The mechanisms involved in the activation of human natural killer cells by staphylococcal enterotoxin B

CELLULAR IMMUNOLOGY 122,108- 12 1 ( 1989)

The Mechanisms Involved in the Activation of Human Natural Killer Cells by Staphylococcal Enterotoxin B’

ARTHUR D. BANKHURS? AND TURGIT IMIR~

Department ofMedicine, University of New Mexico, School ofMedicine, Albuquerque, New Mexico 87131

Received November 7,1988; accepted April 24, I989

The induction of enhanced natural cytotoxicity from human peripheral mononuclear cells by staphylococcal enterotoxin B (SEB) was examined. The activated killer cytotoxicity (AKC) was maximum at 16 hr with 1 m&ml SEB. The precursor and effector cells of AKC were determined to be primarily CD5 negative, CD8 negative, CD 16 positive cells. Monocytes and interleukin- 1 played no role’ in the generation of AKC. However, a major role for interleukin-2 (IL- 2) in AKC was shown by the inhibition of AKC when anti-IL-2 antibody or cyclosporin was added to the induction cultures. SEB rapidly induced the production of IL-2 from glass nonadherent cells by 6 hr and reached peak levels by 24 hr ( 162 U/ml). IL-2 induced by SEB in these induction cultures was preferentially produced by CD16 positive cells. Even though interferon- y (IFN-7) production was induced in these cultures, no role for IFN could be shown in SEE induced AKC. 8 1989 Academic ~m.s, hc.

INTRODUCTION

During the last several years it has been recognized that natural killer (NK)4 cells may play a role in the host defenses against a variety of infections and cancer in addition to their involvement in the regulation of hematopoietic differentiation (1). In addition, nonspecific cytotoxic lymphoid cells may be activated in vitro with a variety of stimuli including interferon (EN), interleukin-2 (IL-2) neuropeptides, al- logeneic cells, simple sugars, lectins, and some microbial products (2-7). These nonspecific activated killer cells (AKC) have been variously described as NK-like cells, anomalous killer cells, and lymphokine-activated killer cells (LAK cells). Generally AKC have an expanded repertoire of cell targets which are sensitive to AKC-mediated lysis including the ability in some instances to lyse fresh autologous tumor cells and autologous lymphoid cells (2,8).

The range of host defense functions mediated by NK cells has been recently expanded by the observation of the selective phagocytosis of gram-positive bacteria by

’ Supported by Grant CA 24873 from the National Institutes of Health. * To whom all correspondence should be addressed. 3 Present address: Gazi University, Faculty of Medicine, Microbiology Department, Besevler, Ankara,

Turkey. 4 Abbreviations used: SEB, staphylococcal enterotoxin B; AKC, activated killer cytotoxicity; IL-2, inter-

leukin-2; IFN-y, interferon-r; LGL, large granular lymphocytes; PMC, human peripheral mononuclear cells; ET, effector:target; GNAC, human glass nonadherent mononuclear cells; NK, natural killer; IL- 1, interleukin- 1; LAK, lymphokine-activated killer; PBS, phosphate-buffered saline.

108

0008-8749189 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

SEB ACTIVATION OF NK CYTOTOXICITY 109

large granular lymphocytes (LGL) (9) and the killing of gram-negative bacteria by highly purified human CD 16 positive cells ( 10). It was appropriate, therefore, to examine whether microbial products may play a role in the biology of NK cells particu- larly with regard to the induction of AKC activity. In the present study, the induction of AKC by staphylococcal enterotoxin B (SEB) was examined. The cell types and mechanism of activation involved in SEB-induced AKC are described.

MATERIALS AND METHODS

Preparation of Peripheral Blood Mononuclear Cells (PMC)

PMC were obtained from fasting healthy donors. Lymphoid cells were prepared by density centrifugation on Ficoll-Hypaque (Pharmacia, Piscataway, NJ), washed three times in phosphate-buffered saline (PBS, pH 7.2), and resuspended in RPM1 1640 (Whittaker, MA; Bioproducts, Walkersville, MD). For certain experiments adherent cells were removed by adherence to glass petri dishes and passage through nylon wool columns (11, 12). Cell viability was greater than 99% as determined by trypan blue exclusion. The proportion of monocytes was determined by histochemi- cal staining with peroxidase and immunofluorescence using an anti-monocyte antibody (anti-Leu M3, Becton-Dickinson, Mountain View, CA) and was less than 0.1% in depleted suspensions. This cell preparation will be referred to as the glass nonadherent cell population (GNAC).

