Activity and dexamethasone sensitivity of natural cytotoxic cell subpopulations

CELLULAR

IMMUNOLOGY

72, 113-121 (1982)

Activity and Dexamethasone Sensitivity Cytotoxic Cell Subpopulations PAUL

Q. PATEK,’

JOHN LESLIE COLLINS,

of Natural

AND MELVIN

COHN

The Salk Institute, P.O. Box 85800, San Diego. Calfornia 92138 Received April 20, 1982; accepted June 9, 1982 Natural cytotoxic (NC) activity is the natural cell-mediated cytotoxic activity of normal spleen cells directed at fibroblast targets. Treatment of spleen cells with dexamethasone, a synthetic glucocorticosteroid, reveals two populations of NC effecters: one population whose cytolytic activity is inhibited by dexamethasone (DEXs) and another which is resistant to inhibition by dexamethasone (DEXn). The short-term culturing of spleen cells results in a shift in the population of splenic NC effecters from predominantly DEXs to predominantly DEXR. This shift also occurs when spleen cells are treated with conditioned medium from which the short-term cultured DEXR populations were derived.

INTRODUCTION Cell-mediated cytotoxic activity against some tumor cells can be detected in lymphocytes from normal mice and other species. This activity or the cells responsible for this activity are referred to as natural killers (l-3), spontaneous killers (4), natural cytotoxic cells (5, 6), anomalous killing (2, 7) activated lymphocyte killing (2), and spontaneous cell-mediated cytotoxicity (8). It is not clear if each of these effector cells, or activities mediated by them, is unique, and if those cells or activities referred to by a common name are the same. It is generally accepted that cytotoxicity against lymphoid and sarcoma targets is mediated by different cell populations (6, 9); however, there is evidence that these populations share some characteristics and may be closely related (1, 6, 9). Although the various classes of natural cell-mediated cytotoxicity are not clearly defined, we will use the term natural cytotoxic cell (NC)? activity to describe the cytotoxicity reported here. There are several reports that the properties of nonimmune cytotoxic activity can be altered by in vitro culturing of spleen cells under various conditions (2, 7, 10) or by treatment of the effecters with mediators such as interleukin-2 (1 l), interferon (3, 12), or tuftsin (13). These alterations include increases in the level of cytotoxic activity as well as changes in the recognition specificity and surface antigens of the effecters. There is evidence that the augmentation of NC activity by interferon results from recruitment of NC cells from a stem cell population that previously had no NC activity (14, 15). Little is known about how other mediators function to alter ’ To whom correspondence should be addressed. * Abbreviations used: NC, natural cytotoxic cell; DEXS, dexamethasone sensitive: DEXR. dexamethasone resistant. 113 0008-8749/82/

130 113-09$02.00/O

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

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PATEK, COLLINS, AND COHN

natural cell-mediated cytotoxic activity; the question remains whether all mediators function by recruiting effecters from a noncytotoxic population or by affecting the original cytotoxic effector population. Here we report the activation of NC activity as a result of either short-term culturing of spleen cells or treatment of spleen cells with conditioned media derived from the short-term cultures. The activated NC population is not the same as the NC population present in freshly dissociated spleen cells in that the activity of the former is dexamethasone resistant while that of the latter is dexamethasone sensitive. MATERIALS AND METHODS Target Cells 1. B/C-N is an anchorage-dependent cloned fibroblastic cell line derived by us from Balb/c fetal tissue; it is relatively resistant to NC lysis (1). 2. 1OME HD A.5 R. 1 ( 10ME) is a cloned transformed cell line derived from B/C-N after treatment with 3-methylcholanthrene- 11,12dihydroepoxide (supplied by NC1 Carcinogenesis Research Program). This transformed variant was selected by growth in 0.3% agarose (i.e., anchorage independent) and then ring cloned; it is relatively sensitive to NC lysis (1). The Chromium Release Assay for NC-Mediated Lysis Spleens from Balb/c mice (Salk Institute colony) were dissociated in balanced salt solution, pH 7.2. Cells were washed once and then resuspended in supplemented RPM1 1640 medium plus 10% FBS, as previously described (16). “0-Labeled targets were prepared as previously described (17). Assays were done in microtiter plates containing 104 “Cr-labeled targets per well in 100 ~1 medium. The assay vessels were gently rocked for approximately 15 hr at 37°C and then centrifuged at 160g for 5 min. Half of the supernate was collected to determine the amount of 5’Cr released. The percentage specific “Cr released was calculated by the formula sample cpm - spontaneous cpm % specific ‘Cr release = total cpm - spontaneous cpm * All data points represent the arithmetic mean of triplicate samples. The spontaneous release of “Cr ranged from 20 to 3 1%. Relative effectiveness of the NC activity in spleen cell populations can be assessed by comparing the number of effector cells required to give an equivalent amount of chromium release, or by comparing the amount of chromium released at any one effector:target ratio. The level of NC activity in a population is a function of the number of effecters and the level of activity of each effector. 5-Day spleen cell cultures, conditioned medium, and conditioned medium-treated spleen cells Nucleated spleen cells ( 10’) were plated in 60-mm tissue culture dishes containing 5 ml of supplemented RPM1 1640 medium plus 10% fetal calf serum (16). After 5 days at 37°C the cells were harvested (0.25-0.4 X 10’ viable cells per dish). The conditioned medium is the supernatant from these cultures. The conditioned me-

