Modulation of human nk cells by interferon and prostaglandin E2

0161-5890.‘82.101341-06503.00.0 Q 1982 Pergamon Press Ltd.

Molrcular Irn~nunologg Vol. 19, No. IO, pp. 1341-1346, 1982 Printed in Great Britain.

MODULATION

OF HUMAN NK CELLS BY INTERFERON AND PROSTAGLANDIN E2* HILLEL

Division

of Immunology,

S. KORENt Duke University (Rrreiced

and KAM H. LEUNG Medical

Center,

Durham,

NC 27710, U.S.A.

5 April 1982)

studies have shown that stimulation of human natural killer (NK) cells by poly I:C does not depend on or require monocytes. In contrast. the presence of monocytes in a mixed population of mononuclear cells stimulated by poly I:C suppresses NK activity. The suppression can be partially overcome if indomethacin (10m6 M) is added to the culture during stimulation. Culture supernatants from poly I:C stimulated monocytes do not have detectable levels of anti-viral activity but contained appreciable amounts of PGE2. Our results offer an explanation as to how NK cells may protect themselves from suppression by PGE*. We have demonstrated that IFN-activated NK cells become resistant to PGE*-mediated suppression; moreover the suppression does not require cells other than the large granular lymphocytes, the major effector cell type for NK. Taken together, the data suggest that stimulation of NK cells is dependent on and regulated by the relative levels of interferon produced by lymphocytes and PGE, produced by monocytes. Abstract-Our

INTRODUCTION

Increasing evidence has suggested that natural killer (NK)$ cells may play an important role in immunosurveillance against tumors (Herberman & Ortaldo, 1981). IFN has been shown to play a central role in the augmentation of NK activity (Bloom, 1980). Known inducers of IFN, most prominently poly I: C, have also been shown to augment NK activity (Zarling et ul., 1980). IFN has been shown to promote the differentiation of pre-NK cells to mature NK cells (Targan & Dorcy, 1980) as well as to augment the cytolytic activity of mature NK cells (Bloom, 1980). Interestingly, NK cells have also been shown to produce IFN in response to various stimuli (Bloom, 1980). The augmentation of NK activity by IFN may be at the pre-NK cell and mature NK cell levels. PGE2, which is produced primarily by mononuclear phagocytes (Kennedy et ul., 1980) and tumor cells (Droller et al., 1979) has been shown to inhibit NK activity (Droller et ul., 1980). It is conceivable that PGEz may protect the tumor from host defense mechanisms.

* This work was supported by NC1 grants CA 23354 and CA29589. t Recipient of Research Career Development Award CA 00581 from the National Cancer Institute. $ Abbreviations: NK, natural killer; IFN, interferon; poly I:C, polyinosinic acid-polycytidylic acid; PC, prostaglandin; FCS, fetal calf serum; E:T. effector cell to target cell ratio; LGL, large granular lymphocytes.

Relatively little is known about the mechanisms involved in the regulation of endogenous or activated NK levels. The focus of this study is an examination of some of the mechanisms involved in the regulation of human NK cells. The understanding of the mechanisms could permit in uivo modulation of NK activity beneficial to the host. Our data demonstrate that: (1) stimulation of mononuclear cells with poly I : C is dependent on and related by the relative levels of IFN produced by lymphocytes and PGE2 produced by monocytes; and (2) that IFN-activated NK cells are relatively resistant to suppression by PGE2.

MATERIALS

Lymphocyte

AND

METHODS

preparation

Heparinized blood, obtained from healthy adult volunteers, 20-38 yr of age, was separated according to the method of Boyum (1968) and depleted of monocytes as described later. Monocyte

depletion

by plastic adherence

Mononuclear cells were incubated in plastic tissue culture flasks in 5”/;, COz for 1 hr at a concentration of 1 x lo6 cells/ml in minimal essential medium (Microbiological Associates, Bethesda, Maryland) with 10% FCS (Flow Laboratories, Rockville, Maryland). This medium will be referred to as MEM-10%. To achieve maximal purity the nonadherent cells 1341

