Activation of human natural killer cells by lipopolysaccharide from Actinobacillus actinomycetemcomitans

Arch8oral Blol.Vol. 34, No. 6, pp. 459-463, 1989 Printed in Great Britain. All rights reserved

0003.9969/89$3.00+ 0.00 Copyright 0 1989Peramon Press plc

ACTIVATION OF HUMAN NATURAL CELLS BY LIPOPOLYSACCHARIDE ACTINOBACILLUS

KILLER FROM

ACTINOMYCETEiWCOit4ITANS

R. A. LINDEMANN’ and F. EILBER~ ‘Section of Oral Diagnosis, Oral Medicine and Oral Pathology, UCLA School of Dentistry and the Dental Research Institute, Los Angeles, CA 90024 and *University of Pennsylvania, Philadelphia, PA 19104, U.S.A. (Accepted I6 November 1988) Summary-Lipopolysacharide (LPS) from the Y4 strain of this bacterium, which is implicated in the pathogenesis of juvenile periodontitis, was incubated with human peripheral blood lymphocytes (PBL) and its action compared to that of LPS from Escherichiaco/i. Both LPS augmented cytotoxicity measured against natural killer (NK) cell-resistant tumour targets within 24 h of incubation. Cytotoxicity was exclusively found in NK-enriched low-density large granular lymphocyte fractions, as separated by Percoll gradient. LPS activated NK cells without stimulating high levels of proliferation. The minimum concentration of A. acrinomycetemcomifans LPS required to activate NK cells was I pg/ml; higher concentrations did not significantly increase this activation. LPS had no synergistic effect on the induction of PBL cytoxicity by interleukin-2. In contrast, LPS pre-activated monocytes inhibited the induction of lymphocyte cytotoxicity by either interleukin-2 or LPS.

INTRODUCITON

Natural killer (NK) cells are cytotoxic effecters capable of lysing certain tumour cell targets without prior sensitization or restriction by the major histocompatibility complex (reviewed by Trinchieri and Perussia, 1984). Proposed in oiuo NK functions are

numerous: however, recent attention has focused on their ability to be activated by lymphokines. In fact, most lymphokine-activated killer (LAK) activity (Grimm, 1982) can be attributed to interleukin-2 (IL-2) activated NK cells (Herberman et al., 1987); IL-2 has been shown to be the only signal required to drive NK cells into LAK effecters (Grimm and Wilson, 1985). Activation of NK cytotoxic potential has also been demonstrated using whole bacteria, especially Gramnegative pathogens (Nencioni et al., 1983; Tarkkanen et al., 1986), and it has been postulated that NK cells may perform an anti-bacterial function in oiuo (Lowell et al., 1980; Nencioni et al., 1983). However, the role of the major outer membrane component of Gram-negative bacteria, lipopolysaccharide (LPS), in NK activation is not agreed upon. LPS has profound potentiating effects on the humoral and cellular immune systems, but when LPS was added with whole Salmonella bacteria normally shown to activate NK cells, inhibition of cytotoxicity resulted (Tarkkanen et al., 1986). Whole Actinobacillus actinomycetemcomitans bacteria, implicated in the pathogenesis of juvenile periodontitis (Hammond and Stevens, 1982), also significantly activated NK cells (Lindemann, 1988; Lindemann, Miyasaki and Wolinsky, 1988). This activation was blocked by polymixin B sulphate, which is known to bind the lipid A portion of LPS (Morrison and Jacobs, 1976). This finding suggests that cell-bound LPS activates NK cells. 459

Because NK cells have been demonstrated in human experimental gingivitis (Wynne et al., 1986) and may be important in periodontal immune responses to pathogenic bacteria or in immune regulation, we have now examined the role of LPS in the induction of NK cytotoxicity by standard cytotoxicity assays. Our aims were to determine: (1) the concentration of LPS necessary to activate NK cells; (2) the time course of activation; (3) the effect of monocytes on NK activation; and (4) the similarities to IL-2 activation. MATERIALS AND METHODS