Enrichment of Eflector Cells

In some experiments effector cells were enriched for LGL on Percoll (Pharmacia) gradients or by negative selection using adherence techniques. The Percoll gradient technique used for the preparation of LGL in this laboratory has been previously described (13). The effector cells were prepared by negative selection techniques performed as follows: GNAC cells were prepared by adherence to glass petri dishes two times and a subsequent passage through a nylon wool column. GNAC (100 X 106) were resuspended in 10 ml of RPM1 1640 containing 10% human AB serum (Irvine Scientific, Irvine, CA). Sheep blood (Colorado Serum, Denver, CO) was diluted 1 OO- fold in pH 7.2 and added to the GNAC. The mixture was incubated for 30 min at 37°C. Following the incubation the mixture was placed on top of a conventional 10 ml Ficoll/Hypaque gradient and centrifuged at 3008 for 30 min. The nonrosetting cells which appeared at the top of the gradient were harvested and resuspended in 1 ml of RPM1 1640 containing 10% AB serum. Two hundred microliters of anti&u 4 (CD3, Becton-Dickinson) and 200 ~1 of anti-Leu M3 (Becton-Dickinson) were added to the cells and incubated on ice with gentle agitation for 30 min. The cells were then placed on a 150 X 15-mm polystyrene petri dish (VWR, Napesville, IL) which had been precoated with goat anti-mouse IgG antibody (Tago, Burlingame, CA). The cells coated with the monoclonal antibodies were diluted to 15 ml in media and incubated at 4°C for 30 min. The nonadherent cells were aspirated from the plates. The phenotype of nonadherent lymphoid cells was verified by immunofluorescence as described below. Ordinarily the percentage of CD 16 positive cells was 80- 85% and no monocytes were present. In some experiments NK-enriched cells were prepared by a negative panning technique (10). GNAC were passed through a nylon wool column and then incubated with monoclonal antibodies (Leu 2, CD8; Leu 3, CD4; and Leu 4, CD3; 20 pl/106 cells) for 30 min at 4°C and then were adhered to

110 BANKHURST AND IMIR

polystyrene plates coated with anti-mouse IgG for 60 min at 4°C. For each experiment, the phenotype of nonadherent (NK-enriched) cells was tested by direct immunofluorescence staining with FITC conjugates and enumerated by either fluorescence microscopy or by FACScan (Becton-Dickinson). The purity of these NK-enriched suspensions was 99% CD 16 positive and < 1% CD3 positive.

Treatment of Eflector Cells

PMC were incubated with several concentrations of SEB (Sigma, St. Louis, MO) for varying time periods. As indicated below the optimal conditions for the induction of AKC was 1 pg of SEB for 16-24 hr. The SEB was homogeneous based on the presence of a single peak in high-performance liquid chromatography on a TSK- G300 SW exclusion column. In various experiments lymphokines and/or antibodies were added to the cell cultures with various combinations of IL-2 (Ultrapure, Electro- nucleonics, Bethesda, MD), interleukin-1 (IL-l, Cistron, Pine Brook, NJ), monoclonal anti-IL-2 (Genzyme, Boston, MA), and rabbit polyclonal anti-1FN-y (Inter- feron Sciences, New Brunswick, NJ). All antibodies were added to effector cells 1 hr before the addition of SEB and remained in the cultures during the incubation with SEB as well as in the cytotoxic assay. Anti-IL-2 receptor antibody was used at 5 pg/ ml which was sufficient to saturate these surface receptors on an equal number of Con A-activated human peripheral blood T-lymphoblasts. The monoclonal anti-IL- 2 and polyclonal anti-IFN-y used were sufficient to neutralize the number of international units per milliliter as specified. Control antibodies which consisted of antibodies of the same isotype with irrelevant specificities were used in all experiments. Cycloheximide (5 pg/ml), actinomycin (1 pg/ml), and mitomycin (50 pg/ml) were obtained from Sigma. Cyclosporin (Sandoz, Basel, Switzerland) was initially dis- solved in 95% ethanol and diluted with medium before use. The concentration of ethanol was always less than 0.01% which had no effect on the assays. Effector cells were treated with metabolic inhibitors for 1 hr at 37°C before exposure to SEB. The cells were washed once before the cytotoxic assay.