HILLEL

1342

S. KOREN

were subjected to the same adherence protocol as described earlier for a second time with the only difference being that the adherence time was 30 min. The nonadherent cells recovered after the second adherence contained less than 1% of monocytes as determined by the specific histochemical stains described later. Monocyte

isolation

An isolation technique recently developed in our laboratory and described in detail elsewhere (Fischer et al., 1981) was used, In short, mononuclear cells suspended at 3 x 105-4 x lo6 cells/ml in RPMI-1640 supplemented with 10% autologous serum were added to autologous serum pretreated plates (60 mm, Falcon plastics, Oxnard, California, catalogue No. 3002) for 1 hr at 37°C. The nonadherent cells were removed and the remaining adherent cells were treated with Versene 1: 5000, 5 ml/plate (Gibco Laboratories, Grand Island, New York) for 15 min at room temperature. The adherent cells were then scraped off the plate with a rubber policeman, collected and washed in RPMI-1640 supplemented with 10% FCS (RPMI-10%). These preparations contained 97-99x of peroxidasepositive monocytes which had only very low levels of spontaneous killing in a 3-hr assay against K-562, HSB or MOLT4. It should be noted that by changing the serum used for suspending the mononuclear cells from loo/;, autologous serum to 10% FCS a relatively high degree of spontaneous killing against the same targets can be achieved (Fischer et al., 1981). Histochemical

determinations

of monocytes

To determine the percentage of monocytes in different cell preparations, slides were prepared from each suspension using a cytocentrifuge (Cytospin, Shanon Southern). The cells were stained with nonspecific esterase (Fischer et al., 1981) and/or Kaplow’s peroxidase stain (Fischer et a/., 1981). A minimum of 300 cells were counted for each preparation. Preparation

of NK-enriched

and

KAM

H. LEUNG

biological Associates) supplemented with 10% FCS and 2 mM L-glutamine, 1 mM pyruvate and 1 x nonessential amino acids (Flow Laboratories). The tumor cells were passed every day and the day preceding an assay. The following human cell lines (Fischer et al., 1981) were cultured and used as target cells: K-562, a myeloid leukemia cell line, and HSB and HOLTA both of which are human T-cell lines. 51Cr release assay for NK Tumor cell targets (5 x 106-20 x 10’) were labeled with Na, 51Cr, (New England Nuclear Co., Boston, Massachusetts). All assays were carried out in triplicate in round-bottom microtiter plates (Linbro, Is-MRC-86TC) in a total volume of 0.2 ml of MEM-10%. In all assays 1 x lo4 targets were added per well. Lymphocyte effector cells were added at various concentrations to obtain appropriate final E:Ts. The microtiter plates were centrifuged for 3 min at 80 g and then incubated for l-3 hr at 37°C in a humidified 5% CO2 incubator. To harvest the assay, plates were centrifuged for 5 min at 500 g, and 100 ~1 of supernatants were removed for counting in a well type gamma counter (Searle, model 1185). Specijic cytotoxicity

The ‘A specific lysis in all experiments was calculated according to the following formula: 0/0specific lysis =

experimental cpm - control cpm x 100. maximum cpm - control cpm

The maximum release is calculated by adding 0.5% Triton X-100 to an aliquot of target cells. Control release is defined as the cpm released from target cells incubated with medium alone; this value was usually 3-5x of the maximum and never exceeded 10%. All data presented are from triplicate determinations with the SEs indicated.

cells

LGL were obtained by Percoll density gradients as previously described (Timonen et al., 1981). Tumor cells

Human lymphoid cell lines were maintained in stationary suspension cultures in 75-cm* plastic flasks in RPMI-1640 medium (Micro-

IFN determination

Supernatants for IFN determinations were recovered after centrifugation of the cultures at 500 g and were subsequently stored at - 70°C until the assay. IFN levels were determined by reduction of the cytopathic effect (CPE) of vesicular stomatisis virus (Have11 & Vilcek, 1972) on a monolayer of HEp2 cells, and IFN levels

Modulation

of Human

were expressed as units per milliliter, standardized with NIH reference human leukocyte IFN (G-023-901-527). The lower limit of sensitivity of this assay was routinely 410 units/ml.

NK Cells

I343

Table 1. The effect of poly I:C and indomethacin on NK activity by unfractionated and nonadherent cells Unfractionate8 TreatmentC

Nonadherentb

% Specific Lysis + S.E.M.