Isolation of lymphocytes Human peripheral blood lymphocytes (PBL) from normal volunteers with healthy periodontal status were obtained by Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, N.J., U.S.A.) densitygradient centrifugation. Adherent cells were depleted by 2 h adherence to plastic tissue culture dishes. Lymphocytes with adherent cells were tested in some experiments. PBL were resuspended in RPM1 (Rosewell-Park Memorial Institute) 1640 medium with 10% human type AB serum. The fraction of monocytes remaining, calculated by universal rosetting reagent (Karavodin and Golub, 1983; Lindemann, Golub and Park, 1987), was l-3%. Lvmohocvtes. ourified as mentioned above, and la;ge graiul& lymphocytes (LGL) were separated by a discontinuous Percoll (Pharmacia) gradient (Timonen, Ortaldo and Herberman, 1981). LPS stimulation LPS was prepared by phenol-water extraction (Westphal and Jann, 1965) of the A. actinomycetemcomitans Y4 strain. The preparation was purified by precipitation with alcohol and sedimentation in an

460

R. A.

LINDEMANN

ultracentrifuge at 110,OOOg for 3 h, and was free from contaminating protein or nucleic acid. LPS from E. coli (0.11: B4, Sigma Chemical Co., St Louis, MO, U.S.A.) was tested in some experiments as a representative enteric bacterial LPS. Lipoteichoic acid (LTA, Sigma, L2.515) from Staphylococcus aureus was tested as an immunogenic cell-wall component of Gram-positive bacteria in some experiments. LPS and LTA were incubated with lymphocytes at varying concentrations for up to one week. Cytotoxicity was measured in standard S’Chromium-release assays (described below).

and F.

a9 15 si 1 -I

Anti-IL-2 antiserum was provided by Amgen (Thousand Oaks, Calif., U.S.A.). A 1: 1000 dilution of this antiserum was shown to inhibit NK cytotoxicity induced by lOOU/ml of ala-125 rIL-2 (Amgen) against M 14 melanoma targets (see below). DNA synthesis

Effector cells (10’ cells/well in 200 ~1 RPM1 with 10% AB serum) were seeded in microwells and cultured with medium only, LPS, LTA, or IL-2 for the indicated period of time. Each well was pulsed with 1 PCi of [methyl-3H]-thymidine ([3H]-TdR; New England Nuclear, Boston, Mass., U.S.A.) for 5 h and was then harvested onto filter paper with a Ph.D. harvester (Cambridge Technology, Cambridge, Mass., U.S.A.) The paper was processed for liquid scintillation counting. Each assay was done in triplicate.

q

5

2,3

495

697

Fraction

Fig. 1. Cytotoxic activation of Percoll-separated PBL fractions by 1pg/ml LPS from E. coli, A. actinomycetemcomitans Y4 and 1 pg/ml LTA from Staph. aureus measured against Ml4 melanoma targets (24-h incubation). Vertical bars are the SEM; LU-lytic units as explained in text.

values into lytic units (LU; Pross et al., 1981). LUs are defined as that number of cells required to cause a specified amount of target lysis (in this case 30%), usually expressed as LU/106 lymphocytes. This method allows a more accurate comparison between lymphocyte donors. The paired t-test (two-tailed) was then applied to determine significance of LU (30%) values. RESULTS

Target cells

The NK-sensitive human erythroleukaemic cell line, K562, and the NK-resistant cell line, UCLA SO-M 14 (M 14, melanoma; Zielske and Golub, 1976), were used as targets in the cytotoxicity assays. Target cells (5 x IO6 in 1 ml RPM1 with 10% fetal calf serum) were labelled with 25OpCi “Cr for 1 h at 37°C. Cytotoxicity

PEL control

10

0

Antibodies

EILBER

assay

K562 or Ml4 target cells (5 x 10’) were mixed with

effector cells (approx. 50: 1,25: 1 and 12.5: 1 ratios) in round bottom microtitre plates containing 200 ~1 of RPM1 1640 with 10% AB serum. The plates were centrifuged at 65 g for 4 min to initiate cell-to-cell contact and then incubated for 4 h at 37°C in a humidified incubator with 5%C02. At the end of the assay, 100 ~1 of supernatant was harvested from each well and counted for 51Cr released from target cells. Each assay was performed in quadruplicate. Cytotoxicity was defined as % specific S’Cr released or [(experimental release - spontaneous release)/ (maximum release - spontaneous release)] x 100%. Experimental release was defined as the counts of Wr released from target cells caused by effector cells as measured by counts/min. Maximal release was the “Cr released from target cells induced by 2% Nonidet P-40 detergent. Spontaneous release was the Wr released from target cells incubated alone. Statistics