DEXAMETHASONE

SENSITIVITY OF NC CELLS

115

60

I

0

2.5 CONCENTRATTKIN OF CEXAMETHASONE I X KT6M 1

,

5.0

FIG. 1. Dose-dependent inhibition of NC activity in freshly dissociated spleen cells by dexamethasone. Spleen cell: 1OME target ratios were: 40: I (V), 20: 1 (O), 10:1 (0).

dium can be stored at 4°C and will maintain its NC-augmentation potential for more than a year. The conditioned medium-treated spleen cells were prepared by incubating 2-10 X lo6 freshly dissociated spleen cells per milliliter of conditioned medium at 37°C. After 5-10 hr the cells are harvested and washed once before being assayedfor NC activity (greater than 80% of the cells were recovered). Spleen cells or targets were treated for 5 to 10 hr because this culture period results in the maximum decreasein NC activity of untreated cells and therefore the lowest background level of unstimulated NC activity (see Fig. 3). RESULTS

A Major Proportion of the NCActivity in Freshly DissociatedSpleenCells Is Sensitive to Dexamethasone(DEX’) Figure 1 shows the NC-mediated lysis of 1OME and the dose-dependent inhibition of that activity by dexamethasone. In this experiment dexamethasone (Sigma, St. Louis, MO.) was added at the beginning of a 14-hr NC assay. At a dexamethasone concentration of 2.5 X 10m8M the NC activity of fresh spleen cells was markedly inhibited (greater than 50% inhibition). In order to demonstrate whether the reduction in “Cr release resulted from a reduction in NC effector cell activity or from an increase in target cell resistance to NC activity, we treated spleen cells or target cells with dexamethasone prior to their addition to the NC assay. Spleen cells and targets were incubated in medium or medium containing dexamethasone (2.5 X lop8 A4) for 30 min at 37°C and then washed once before they were used in the NC assay. The results (Table 1) show that the effect of dexamethasone is to inhibit the activity of the NC effector cells. Dexamethasone pretreatment of the targets did not alter the NC activity. Because there is similar inhibition of NC activity by pretreatment of effecters with dexamethasone

116

PATEK, COLLINS, AND COHN TABLE 1 NC Assay Using Dexamethasone-Pretreated Targets ( 1OME) and/or Spleen Cells Pretreatment with 2.5 X IO-’ M dexamethasone Freshly dissociated spleen cells

1OME Target

+ +

+ +

Percentage specific “Cr release at spleen cell: IOME IO:1 23 12 (48) 23 (0) 9 (61)

20: 1 34 15 (55) 34 (0) 13 (62)

40: 1 43 24 (45) 39 (9) 18 (58)

a Percent inhibition by dexamethasone in parentheses.

and the continuous presence of dexamethasone during the assay period, we assume the effect of the drug is rapid and irreversible within the assay period. A Major Proportion of the NC Activity in S-Day Cultures of Spleen Cells Is Resistant to Dexamethasone (DEXR) Spleen cells, from immunologically naive mice, maintained in medium for 5 days have been shown to exhibit significantly higher NC activity per cell than freshly dissociated spleen cells (2, 7). Figure 2 shows that this increase in total NC activity per cell in the cultured spleen cells is accompanied by an increase in DEXR NC activity. The DEXR activity in the 5-day cultured spleen accounts for the majority

5DPYFlIENCELl-S + Ex (IO-W)

FRESH GREEN CELLS + OEX (lo-~ M)

SREEN

CELLS : TARGETS

FIG. 2. Effect of 2.5 X lo-@ M dexamethasone on NC activity in freshly dissociated spleen cells and spleen cells cultured for 5 days. The target is IOME. The bars represent the standard deviations of triplicate samples.