Radioimmunoassay (RZA) for PGE

None

PGs were quantified using the RIA of Jaffe et al. (1973) as previously reported (Koren et al., 1981). All dilutions were made in an assay buffer of phosphate-buffered saline containing 1 mg/ml sodium azide and 1 mg/ml bovine y-globulin (Sigma Chemical Co., St. Louis, Missouri). Rabbit anti-PGE, was purchased from Sigma Chemical Co. Standards were obtained from Upjohn Co. (Kalamazoo, Michigan). The absolute level of sensitivity (B/B,, = 0.9) of the RIA was 1.5 x lo- lo M. All samples were tested directly, without extraction, since we found that there was no significant effect of MEM-10% on antibody binding in the RIA.

Poly I:C (100ug/ml)

1251

Indo (10-~F1)

11+1 _

751

Poly I:C+ Indo

1722

24+2 -

751 22+3 _

"Unfractionated cellscontained 31"/,of peroxidasepositivecells. bPlastic nonadherent cellscontained I"/;, of perox-

idase-positive cells. ’ Unfractionated or nonadherent cells were incubated with poly f:C, indomethacin or their combination for 18 hr, washed extensively and assayed for their cytotoxicity against HSB in a 2-hr assay; E:T = 1O:l.

lated by poly I:C produce PG. and that inhibition of its synthesis by indomethacin causes further augmentation of the cytotoxic activity. Identijcation of NK-modulating factors in the supernatants of poly I: C stimulated cells

Reagents Poly 1:C (catalogue No. P4136) and indomethacin (catalogue No. l-7378) were purchased from Sigma Chemical Co. PGEr and PGF,, were kindly provided by Dr J. Pike (Upjohn Co.). Human fibroblast IFN was obtained from HEM (Rockville, Maryland). PGE, was purchased from Upjohn Co. RESULTS

The &ect of monocytes on activation of NK cells by poly I: c To evaluate the possible involvement of monocytes and PGs in the activation of NK cells by poly 1:C we tested the effect of indomethacin, an inhibitor of cycle-oxygenase. Unfractionated mononuclear cells and plastic nonadherent cells were incubated with poly 1:C (lOOpg/ml) or indomethacin (10e6 M), or with both for 6 hr. The cells were then washed twice and assayed for cytotoxicity against HSB targets. The results presented in Table 1 show that nonadherent cells stimulated with poly 1:C reached higher levels of cytotoxicity (22%) than unfractionated mononuclear cells (12%). The addition of indomethacin to nonadherent cells stimulated with poly 1:C did not change their NK activity significantly; however, the NK activity of the unfractionated cells stimulated with poly 1:C and indomethacin was augmented (12 vs 17%). The results suggest that the monocyte-containing populations stimuM.I.M.M 19’10-1

75

To determine the nature of NK modulatory factors obtained from poly I : C stimulated cells, a two-step procedure was used to obtain those supernant free of poly 1:C from mononuclear cell populations. Mononuclear cells were pulsed with poly 1:C (100 pg/ml) in the presence of indomethacin (10m6 M) for 618 hr, washed extensively to remove poly 1:C and then incubated for an additional 18 hr. The cell-free supernatants were then tested for their effect on NK activity by nonadherent cells. Determinations of anti-viral activity and PGE2 content of the supernatants were also performed. The data presented in Fig. 1 show that the supernatants of unstimulated or poly I: C stimulated monocytes added to nonadherent cells 18 hr prior to the NK assay did not enhance NK activity. In contrast, supernatants of nonadherent cells stimulated with poly 1:C augmented NK activity significantly. Interestingly, supernatants of unstimulated unfractionated cells and nonadherent cells had augmenting activity as well. The supernatants of all cell populations treated with indomethacin alone or indomethacin and poly I: C clearly augmented NK activity. A summary of the IFN and PGE levels found in supernatants of the three cell populations in the absence and presence of poly I : C is presented in Table 2. Supernatants of both untreated or poly 1:C stimulated monocytes had low levels of IFN. In contrast, super-

HILLEL

1344

x g

S. KOREN

10

n" L x "