Significance of cytotoxicity assays was determined by first converting the three effector-to-target ratio

LPS activation

of PBL

Previous experiments had demonstrated that primarily NK cells were activated by whole bacteria to kill K562 and Ml4 targets (Lindemann, 1988; Lindemann et al., 1988). Those experiments had also suggested that cell surface LPS was responsible for the cytotoxic activation. To determine if the same ceil would respond to extracted LPS, PBL (Percoll gradient) fractions were tested: NK-enriched low-density fractions 2 and 3 were highly responsive to activation by both LPS and LTA; in contrast, high-density fractions 4, 5, 6 and 7 were unresponsive (Fig. 1). To determine the minimum and optimum LPS concentration necessary to activate NK cells, PBL depleted of adherent cells were incubated with multiple concentrations of LPS and tested for cytotoxicity after 24 h culture. Figure 2 shows that a significant increase (p < 0.05) in cytotoxicity above control values occurred at the 1 pg/ml dose. Concentrations above this dose had no additional effect on cytotoxicity. The kinetics of LPS and LTA activation were tested by adding in 1 pg/ml concentration of these agents to PBL depleted of adherent cells during a l-week incubation period and measuring cytotoxicity against M 14 targets. Cytotoxicity was measured on days 1, 4 and 7 (Fig. 3); it peaked 1 day after culture. By days 3-4, there was virtually no significant cytotoxic enhancement over that of unstimulated controls. To define better the time required for activation, PBL were incubated with LPS for 24 h, and cyto-

LPS NK activation 35

30-

af

461

25

-

20

-

30 F 25 -

T

15-

-m-

E.coli

-.-

LTA

-

Y4

+

Control

% 2 -I

I0 5O-

-5~

I

control

0.001

’ 0.01

I

I

I

I

0.1

1.0

10

100

pg/ml

LPS

Fig. 2. Effect of A. acfmomycetemcomitans Y4 LPS concentration on development of PBL cytotoxicity after 24-h culture measured against MI4 melanoma targets for three subjects; control is unstimulated PBL. The vertical bars are

2

0

4

on DNA

synthesis

IL-2 is known to induce PBL DNA synthesis which

may be necessary for the concurrent development of cytotoxicity. Therefore the ability of LPS and LTA to induce DNA synthesis was tested. Proliferation after LPS stimulation was measured by [)H]-TdR incorporation and compared to IL-2 induced proliferation 4 and 7 days after culture. Table 2 indicates that moderate stimulation of DNA synthesis was detected for E. coli LPS and LTA on day 4, but A. actinomycetemcomitans Y4 LPS-treated PBL elevated thymidine uptake only slightly above background. Pre-exposure

of PBL to LPS

As IL-2 activated NK cells have been shown to be the major cytotoxic effecters of LAK activity, the effects of LPS on LAK development were investigated. LPS was added to whole PBL for 1 h and then

T _

8

15-

8 3

10-

5-

L 0

I 4

6

12

16

20

Culture

*Lytic units-the lysis.

26

the cells were thoroughly washed. Lymphocytes were cultured with either IL-2 or LPS. Pre-exposure of PBL to LPS led to inhibition of LAK cytotoxicity against K562 targets and reduced LPS activation at 24 h (Table 3). When PBL were depleted of adherent cells, the l-h pre-exposure to LPS had no effect on the subsequent development of LAK or LPS cytotoxicity (Table 3). The relationship between LPS-induced and IL-2induced cytotoxicity was investigated. When LPS and IL-2 were maintained in culture over a 24-h period, the combined cytotoxic effects were either equal to the IL-2 effect or were additive; they were never found to exert a synergistic effect on cytotoxicity. LAK development occurred in the presence or

Untreated Y4, 1 pg/ml)

24

(Ill Fig. 4. The kinetics of PBL cytotoxic activation in lytic units (LU) during a 24-h incubation measured against Ml4 melanoma targets for three subjects. Vertical bars are the SEM.