DEXAMETHASONE

SENSITIVITY

OF NC CELLS

117

of the activity of this population and is greater than the combined DEXR and DEXS activity of fresh spleen cells. The level of DEXR and DEXS NC activity as a function of the number of days in culture is shown in Fig. 3. In this experiment spleen cells were placed in culture for varying lengths of time before assaying their NC activity in the presence and absence of dexamethasone. The level of DEXR NC activity is measured as the percentage specific “Cr release in the presence of dexamethasone, while the level of DEXS NC activity is calculated by subtracting the DEXR activity from the total NC activity. Initially the activity of the DEXR population is relatively low compared to that of the DEXS population; thereafter, the DEXR activity increases throughout the 5-day culture period. In contrast, initially the DEXS NC activity is high, and then rapidly declines to a plateau which is approximately half the initial level. Initial decreasesfollowed by increases in total NC activity as a result of culturing spleen cells in vitro have been observed by others (7). Here we show that the increase in total NC activity over the 5-day culture period is a result of the increase in DEXR NC activity, and in conjunction with the increase in DEXR NC activity is a decrease in the DEXS NC activity.

003 I

2 tXiC3

3

4

5

IN CULTURE

FIG. 3. Kinetics of the NC activity against IOME, and the sensitivity of that activity to dexamethasone. Total NC activity (V), DEXR (IO-* M) NC activity (o), and DEXs NC activity (calculated by subtracting the DEXR activity from the total activity) (0). The bars represent the standard deviations of triplicate samples. Effector:target is 40: 1,

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PATEK, COLLINS, AND COHN TABLE 2

Effect of Conditioned Media on the Dexamethasone Sensitivity of NC Activity against 1OME Percentage specific Wr release DEXR NC activity at spleen cell: 1OME (2.5 X lo-* M DEX)

Total NC activity at spleen cell: 1OME Spleen cell population Freshly dissociated, assayed immediately Incubated 10 hr in conditioned medium, then assayed Incubated 10 hr in nonconditioned medium, then assayed

20: 1

5:l

1O:l

20: 1

5:l

1O:l

28

37

49

13 (54)”

19 (49)

22 (55)

57

67

72

49 (14)

57 (15)

63 (12)

21

26

38

7 (67)

11 (58)

23 (39)

’ Percentage inhibition by dexamethasone in parentheses.

The Conditioned Culture Medium of S-Day Spleen Cells Increasesthe Rate of Appearance of DEXR NC Activity of Freshly Dissociated Spleen Cells We tested whether some factor released into the culture medium during the 5day incubation period was responsible for the increase in DEXR NC activity over the 5-day culture period. To do this, freshly dissociated spleen cells were incubated for 5-10 hr either in the culture medium from which the 5-day spleen cells were recovered or in medium. Freshly dissociated spleen cells incubated in this conditioned medium show more NC activity than similarly treated spleen cells incubated in medium (Table 2 and Fig. 4). The higher total NC activity of the conditioned medium-treated spleen cells is, like that of the cultured spleen cells, largely attributable to an increase in DEXR NC activity with only a modest loss of the DEXS NC activity (Table 2). Figure 4 shows that culturing spleen cells for 5 days or treating freshly dissociated spleen cells with the conditioned media from the cultured spleen cells increases the total amount of NC activity; however, the relative sensitivity of an NC-sensitive target (10ME) and an NC-resistant target (B/C-N) remains unchanged. DISCUSSION We describe here two populations of NC effector cells whose activity can be distinguished by their differential sensitivity to dexamethasone. We also describe culture conditions and a supernatant factor which affect both the level of spleen cell NC activity and the level of NC activity that is sensitive to dexamethasone. Dexamethasone has been shown to be directly toxic to some lymphoid cells (18). How dexamethasone reduces NC activity is not known, and could result from any one or combination of the following: (i) inactivation or death of the NC effector cell; (ii) a differential effect on the effector activity, e.g., inhibition of the delivery of NC lytic signals from effecters or inhibition of the effector cell’s recognitive functions; (iii) the blocking of the differentiation of pre-NC cells to NC effecters; or (iv) the induction of suppressor cells which inhibit NC effecters. The fact that the inhibition