-

-

__

-

+

+

+

IFN pretreatment

-

+

PGEZintheassoys

and

KAM

H. LEUNG

with poly 1:C contained similar PGE levels (4.2 + 1.2 x lo-’ M) as those from stimulated unfractionated cultures (2.1 + 1.0 x 10m8 M), but contained about lo-fold higher PGE levels than supernatants of stimulated nonadherent cells (5.4 k 2.0 x lop9 M). The results suggest that human monocytes stimulated by poly I:C produce substantial amounts of PGE but no IFN, whereas nonadherent cells stimulated with poly 1:C produce high levels of IFN but very small amounts of PGE. Actiuuted NK cells are refractory hy PGE,

to suppression

To examine if PGEz can also inhibit cytolyactivity of IFN-activated cells, the cytolytic activity of nonadherent lymphocytes cultured with IFN or poly 1:C for 18 hr was assessed in the presence or absence of PGE, in the NK assay. The data presented in Table 3 show that PGEz (30-3000 nM) inhibited endogenous NK activity in a dose-dependent manner. In contrast, poly 1:C (100 pgjml) activated cells partially lost their sensitivity to PGE,-mediated suppression. For instance, a PGE, concentration of 300 nM resulted in a 42%) inhibition of unstimulated cells while stimulated cells were inhibited by only 15% with the same concentration of PGE2. IFN (1000 pg/ml) also caused a similar loss of PGE,-mediated suppression. The data therefore suggest that activated NK cells acquire partial resistance to suppression by PGE2. tic

Fig. 1. The effect of supernatants obtained from poly I:C stimulated mononuclear cell populations on NK activity. Purified monocytes. plastic nonadherent cells and unfractionated mononuclear cells were incubated alone. with poly I:C (100 bcg;ml), indomethacin (IO. 6 M) or with both poly 1: C and indomethacin. Cell cultures were washed after an initial incubation of 618 hr to remove any poly I:C. and were further incubated for a period of IS hr to obtain the various supernatants. The poly I:C free supernatants were incubated with nonadherent cells overnight. The cultures were then washed and tested against K562 target cells. Cytotoxic data obtained at E:T of 1O:l are presented.

natants of poly I:C stimulated unfractionated or nonadherent cells had increased levels of IFN (l&200 pg/ml) when compared to those of untreated cells (l&40 pgjml). Supernatants of the stimulated cells contained in general higher levels of PGE than those of the unstimulated cells. Supernatants of monocytes stimulated Table 2. Interferon

The loss of PGE,-me-diated other cell types We next

sensitivity of NK cells to suppression does not require

determined

whether

and PGE levels in supernatant of poly I:C stimulated mononuclear cell fractions

SOURCE OF SUPERNATEa

IFNb (Units/ml)

PGEC

MONOCYTES

do-20

1.921.7

MONOCYTES

+ POLY


UNF UNF + POLY

I:C

I:C

10-200 do-20

NA NA + POLY

I:C

* Supernatants

100-1200

hM) 40.2k1.2 3.5+3.2 21.0+1.0 2.4+2.6 5.4t4.0 -

of monocytes, whole mononuclear cells (UNF) and nonadherent cells (NA) which were cultured in the presence or absence of poly 1:C (lOO~g/ml) for 6 hr followed by an additional 18 hr incubation in the absence of poly 1:C. b Range of five experiments; assay sensitivity ranged between 5 and IO pgjml. ‘Range of PGE levels in culture supernatants from five experiments. Determinations made by RIA.

activation

of

Modulation

of Human

Table 3. The effect of PGE,

NK Cells

on activated PGG (nM)

Treatment

0

26.5tZ.O

I:C

56.854.4

21.2+1.0

54.352.5

Release

+

15.59.4

S.E.M. 11.450.7

(42)

53.2t6.2

(57)

48.5+4.a

(6) IFN

3,000

300 "Cr

(20) Poly

NK cells

in Assay

30 % Specific

None

I345

39.852.8

(15)

49.022.1

(30)

39.452.8

(10)

31.8+2.0

(27)

(41)

Nonadherent cells were incubated with poly I:C (Iot3~g/ml), or human fibroblast interferon (IFNB) (1000 ng/ml) for 18 hr. washed and assayed for NK activity in the presence or absence of PGE,. The E:T ratio was 10: 1 in a 2-hr cytotoxicity assay. The numbers in parentheses represent per cents of inhibition by PGE2.