Target Cells and Measurement of Cytotoxicity

K562 and Daudi cells were maintained in continuous culture. Between 0.5 and 1.5 X lo6 target cells were labeled with 0.1 ml of Naz 51CrG4 (Amersham Corp., Arlington Heights, IL) for 1 hr at 37°C. After two washes, K562 cells were resuspended at lo5 cells/ml and 0.1 ml was pipetted into conical-bottom microtiter plates. Effector cells in 0.1 ml of medium (RPM1 1640, 10% AB serum, 1% glutamine, 1% penicillin- streptomycin) were added to the wash to provide various E:T cell ratios. After incubation at 37°C for 4 hr, 0.1 ml of supernatant was collected from each well and counted in a gamma counter. Percentage of cytotoxicity for each individual assay was determined by percentage cytotoxicity = [(test cpm - spontaneous cpm/(total cpm - spontaneous cpm)] X 100. In some experiments results were expressed as lytic units (L.U.) determined by linear regression analysis; one lytic unit was the equivalent of the cytolytic activity required to kill 33% of 1 O4 susceptible target cells.

Identijcation of Efector Cell Phenotype

The following monoclonal antibodies (Becton-Dickinson) directed against the surface antigens indicated were used in this study: CD5 (Leu 1, pan-T antigen); CD16


(Leu 11, Fe-r receptor on large granular lymphocytes and neutrophils); NKH- 1 (Leu 19, reactive with most large granular lymphocytes); and CD8 (Leu 2a, reactive with cytotoxic/suppressor T cells). Effector cells were washed twice in RPM1 1640 and resuspended at 1 X lO’/ml at 4°C. The previously determined optimal dilution of monoclonal antibody was added directly to the cells (generally 1:50 dilution). The cells were incubated for 30 min at 4°C and pelleted. Nontoxic rabbit complement (Low-Tox-H Complement, Cedarlane Laboratories, Hornby, Ontario) was added to the cell pellet for 15 min at 4°C (optimal concentration of complement was a 1:3 dilution after a prior absorption on GNAC to remove any residual nonspecific cytotoxicity). Antibody-dependent complement-mediated lysis was performed for 30 min at 37°C after which the cells were washed once in RPM1 1640, resuspended, and counted for viability using trypan blue exclusion. Complement alone did not cause cell lysis and the phenotype of the cell suspensions was examined by visual immunofluorescence to assure the complete elimination of a specific phenotype.

Flow Cytometric Analysis

A Coulter Epics V (Coulter, Hialeah, FL) equipped with dual argon lasers with an MDADS computer was used for flow cytometric cell analysis. Forward and 90” light scatter analysis was used to detect debris and relative cell size. Clear separation of light scatter profile and the presence of immunofluorescence was obtained by gate settings determined by the operator. Generally, all staining of precursor or effector cells with fluorescent antibodies in preparation for flow cytometric separation was done at 4°C for 45 min. All dilutions, washings, and incubations were performed in cold PBS containing 0.1% sodium azide. The immunofluorescent antibodies utilized were directly labeled with fluorescein isothiocyanate obtained from the commercial sources indicated above. The antibodies were titered for optimal fluorescence prior to use. Isotype-matched fluorochrome-conjugated myeloma proteins or nonreactive monoclonal antibodies were used to control for Fc-related binding.

Measurement of Lymphokines

IL-2 activity was determined using a modification of the method described by War- ren and Pembrey ( 14). Briefly, PMC were cultured at 37°C for 4 days at 1 X 1 O6 cells/ ml with 20 Kg conconavalin A/ml in RPM1 1640 containing 10% heat-inactivated human AB serum, 2 rnJ4 L-glutamine, 50 mg gentamicin/ml, and 0.1 mM 2-mercap- toethanol. After 4 days in culture the lymphoblasts are dependent on IL-2 for contin- ued growth. The cells were then washed three times in medium without Con A and 2 X lo4 lymphoblasts per well were seeded into 96-well, flat-bottomed microtiter plates and incubated 48 hr in the medium described above with log, dilutions of the test samples in a volume of 0.2 ml. Six hours before harvest, each well was pulsed with 1 &i [3H]methyl thymidine (ICN, Irvine, CA). Cells were then harvested onto glass fiber filters using a Skatron Cell Harvestor (Bioproducts) and assessed for thymidine uptake by liquid scintillation counting. An IL-2 standard was run in parallel with the samples.