Table 1. ‘The effect of anti-IL-2 antiserum on LPS, LTA, and IL-2-induced lymphocyte cytotoxicity against melanoma Ml4 targets, expressed in lytic units* (at 24 h)

Control LPS (A. actinomycetemcomirans LPS (E. co/i, 1 pg/ml) LTA (Staph. aureus, 1 pg/ml) IL-2 (50 l-J/ml)

6

25

20 c

LPS efect

-

Day Fig. 3. The kinetics of PBL cytotoxic activation over a l-week incubation measured in mean lytic units (LU) against Ml4 melanoma targets for five subjects. For LPS and LTA, 1U/ml concentrations were used for activation; control is unstimulated PBL. Vertical bars are the SEM.

the standard deviation of the mean; LU-lytic units as explained in text. *Significantly different (p < 0.05, paired r-test) from the control.

toxicity was measured at several times. Activation required approx. 8-10 h and peak cytotoxicity occurred between 16 and 20 h of incubation for all subjects tested (Fig. 4). To determine if LPS induced IL-2 release which caused the measured activation, an antiserum to IL-2 was added to the cultures. Table 1 shows that the antibodies blocked lymphocyte activation by IL-2 but had no effect on cytotoxicity induced by LPS or LTA.

6

0.79 f 0.50 5.43 f 0.98 6.72 + 1.21 8.35 f 1.76 16.54+2.11

Anti-IL-2 0.65 k 6.20 f 5.99 * 7.87 f 4.32 f

0.63 1.15 1.77 1.43 1.54

number of cells per 1 x IO6lymphocytes required to cause a 30% target cell

R. A. LINDEMANN and F. EILBER

462

Table 2. Effect of 1 pg/ml LPS and LTA on PBL DNA synthesis 1,4 and 7 days after culture Condition IL-2 (100 U/ml) LPS (E. coli) LPS (A. actinomyceremcomilans Y4) LTA (Staph. aureus) Control