DEXAMETHASONE

119

SENSITIVITY OF NC CELLS

of NC activity occurs at concentrations of dexamethasone in the range of the binding constant of known murine dexamethasone receptors ( 19) indicates that the inhibition probably occurs as a result of binding to a dexamethasone receptor. The physiological role of this DEXS system is not known, although the sensitivity of the immune system to glucocorticosteroids is well established ( 18). The NC activity of freshly dissociated spleen cells is primarily DEXS with a minor fraction of the activity being DEXR (Figs. 1 and 2). When spleen cells are cultured for 5 days or treated with conditioned medium from which the 5-day spleen cells were recovered, there is an increase in total NC activity per spleen cell and this activity is predominantly DEXR (Fig. 2). The rate of generation of the DEXR NC effecters over the 5 days of culture shows that the DEXR NC activity per viable spleen cell in culture increased with time (Fig. 3). The increase in DEXR NC activity which results from either culturing spleen cells or treating them with conditioned medium could be caused by one or any combination of the following: (i) an increase in the number of DEXR NC cells as a result of proliferation of this population; (ii) an increase in the level of NC activity per preexisting DEXR NC cell; (iii) an enrichment of the DEXR NC activity by the selective loss of other cells, this loss

70 -

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51KM

204

FRESH-Y DSSOC4AlEO

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404 YI

IO4

204

404 w K)1

204

404

FRESCY u3souAlED StwunxJREo sxENcaJs~TEowllH 3PLEm cELs:lal?oEls molm EoIlJM : TAR6El-6

FIG. 4. NC activity in freshly dissociated spleen cells (A), freshly dissociated spleen cells treated with conditioned medium for 10 hr (B), and spleen cells cultured for 5 days (C). The targets were IOME (0) and B/C-N (0).

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PATEK, COLLINS, AND COHN

possibly including the DEXS NC cells; (iv) an acquisition of DEXR NC activity by cells that previously had no NC activity; (v) by the loss of cells that suppress NC activity; or (vi) by conversion of the DEXS NC population to DEXR (e.g., loss of dexamethasone receptors). Since spleen cells cultured for 5 days produce, and are therefore cultured in conditioned medium, one might assume that the same mechanism operates to increase the DEXR NC activity in both the 5-day cultured spleen cells and the conditioned medium-treated spleen cells. Because the DEXR activity of freshly dissociated spleen cells is increased within hours after treatment with conditioned medium this increase in DEXR NC activity cannot result from proliferation. In addition, since there is no significant loss of cells during the short treatment period, there can be no enrichment of DEXR NC activity by loss of other cells. This leaves us with the possibilities that DEXR activity must be the consequence of either an increase in the level of NC activity per preexisting DEXR NC cell, the acquisition of DEXR NC activity by cells which previously did not have NC activity, or the loss of NC suppressor cell activity. If dexamethasone induces NC suppressor cells in vitro [as they are induced in vivo by hydrocortisone (20)] then the conditioned media must contain a contrasuppressive factor. At present we do not know what factor(s) in the conditioned media is responsible for the increase in the DEXR NC activity. We do know that interferon, a known activator of NC activity (3, 12), is not detectable in our conditioned media as determined by vesticular stomatitus virus plaque reduction on L929 cells (21). Lowmolecular-weight factors, such as cyclic nucleotides (22) and tuftsin (13) (a tetrapeptide released after enzymatic cleavage of immunoglobulin G), have been shown to enhance NC activity. Although the dexamethasone sensitivity of NC cells activated by these factors is not known, it is possible that one of them is the active factor in the 5-day conditioned medium. ACKNOWLEDGMENTS We acknowledge the contribution of the Armand Hammer Cancer Workshops and the technical assistance of Ms. Scotti Brauer. We are grateful to Drs. Carl Wust and Stuart Cohen for interferon determinations. This work was supported by NC1 Grant CA-19754.

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DEXAMETHASONE 14. 15. 16. 17. 18. 19. 20. 21. 22.

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