IFN-7 was determined in a 96-well microtiter plate by a cytopathic inhibition assay as described previously ( 15). The samples were serially diluted and placed into the appropriate microtiter well. Each plate contained as a control a reference preparation of IFN--, (NIH, Bethesda, MD). Each well was seeded with WISH cells (American Type Culture Collection, CCL25) and challenged with vesicular stomatitis virus. The


TABLE 1

The Induction of AKC Cytotoxicity against KS62 and Daudi Target Cells by SEB

Time WI

0 4

16 24

Concentration of SEB” (pg/ml)

0 lo-* 10-l 1 10

K562 targets

26.1 + l.2b 24.6 f 2.0 25.3 f 1.0 27.2 + 2.0 26.1? 1.0 28.6 + 1.0 31.2k0.6 31.7 f 1.0 31.3 +0.8 42.1 + 2.0 27.2 k 1.8 32.1 + 0.5 44.2 + 2.0 72.1 f 2.8 68.1 + 3.1 23.2 + 1.4 72.2 + 2.0 71.0k2.4 73.2 + 2.0 75.1 + 1.5

Daudi targets

0 4.1 f 0.5b 4.1 + 1.0 5.0 + 1.0 4.2 f 0.5 4.1 kO.5 4 5.2 + 2.2 16.1 f 2.2 21.8 kO.7 31.Ok5.1 32.1 + 1.1

16 5.2 + 0.4 22.3 + 2.4 34.9 + 2.4 61.0 f 1.0 62 + 1.5 24 9.0 + 0.3 30.1 f 1.6 41.0* 3.1 62.2 + 1.2 64.1 -c 1.8

p The SEB was incubated with effector cells for the times indicated. b Percentage cytotoxicity is expressed as mean f SE at 25: 1, effector:target cell ratio. The experiment

shown is one of three experiments with similar results.

titer in units per millimeter was calculated for each sample by comparison with the dilution of the standard control sample. The identity of the IFN--y was confirmed by neutralization with anti-IFN-y antibody.

RESULTS

The Induction ofAKC: Dose Response and Kinetics of Induction

Initial experiments examined the optimal culture conditions for the induction of AKC by SEB. Various concentrations of SEB were added to PMC for time intervals which ranged from 1 to 24 hr (Table 1). Little cytotoxicity against NK-resistant Daudi target cells is present at 0 time or in the absence of SEB. Optimal cytotoxicity against Daudi cells was seen at 16 hr with concentrations of SEB of at least 1 fig/ml. SEB treatment also increased the cytolytic activity against the sensitive K562 target cells, with peak cytotoxicity also occurring at concentrations of at least 1 &ml for 16 hr. When effector cells were incubated with SEB (1 &ml) for more than 24 hr a decrease in activity against both the NK-sensitive (K562) and NK-resistant (Daudi) target cells was seen (data not shown). The optimal conditions for SEB induction of AKC was established with cells incubated with 1 pg SEB for 18-24 hr.

The Phenotype of the Precursor and Eflector Cells of SEB-Induced Cytotoxicity

The phenotypes of the precursor and effector cells of AKC were investigated by the removal of selected subpopulations of GNAC cells using monoclonal antibodies and complement. In Fig. 1 it can be seen that complement-mediated cytotoxicity with the Leu 7 and Leu 11 antibodies of precursor cells on Day 0 prior to incubation of cells in vitro with SEB led to a marked decrease in AKC. In addition when GNAC were fractionated on Percoll gradients prior to the addition of SEB as seen in Table

100

60

60

40

20

60

60


K562 Daudi

1 Exp. 1

Exp. 4

251 12:l 8:l

E/T RATIO

FIG. 1. The effect of the removal of specific precursor cells on SEB-induced AKC. Specific cell populations indicated were removed by complement-mediated lysis on Day 0 prior to the incubation of the residual population with SEB (1 mg/ml for 24 hr). The phenotypic profile of GNAC was as follows: CD4 = 72%, CD8 = 28%, Leu 7 = 20%, and CD16 = 15%. The absence of any residual cells reactive with the relevant antibodies after complement-mediated lysis was verified by immunofluorescence. The standard error of all determinations was less than 3%. The experiment shown is representative of three performed. The E:T ratio expressed is that prior to complement-mediated removal of selected subpopulations.

