitro concentration of compound, estrogen metabolites modulated nonspecific cell-mediated growth inhibition, a lymphocyte effector cell function, and lymphocyte blastogenic response to PHA in an identical manner (Fig. 2). As previously suggested for the correlative effects of estrogens on blastogenesis and PHA-induced lymphocyte agglutination (Pfeifer and Patterson, 1985), an early event of cell activation, this modulation suggests that effector cell function is altered early on at the cell surface in the lytic interaction with target cell. It is now recognized that membrane alterations associated with lymphocyte activation such as the projection of microvilli and the clustering of microfilaments at certain points on the cell surface are also associated with the expression of contact-mediated killing of nucleated mammalian target cells by cytotoxic T cells, antibody-dependent K cells and N K cells (Loor, 1980; Sanderson, 1981; Ryser and Vassalli, 1981; Katz et al., 1982; Weltzin et al., 1983; Bubbers and Henney, 1975). The catechol estrogen 2-OH estrone, a major metabolite of 17-fl estradiol (Fishman and Martucci, 1980), is a potential substrate for oxidation to reactive quinone intermediates. Predictably, 2-OH estrone demonstrated a significantly greater potency at suppressing nonspecific cell-mediated growth inhibition and blastogenesis in vitro than the parent compound, 17-fl estradiol, and other metabolites (Fig. 2). At the lowest inhibitory concentrations, this sublethal suppression of function presumably reflects the particular reactivity of sulfhydryl groups associated with cytoskeletal proteins such as actin and tubulin, and which are prerequisites for the assembly of microtubules and micro filaments (for reviews, see Pfeifer and Irons, 1983; 1984). Such a mechanism 'has been suggested for immunotoxicity demonstrated by quinone oxidation products of hydroquinone and catechol, major metabolites of benzene (Pfeifer and Irons, 1981; Irons et al., 1981; Wierda and Irons, 1982). However, at dosing regimens of 2-OH estrone

that significantly inhibited splenic lymphocyte PHA-induced blastogenesis in vivo, nonspecific effector cell function remained unimpaired (Table IV). Although chronic administration of estrogens may suppress N K function (Seaman et al., 1979b), the short-term exposure reported here suggested the lymphocyte proliferative response to be more sensitive to inhibition than the effector cell function. The in vitro enhancement of N K cell-mediated growth inhibition by estrone and 2-OCHa estrone is presumably related to the membrane partitioning effects of these metabolites implicated in the enhancement of the lymphocyte response to suboptimal agglutinating concentrations of P H A (Pfeifer and Patterson, 1985). It appears likely that the hypocellularity and suppression of lymphocyte proliferative responses and N K cell function after estrogen exposure is linked to the production of catechol metabolites; these compounds may then undergo oxidation to reactive quinone intermediates in appropriate target tissues. Immunopharmacological responses to estrogens in man may be of most significance to patients with breast or prostatic cancer being treated with diethylstilbestrol (Ablin et al., 1976). TABLE IV Effect of 2-OH estrone on nonspecificcell-mediatedgrowth inhibition after in vivo exposure Groupa

Control Corn oil 2-OH Estrone

Growth inhibition for an EC/TC ratio of: 50

100

46 51 47

66 77* 74

a 4 mice per group were injected i.p. daily for 2 days with 8 mg/kg 2-OH estrone dissolvedin corn oil. 48 h later triplicate cultures of pooled, ACT-purifiedspleencell suspensions were assayed for nonspecificgrowth inhibition as describedin Materials and Methods. At this exposure, the blastogenic response of spleniclymphocytesfrom 2-OH estrone-treatedanimals was 36% of the corn oil control response; no significant differencewas observedfor 17-flestradiolanimals (Pfeiferand Patterson, 1985). * Statistically significantdifferencefor the corn oil group relative to untreated animals.

134

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