NK cells and loss of susceptibility to PGE,-mediated suppression as a result of activation with IFN involved more than one cell type. Unfractionated cells and NK-enriched cells, or LGL obtained by Percoll gradient sedimentation, were utilized. Cells were incubated overnight in the presence or absence of IFNa and tested the following day in a cytotoxicity assay in the presence or absence of PGE, (3OOnM). Figure 2 shows that the endogenous NK activity of the two lymphocyte preparations could be inhibited by PGE, on day 0 as well as after 18 hr of incubation. The LGL population had a relatively higher NK activity than unfractionated cells and the NK activity of the two groups of cells was further augmented by IFNP. Such activated cells lost their sensitivity to PGE,-mediated suppression. The data indicate that IFN can act directly on NK cells (LGL) to induce the loss of sensitivity of PGE,-mediated suppression. DISCUSSION

Recent data suggest that NK activity is, like most other immunological responses, under the regulatory influences of a variety of substances, most prominently those of IFN (Bloom, 1980). IFN inducers (Zarling et ul., 1980) and PGE2 (Droller et al., 1978; Koren et al, 1981). Our studies presented here have indeed established a biological link between the cells producing some of these regulatory molecules and the cells primarily involved in mediating NK activity, namely LGL. When mononuclear cells were stimulated with poly I:C it was noted that NK augmentation was less pronounced than that by nonadherent cells (depleted of monocytes). Moreover, the presence of indomethacin (lo-‘M) during the stimulation of

mononuclear cells with poly I:C resulted in further augmentation of NK levels. These data suggested the involvement of PGE, in the stimulation of monocyte-containing cultures. Further evidence for the involvement of PGE2 in poly I:C stimulated cultures came from experiments where supernatants of poly I:C stimulated mononuclear cell fractions were tested for their effect on NK levels of nonadherent cells (Fig. 1). Those experiments demonstrated that unstimulated or poly I:C stimulated monocytes generated supernatants with NK-suppressive activities. The presence of indomethacin in those cultures, however,

90

2

80

f Z ’ F =

60

t

40

E

30

70

50

“, 20 z d IO 0

' 0 'PIC'lndol,,,l' 0 'PIG'hxb'f'i~'' 0 'PIC'Indo'W;~ MONOCYTE

SUPS

UNFCELLS

SUPS

NA CELLSSUPS

Fig. 2. The effect of PGE, on interferon-activated mononuclear cell populations. Unfractionated mononuclear cells and large granular lymphocytes (LGL) obtained from a five-step discontinuous Percoll density gradient were incubated alone, or with interferon (lOO~g/ml) for 18 hr. The cells were then washed and assayed in the presence or absence of 300 nM PGE?. The E:T ratio was 5: 1 in a 1-hr cytotoxicity assay against K562. The numbers in paren_ theses represent per cents 01 inhibition.

I346

HILLEL

S. KOREN I and

resulted in supernatants capable of augmenting NK activity against K-562 targets. In contrast, supernants generated from stimulated nonadherent cells augmented NK activity by 7&110x depending on whether or not indomethacin was included in those cultures. The further increase seen in the activity of the poly I: C stimulated lymphocytes in the presence of indomethacin would suggest that a small number of monocytes capable of producing PGE2 was still present in the nonadherent population. Direct measurements of PGE, and IFN levels in the supernatants of the different mononuclear fractions incubated under various conditions indicated by a direct relationship between those levels and the NK activity of the nonadherent cells treated with those supernatants. For instance, poly I :C stimulated monocytes produced low levels of IFN but significant levels of PGE,, in line with their NKsuppressive effect (Fig. 1). Though monocytes have previously been shown to be a major source of PGEz (Kennedy et al., 1980) poly I: C has not previously been shown to be a PGE2 inducer. In marked contrast, poly I: C stimulated nonadherent cells had high titres of antiviral activity (lo&1200 units/ml) but only very low PGE, levels. Further experiments on the sensitivity of NK cells to PGEz have shown that IFN or poly 1:C activated cells are partially resistant to the effects of PGE2 (Table 3). For example cells activated by IFN doubled their NK activity (from 27 to 54%) but were only 27% inhibited by the addition of 300nM PGE2 to the assay compared to 427: inhibition of unstimulated cells. This partial loss of inhibition was not due to endogenous PGEz production since the addition of indomethacin to the cultures stimulated with poly 1:C did not prevent the partial loss of sensitivity to PGE2 (data not shown). It was of further interest to investigate whether cells other than those mediating NK activity are required in the acquisition of resistance to PGE,. Experiments performed with LGL obtained by Percoll density gradients demonstrated that both activation by IFN and loss of sensitivity to suppression by PGE, are most probably autonomous functions of LGL (Fig. 2) independent of monocytes. The data presented in this paper suggest that the regulation of NK activity is under the influence of IFN and PGE*. Poly I:C, known for its ability to induce IFN, is also capable of inducing PGEl in human monocytes. This