2, it was shown that the great majority of AKC activity was found in those fractions which were enriched for LGL and the LGL phenotype marker CD16. Only minor AKC activity was found in the fractions which contained few LGLs or CD 16 positive cells. The removal of CD5 and CD8 positive cells did not decrease cytotoxicity. Like- wise when the cells were similarly subjected to complement-mediated lysis after 24 hr incubation with SEB (Fig. 2) only the Leu 7 and anti-CD 16 antibodies caused a marked reduction in AKC. Since it appeared that a CD 16 positive cell was responsible for AKC-mediated cytotoxicity, further verification of the punitive effector cell was investigated. CD 16 positive cells were enriched by negative selection adherence techniques. Figure 3 shows that AKC was markedly increased by enrichment for CD 16 positive cells. These enriched cell suspensions were also devoid of monocytes which supports the fact that monocytes did not function as obligatory accessory cells for cytotoxicity induced by SEB. In an effort to further exclude the involvement of cytokines secreted by monocytes in AKC cytotoxicity induced by SEB, purified IL- 1


TABLE 2

Cytotoxicity Induced by SEB in Various Cell Fractions Prepared on Percoll Gradients

Fraction” 25:l

K562 b

12:l 6:l 25:l

Daudi b

12:l 6:l

-SEB’ 1 41.1 + 1.1 24.2 + 1.3 11.2k 1.0 34.2k0.7 18.2 f 0.4 10.8 f 1.1 2 43.1 + 1.6 24.8 + 1.7 13kO.8 33.9 f 0.9 17.2f 11.1 9.2 k 1.4 3 17.2 -t 1.5 8.2 f 1.4 5.3 zk 0.9 13.6 + 1.6 7.2f 1.0 4.1 + 1.2 4 5.2 f 0.3 2.1 + 0.1 1.9kO.l 3.1 +0.1 2+ 0.1 l.OkO

+SEBC 1 100 + 1.3 82.4 f 1.8 47.6 + 1.5 91.2k2.1 61.2+ 1.9 35.5+ 1.8 2 100 + 1.1 80.2k 1.7 49.22 1.3 93.2+ 1.6 49.2+ 1.8 36.5 + 0.9 3 31.8 + 2.0 16.4 + 1.5 9.2 + 0.7 20.1 + 0.8 11.1 + 1.1 6.2 + 0.5 4 3.1 +0.6 3.1 +0.3 0 1.0+0 0 0

a Fraction 1 represents a 37.5% Percoll fraction while other fractions are increments of 2.5%. The cell composition of each Percoll gradient fraction was as follows: Fraction 1 = 8 1% LGL, 9 1% Leu 11; Fraction 2 = 85% LGL, 90% Leu 11; Fraction 3 = 5% LGL, 1% Leu 11; Fraction 4 = 2% LGL, 1% Leu 11.

b Results are expressed as means + SE at each ratio. This experiment is representative of three performed. c The cell viability was 98% in all cultures and this viability did not change with or without exposure to

SEB.

(free of endotoxin) was added to nonadherent lymphoid cell suspensions for 18 hr. There was no significant change in anti-Daudi or anti-K562 cytotoxicity after the addition of IL- 1 over a wide range of IL- 1 concentrations (l-200 units/ml, data not shown).

The Role of IL-2 and IFN-y

In order to assess if IL2 or IFN--r may play a role in the induction of AKC by SEB, anti-IL2, anti-IFN y, and control antibodies were added to the SEB induction cultures. Control antibodies consisted of antibodies of identical isotypes and irrelevant specificities and had no effect on either spontaneous or activated cytotoxicity. It can be seen in Fig. 4 that SEB-induced AKC was inhibited in the presence of anti- IL-2 but not in the presence of anti-IFN-y. To verify that sufficient antibody was utilized parallel experiments were conducted in which appropriate amounts of IL-2 or IEN- were added to verify the neutralizing effect of the anti-IL-2 and anti-IFN-y antibodies. The addition of cyclosporin, an inhibitor of IL-2 production also blocked the SEB-induced augmentation of cytotoxicity against both K562 and Daudi targets (Fig. 5). There was no loss of cell viability up to 5 pg/ml of cyclosporin. Similar results were seen when cycloheximide, an inhibitor of protein synthesis, was added to the cultures (data not shown).