Day 1

(counts/min f SD) Day 4

Day I

315 *43 328 f 67 309 &-87

32,919 k 3888 5957 k 1582 1155 k 132

31,658 f 4290 2187 + 672 870 k 197

429 +C50

7418 & 1547

1065 k 313

387 + 38

457 & 27

493 k 69

not responsible for inducing the observed cytotoxicity. It also suggests that IL-2 was not present in sufficient concentration to stimulate cell division; the lack of DNA synthesis supports this observation. DISCUSSION High concentrations of LPS appeared to be required to activate NK cells (approx. 1 pg/ml). HowLPS is a potent immunoregulating agent which affects both humoral and cellular immune reever, this may be due to the relative sensitivity of the assay system. It is possible that lower LPS concensponses. Macrophages are highly sensitive to LPS trations may have an activating effect in oiuo. With and it can potentiate their tumouricidal activity (Doe Gram-negative septicaemia, LPS blood levels have and Henson, 1978). Our study has demonstrated that been reported to be in the 0.5-5.0 ng/ml range (Levin human NK cells can also undergo cytotoxic actiet al., 1970). Presumably, in confined tissue spaces vation by LPS, but that interactions between LPS, without the dilutional effect of peripheral blood, LPS adherent accessory cells and NK cells may modify concentrations could be higher. Alternatively, macrothis. phages are exquisitively sensitive to LPS. Ding and NK activation kinetics by LPS appear identical to Nathan (1987) have demonstrated that as little as those of whole Gram-negative bacteria (Lindemann l-10 pg/ml renders peritoneal macrophages refracet al., 1988) because cytotoxicity peaked I day after culture. This finding further supports the idea that tory to subsequent activation by interferon-y or tumour necrosis factor-a. Our results also illustrate surface-bound LPS is responsible for the activation the activation differences between macrophages demonstrated with whole bacteria. The question (monocytes) and NK cells. By pre-exposing PBL to arises as to why LPS cytotoxic activation declined rapidly after approx. 24 h. It was shown that LPS LPS for 1 h and then washing off the LPS, subsequent minimally stimulated DNA synthesis during a 7-day activation by IL-2 or LPS was inhibited; when adherent cells were removed such inhibition was not culture period. The inability to maintain cytotoxicity in LPS-PBL cultures may be due to lack of prolif- detected. This suggests that LPS preferentially eration of cells specific for effector function. Prolif- activates monocytes and that the effects of LPS infection may be to inhibit eration and cytotoxicity have been linked in LAK cell in Gram-negative activity by Grimm and Wilson (1985). Another pos- NK activity through the down-regulating effects of sible explanation for the observed lack of DNA macrophages (Aderem et al., 1986). These findings are in contrast to those of Tarksynthesis may be that LPS does not induce or only kanen et al. (1986), who demonstrated that LPS had modestly induces IL-2 release. When anti-IL-2 antian inhibitory effect on activation. This difference may serum was incubated with LPS and PBL, cytotoxicity was unaffected. This suggests that IL-2 release was be explained by differing concentrations of LPS: Tarkkanen used 20-30 pg/ml concentrations of Salmonella LPS, whereas 1 pg/ml was shown to be as Table 3. The effects on cytotoxicity when lymphocytes were effective as higher concentrations in our study. pre-exposed to LPS, when LPS was incubated in combinaDifferences between LPS from enteric and oral bactetion with IL-2 ria may also explain these contradictory findings: Cytotoxicity (LU 30%). however, we noted that LPS concentrations above Experimental 1 pg/ml were commonly less effective or inhibitory to groups PBMC PBL activation than a concentration of 1 pg/ml. 1.65 k 0.59 0.03 f 0.02 Untreated The cytotoxic activation effects of A. actinomy16.99 k 1.66 9.12 k 2.91 LPS (1 &ml) cetemcomitans LPS were similar to those of E. co/i, 46.34 f 5.23 22.65 + 3.05 IL-2 (100 U/ml) despite any differences between oral and enteric 55.80 & 7.05 34.97 + 2.89 LPS/IL-2 bacterial LPS. We also compared LPS with LTA, a 41.80 f 3.00 5.98 i: 1.11 Pre-LPS/IL-2t prominent antigenic cell-wall component of Gram12.40 f I .76 3.43 * 0.79 Pre-LPS/LPSt positive bacteria. The effects of LTA on NK cells *LU 30%-lytic units. The number of lymphocytes per 106 have not, as far as we are aware, been reported and, required to cause a 30% target cell lysis (K562). interestingly, LTA from Staph. aweuS also activated tPre--pre-exposure of PBL for 1 h with I U/ml LPS from NK cells. The LTA effect on activation kinetics was A. actinomyceremcomirans Y4. After washing, 100U/ml also similar to that of LPS, the primary cytotoxic IL-~ or 1 fig/ml LPS was added for 24h. effect being measured 1 day after culture. LTA PBMC-peripheral blood mononuclear cells. PBLexerted a modest effect on proliferation, yet it was peripheral blood lymphocytes depleted of adherent significantly lower than IL-2 induced DNA synthesis. mononuclear cells. absence of adherent cells, from adherent cell-depleted

but was always higher populations (Table 3).

LPS NK activation LPS activation was compared to IL-2 activation: IL-2 induced higher cytotoxicity, prolonged cytotoxicity and cell division. When IL-2 and LPS were combined in culture, cytotoxic effects were either equal to IL-2 alone or additive; they were never synergistic. This finding suggests that they activate via different pathways and that the IL-2 pathway is more efficient in promoting cytotoxicity. The mechanism whereby LPS activates NK cytotoxicity is poorly understood. Salata et al. (1984) observed that LPS enhanced NK activity by increasing NK binding to tumour targets and by promoting more efficient killing. Inter-relationships between immune cells during the course of Gram-negative bacterial infection may depend on the relative numbers of effecters present. Furthermore, potentially opposing effects may occur depending on which cell type first encounters and processes LPS. Because of the preferential activation of macrophages, NK anti-bacteria1 immune responses

could be inhibited

by LPS.

Acknowledgements-We thank Dr Sidney Golub for helpful discussion, Dr Hungui Shau for his critical reading of the manuscript and Angela Layton Gadsby for technical assistance. The Y4 strain of A. actinomycetemcomirans was generously provided by Dr A. Nowotny, University of Pennysylvania. REFERENCES

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