KAM

H. LEUNG

finding might have clinical implications since various forms of poly I: C are used in cancer therapy (Zarling et al., 1980). The fact that activated NK cells in contrast to endogenous NK cells are partially resistant to suppression by PGE2 is intriguing. The mechanism for the loss of sensitivity may be due to loss of receptors for PGE, and/or related to the intracellular levels of cyclic AMP. Those possibilities are currently being investigated in our laboratory. Biologically, the loss of sensitivity to suppression by PGEz may be an important mechanism by which NK cells can avoid the negative effects of PGE, which may be of tumor (Droller et al., 1979) and/or monocyte origin (Kennedy et al., 1980).

Acknowledyement.~ -The authors with to thank M. Hayes for her secretarial assistance.

MS Connie

REFERENCES Boyum A. (1968) Isolation of mononuclear cells and granulocytes for human blood. &and. J. c/in. Lab. Incest. 21 (Suppl. 97) 77-90. Bloom B. R. (1980) Interferons and the immune system, Nature, London. 284, 593-595. Droller M. J., Lindgren J. A., Claessein H. A. & Perlmann P. (1979) Production of prostaglandin E, by bladder tumor cells in tissue culture and a possible mechanism of lvmnhocvte inhibition. Cell Immun. 47. 261-273. Droller M.. J., Schneider M. U. & Perlmann P. (1978) A possible role of prostaglandins in the inhibition of natural and antibody-dependent cell-mediated cytotoxicity against tumor cells. CeX Zmmun. 39, 1655177. Fischer D. G., Hubbard W. J. & Koren H. S. (1981) Tumor cell killing by freshly isolated peripheral blood monocytes. Cell Immun. 58, 42&435. Havell E. A. & Vilcek J. (1972) Production of high-titered interferon in cultures of human diploid cells. Anrirnicrob. Ag. Chemother. 2, 47f+E32. Herberman R. B. & Ortaldo J. R. (1981) Natural killer cells: their role in defenses against disease. Sciencr 214, 24-30. Jaffe B. M., Behrman H. A. & Parker C. W. (1973) Radioimmunoassay measurement of prostaglandins E, A, and F in human plasma. J. c/in. Incrst. 52, 398-405. Kennedy M. S., Stobo J. D. & Goldyne M. E. (1980) In vitro synthesis Kennedy M. S., Stobo J. D. & Goldyne M. E. (1980) In vitro synthesis of prostaglandins and related lipids by populations of human peripheral blood monocytes. Prosraglmdim 20, 135 145. Koren HI S., Anderson S. J., Fisher D. G., Copeland C. S. & Jensen P. J. (1981) Regulation of human natural killing: I. The role of monocytes, interferon, and prostaglandins. J. Immun. 127, 200772013. Targan S. & Dorcy F. (1980) Interferon activation of prespontaneous killer: (pre-SK) cells and alteration in kinetics of lysis of both ‘pre-SK’ and active SK cells. J. Immun. 124, 215772161. Zarling J. M., Schlais L.. Eskra J. J., Greene P. 0. P., Ts’o and Carter W. (1980) Augmentation of human natural killer cell activity by polyinosinic acid-polycytidylic acid and nontoxic mismatched analogues. J. Immun. 125,