The Production of Cytokines by SEB-Stimulated Cells

Since IL-2 appeared to play a role in the induction of AKC by SEB, it was important to verify that sufficient amounts of IL-2 were produced in the supematants of SEB-treated cultures to account for the inhibition by anti-IL-2 antibody seen in previous experiments. In Table 3 it can be seen that sufficient IL-2 was produced in culture after 24 hr with SEB to account for the inhibition seen with various concentrations


K562 Daudi

100 r Exp. 1

c

Exp. 1

60

60

40

20

0

60

Exp. 2

-Ld

c Exp. 2

251 121 6:l

E/T RATIO

FIG. 2. The effector cells involved in SEB-induced AKC. GNAC were incubated with SEB (1 mg/ml for 24 hr) prior to incubation of the activated cell population with monoclonal antibodies followed by complement-mediated lysis. The average number of cells hilled by complement-mediated lysis with the various antibodies were similar to those described under Fig. 1. The standard error of all determinations was less than 2.8%. The experiment shown is representative of three performed.

of anti-IL-2 antibody. Although IFN--y is also produced, previous experiments did not demonstrate a role for IFN-y in the induction of AKC by SEB. The kinetics of IL-2 production is shown in Fig. 6. Even at 6 hr significant IL-2 production (30 units/ ml) can be detected. The kinetics of IEN- production was slower in that no 1FN-y could be detected at 6 hr and only small amounts (less than 10 U/ml) were detected at 12 hr (data not shown).

IL-2 Induced by SEB Is Produced Preferentially by CD16 Positive Cells

In Table 4 it can be seen that the IL-2 induced by SEB was produced by CD 16 positive cells in the GNAC suspension. For example, in Experiment 1 the difference between GNAC + SEB (44 k 3.2) and GNAC - CD16 positive cells + SEB (0.26 f 0.1) was highly significant (P < 0.00 1).


60

Daudi Lou 11

25: 1 25: 1 121 6:l 121 6:l 25:l 25:l 12:l 6:l 12:l 6:l

E/T RATIO FIG. 3. The cytotoxicity induced by SEB in cell suspensions enriched for leu 11. Cytotoxicity against

K562 versus Daudi was compared between GNAC versus cell suspensions enriched for CD 16 positive cells. A representative experiment is shown of three performed. Results are expressed as the mean percentage cytotoxicity. Standard error was always less than 3.0%. The phenotype of the cell suspensions was as follows: GNAC: CD16 = 1 l%, Leu 1 = 73%, Leu 19 = 10%; CD16-enriched cell suspension: CD16 = 82%, CD3 = l%, Leu 19 = 80%, Leu MS = 0%.

Highly Purified Suspensions of CD16 Positive Cells Are Activated by SEB without Participation of Non-CD1 6 Cells

In order to demonstrate that CD 16 positive cells can be activated in the absence of other cells which have the potential to produce IL-2, highly purified suspensions of CD 16 positive (>99% pure) were prepared using negative selection “panning” techniques. In Table 5 can be seen two representative experiments which show that these CD 16 positive cells respond to SEB in the absence of T and other non-NK cell subpopulations.

DISCUSSION

Activated natural killer cells could be induced rapidly by incubation of human LGL, CD 16 positive peripheral cell populations with SEB. The activation was maximum within 16 hr and endogenous IL-2 production by CD 16 positive cells appeared to be critical to the major portion of the induced cytotoxicity.

Enterotoxins are a group of bacterial exotoxins released by some viable gram-negative and gram-positive bacteria that are distinguishable from lipopolysaccharide endotoxin. SEB is one of several serologically distinct enterotoxins produced by Staphy- lococcus aureus. Aside from the effect of SEB on the stimulation of fluid and electro-

140

120

2c

C I! B In 0’

SEB ACTIVATION OF NK CYTOTOXICITY

i

K562

L

FIG. 4. The effect of anti-IL-2 and anti-IFN-y antibodies on SEB-induced AKC. GNAC were incubated with SEB (1 g/ml) with or without antibodies for 24 hr. Results are reported as lytic units for a representative experiment of three performed. Variance (SE) was always less than 3%.

c 5 100 K562 Daudi x e 80

[ 60

bo

E i= c 20

z

’ M 50 25 1.25 0.6 0.3 0.03 50 2.5 1.25 0.6 0.3 0.03

Concentration of Cydosporin (micrograms/ml)

CYCLOSPORIN FIG. 5. The inhibition of SEB-induced augmentation of cytotoxicity by cyclosporin. GNAC were incu-

bated with SEB with or without cyclosporin at various concentrations. Results are expressed as percentage inhibition of the increase in cytotoxicity against K562 and Daudi seen after the addition of cyclosporin. SE was always less than 3%. This experiment is representative of three experiments.


TABLE 3

The Induction by SEB of Lympokines in 24-hr Culture Supematants

IL-2 IFN-7

Expt 1 Expt 2 Expt 1 Expt 2

GNAC (control) 0.2kO.l 0.8 f 0.1 0 0 GNAC ? SEB 162 + 5.0 78.0 + 2.0 120 + 6.0 38.8 -c 4.0

Note. Results are expressed as international units per milliliter, mean f SE. The preparation of cells and stimulation with SEB are described under Materials and Methods.

lytes from intestinal epithelial cells, there is only fragmentary knowledge concerning its effect on host defense mechanisms. For example, it has been shown that SEB is mitogenic for both mouse and human lymphocytes ( 16, 17) and this mitogenic activity is retained even after detoxification of SEB by formaldehyde. At least two other staphylococcal enterotoxins, staphylococcal enterotoxin A (SEA) and the toxic shock toxin, are also effective mitogens for lymphocytes (18, 19). SEB interacts with lymphocytes by specific ligand-receptor binding (20). The three antigenically distinct staphylococcal enterotoxins (SEA, SEB, SEE) appeared to react with a common receptor. That study did not examine which subpopulation of lymphoid cells were responsible for the SEB binding. The only study which examined the possibility that staphylococcal enterotoxin could regulate NK cells showed that SEA could enhance

80

6 12 18 24

Hours of Incubation with SEB

FIG. 6. The kinetics of IL-2 production induced by SEB. Results are expressed in units per milliliter as a function of time during the incubation of GNAC with SEB. Results are expressed as means + SE of a representative experiment of two performed.


TABLE 4

IL-2 Induced by SEB Is Produced by Leu 1 l+ NK Cells”

Expt 1 Expt 2 Expt 3

GNAC GNAC + SEB GNAC - Leu 11’ cells GNAC - Leu 1 I+ cells + SEB

4* 1.2b 2.4 -c 1.0 2.4+ 1.0 44 f 3.2’ 12Ok2.6’ 192 f 3.4’

0.39 z!I 0.1 0 0 0.26kO.l’ 0’ 0’

L? Leu 1 l+ cells were removed by antibody-dependent complement-mediated lysis as described under Materials and Methods. There were no Leu 1 I+ cells remaining after this treatment. SEB was used at 1 pg/ ml.

h Results are expressed as means + SE, units/per milliliter. ‘The difference between GNAC and GNAC + SEB and between GNAC + SEB and GNAC - Leu 1 l+

cells + SEB were all highly significant (P < 0.001).

NK activity; however, there was no analysis of the effector cell nor an explanation of the mechanism of induction (2 1). The present study clearly shows that SEB preferentially induces the production of IL-2 by NK cells. Although Table 4 supports the possibility that the IL-2 induced by SEB in GNAC is produced by NK cells it is possible that SEB may potentially stimulate other cell types to produce IL-2 under different conditions (for example, the length of exposure to SEB).

Several pieces of data in the present study clearly indicate that IL-2 is critical to the induction of AKC by SEB. When anti-IL-2 antibody was added to the activation cultures, cytotoxicity was markedly diminished. Moreover, SEB-induced AKC was inhibited when cyclosporin or cycloheximide was added to the cultures. Although cycloheximide generally inhibits protein synthesis, cyclosporin appears to inhibit IL- 2 production and/or release as well as block the expression of IL-2 receptors (22-24). Finally, it was shown that the quantities of IL-2 produced in the culture supernatants were commensurate with the quantities of neutralizing antibody shown to block SEB activation. Although the great majority of SEB-induced AKC can be attributed to the endogenous production of IL-2, it should not be overlooked that a minor portion

TABLE 5

Cytotoxicity Induced in Highly Purified NK Cells by SEB”

Target

Experiment K562 Daudi

1 Control 117+35” 41 f 0.36 +SEB 473 f 6.0” 346 f 3.8’

2 Control 70 f 2.4 14 f 0.2 +SEB 327k4.1 303 + 2.6

a The average phenotypic profile of the purified NK cell suspension prepared by negative selection was asfollows:Leu19=99~1%,CD16=99+1%,CD3=0.2+O,LeuM3=0.

b Results are expressed in lytic units. ’ All results comparing control vs SEB with both targets were highly significant (P < 0.0 1).


(25% or less) appears to be independent of IL-2 or IFN-7. Significantly, it did not appear that IFN-y played a major role in the activation process. A recent publication which examined the augmentation of human NK cytotoxicity with IL-2 reported that IL-2 stimulated the production of IFN--r which then indirectly was responsible for the enhancement of cytotoxicity (25). Such an indirect role for IFN--, was not seen in our SEB augmentation of AKC. This discrepancy may be related to at least two factors. First, the previous report used very large amounts of exogenous IL-2 (1000 units). Second, the exogenous IL-2 was added at the onset of the 18 hr incubation. These two parameters are different from the conditions of SEB-induced AKC cytotoxicity employed in our experiments. Furthermore, there are several recent re- ports which support the fact that IFN does not play a role in IL-2-enhanced NK cell cytotoxicity (26-28). Recently it has been shown that IL-l may play a role in the regulation of NK activity (29). In addition, IL-l can be produced by monocytes in response to several microbial products (30, 3 1). Thus, it was important to show that neither IL-l nor adherent cells played a role on the induction of AKC activity by SEB. This was shown by the fact that monocyte-free cell suspensions mediated AKC. Second, exogeneous IL- 1 alone did not induce AKC.

On the basis of several experiments it appears that the precursor and effector cell is a LGL which has the phenotypic markers of NK cells. First, AKC was removed when CD 16 positive cells were removed by complement-mediated lysis from the precursor cells prior to the addition of SEB or if CD 16 positive effector cells were removed at the completion of SEB incubation. Second, an enhancement of AKC was seen in cell suspensions enriched for CD16 positive cells by negative selection techniques. Third, the precursors of AKC were derived from the Percoll fractions which contained LGLs and CD 16 positive cells. The validity of these observations is supported by another laboratory which showed that augmentation of cytotoxicity against NK-sensitive targets by whole-fixed bacteria was mediated primarily by cells which have the NK marker, Leu 19 (32).

In order to exclude the possibility that T or other non-NK cells which potentially have the ability to produce IL-2 do not play an obligatory role in SEB-induced activation of NK cells, very highly purified CD 16 positive cell suspensions were prepared by negative selection techniques. Such cell suspensions were 99% pure CD 16 positive cells and devoid of significant other cell types. These results unequivocally showed that non-CD16 cell populations were not required for CD16 positive cell activation by SEB.

The observation that bacteria enterotoxins may regulate NK activity may have considerable in vivo relevance in terms of spontaneously occurring natural defense mechanisms. For example, the rapid induction of phagocytosis by LGL may be an important initial defense against microbial invasion. It has already been shown that NK cells can phagocytose and/or kill bacteria (9, 10,33,34) and in addition, bacteria may play an important role in the maintenance of normal NK cytotoxic activity. For example, studies in germ-free piglets have shown an absence of NK activity which subsequently appears in adult animals. These findings would be consistent with an antigen-driven activation of NK effector cells (3 5, 36).

In summary, the present studies show for the first time that the mechanism by which a bacterial enterotoxin, SEB, can rapidly induce the formation of NK cells with an expanded target cell reportoire is primarily via the endogenous production of IL-2 by CD16 positive cells. Such a regulation of AKC by commonly occurring


bacterial toxins may potentially play an important role in antimicrobial defenses and immunosurveillance against the development of neoplasms.

ACKNOWLEDGMENTS

The authors thank Christopher Henze, Ray Mills, and Michael Lennon for their expert technical assis- tance and Ms. Debbie Chavez for the preparation of the manuscript.

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