Immunology 1990 70 106-110

A central role for phosphoinositide hydrolysis in activating the lytic mechanism of human natural killer cells S. C. CHOW & M. JONDAL Department of Immunology, Karolinska Institute, Stockholm, Sweden

Acceptedfor publication 10 January 1990

SUMMARY Using neomycin, which inhibits phosphoinositide breakdown, cytotoxicity mediated by natural killer (NK) cells was suppressed in a dose-dependent manner, with complete inhibition at 16 mM. Generation of inositol phosphates in effector cells after target cell binding was inhibited in the presence ofneomycin. The formation of effector to target cell conjugates was not affected. Neomycininduced inhibition of NK killing was abolished when TPA was added to the cytotoxic assays. This reconstitution was dependent upon extracellular Ca2+. When the intracellular free Ca2+ level in effector cells was reduced from 73 + 11 nm to 43 + 3 nm (n = 4) using the Ca2+ indicator dye, Quin 2, NK killing was markedly reduced. Inhibiting the enzyme diacylglycerol (DG) kinase in effector cells with 10 gM R59022 (DG kinase inhibitor) potentiates NK killing, suggesting an increase in protein kinase C (PKC) activity due to accumulation of DG. The PKC inhibitor, H-7, suppressed NK killing in a concentration-dependent manner. These results demonstrate that phosphoinositide metabolism is an early event and its derived second messengers play a central role in activating the lytic mechanism of NK cells. INTRODUCTION Natural killer (NK) cell-mediated cytotoxicity is triggered by the interaction of effector cell receptors with putative target structures on target tumour cells. So far, neither NK cell receptors nor target structures have been identified. However, a number of adhesion molecules like LFA-1, ICAM-1, LFA-3 and CD 18 are known to be involved in effector to target cell conjugation (Sitkovsky, 1988; Uggla et al., 1989; Axberg et al., 1987). These adhesion molecules may, by themselves or in association with other membrane receptors, interact and generate biochemical signals that activate the lytic mechanism of NK cells. Evidence from previous work (Chow et al., 1988; Windebank et al., 1989) strongly suggests that transmembrane signalling through phosphoinositide-derived second messengers may provide the biochemical signals required to activate the lytic mechanism. Hydrolysis of phosphoinositides by phospholipase C (PLC) releases two second messengers, inositol 1,4,5-trisphosphate (1P3) and diacylglycerol (DG). IP3 mobilizes intracellular Ca2+ from endoplasmic reticulum (ER) and DG activates protein kinase C (PKC), resulting in the phosphorylation of specific

proteins that regulate cellular responses (Bishop & Bell, 1988; Nishizuka, 1984; Abdel.latif, 1986; Majerus et al., 1986). Different killing mechanisms may be involved in NK cellmediated cytotoxicity and could vary with different target cell types. Degranulation and secretion of pore-forming molecules (performs) stored as cytoplasmic granules may represent one of several potential killing mechanisms (Young & Liu, 1988). Killing may also depend upon secretion of cytotoxins or 'apoptosis', the activation of a target cell suicide programme which leads to DNA fragmentation in target cells (Gromkowski et al., 1986; Duke, Cohen & Chervenack 1986). All these mechanisms are dependent upon Ca2+, although some cloned T cells can still kill in the absence of Ca2+ (Clark et al., 1988). Phosphoinositide-derived second messengers may provide the positive transduction signals required for activating the lytic mechanism in NK cells. In contrast, another second messenger system, the cyclic nucleotides, seems to provide negative signals by inhibiting NK killing through impairment of target cell recognition (Ullberg et al., 1983). In the present study, the role of phosphoinositide hydrolysis in activating the lytic mechanism of NK cells was further characterized.

MATERIALS AND METHODS

Abbreviations: AM, acetoxymethyl ester; DG, diacylglycerol; FCS, fetal calf serum; H-7, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine; IP3, inositol 1,4,5-trisphosphate; NK, natural killer; PA, phosphatidic acid; PKC, protein kinase C; PLC, phospholipase C; TPA, 12-0tetradecanoylphorbol- 13-acetate. Correspondence: Dr S. C. Chow, Dept. of Immunology, Karolinska Institute, Box 60400, 104 01 Stockholm, Sweden.

Chemicals Lymphoprep was obtained from Nycomed AS, Oslo, Norway. 12-O-tetradecanoylphorbol-13-acetate (TPA), Quin 2/AM and

1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H-7) were obtained from Sigma Chemicals, St Louis, MO. Neomycin

106

Natural killer cells

107

sulphate was from Welcome, Beckenham, Kent, U.K. R59022 was from Janssen Chemicals, Belgium. Na25"CrO4 was from NEM and [3H]inositol was from American Radiolabelled Chemicals Inc. (St Louis, MO). 20

Isolation and treatment of effector lymphocytes Normal peripheral blood lymphocytes from healthy donors were isolated by density centrifugation on Lymphoprep. Nonadherent lymphocytes (effector cells) were obtained by passage through scrubbed nylon-wool columns and suspended in RPMI-1640 medium with 10% fetal calf serum (FCS). Quin 2-loaded cells were obtained by incubating non-adherent lymphocytes with 50 gM Quin 2/AM at 370 for I hr. The cells were washed twice before use in NK assays or cytosolic Ca2+ determination (Treves et al., 1987).

Cell lines and labelling of target cells K562 (erythroleukaemia), MOLT-4 (T-cell leukaemia) and JURKAT (T-cell leukaemia) were maintained in suspension culture using RPMI-1640 medium with 10% FCS and antibiotics (100 U/ml penicillin and 50 pg/ml streptomycin). To label cells, 50 pCi of Na251CrO4 were added to approximately 2 x 106 target cells in 0 3 ml, incubated for 1 hr at 370, washed and incubated for a further 20 min to lower the spontaneous release. The cells were washed again and diluted to appropriate concentration for 5'Cr-release assays. The radioactivity released due to cell lysis was calculated using the standard formula (Ullberg & Jondal, 1981). Measurement of inositol phosphate release This was essentially the same as previously reported (Chow et al., 1988). [3H]myoinositol labelled effector cells (4 x 107 cells each) were added to K562 cells (8 x 106 cells), mixed and centrifuged down to facilitate conjugation. The pellet was resuspended by gentle vortexing and incubated at 37°. The assay was stopped by adding ice-cold 10% perchloric acid after 15 min. The supernatants were processed as described by Downes, Hawkins & Irvine (1986).

Effector-target cell conjugation assay Effector and target cells were centrifuged at 1:1 ratio for 5 min at 100 g and incubated at 370 for 10 min. The pellet was resuspended gently and the effector-target cell conjugates were scored using a haemocytometer. At least 200 cells per sample were counted. RESULTS Inhibition of NK-cell mediated cytotoxicity by neomycin

Using various concentration of neomycin, NK killing against two target cell lines, K562 and JURKAT, was inhibited in a dose-dependent manner (Fig. 1). Complete inhibition against both target cell lines was observed at 16 mm of neomycin. This concentration was not toxic on either effector or target cells after 3 hr in a control incubation (data not shown), and does not affect effector to target cell binding (Table 1). Effect of neomycin on the formation of inositol phosphates in effector cells Previous study (Chow et al., 1988) showed that phosphoinosit-

0)

C 1 2 4 8 16 Neomycin conc. (mM)

Figure 1. Inhibition of NK cell-mediated cytotoxicity by neomycin. Neomycin was added to effector cells prior to the addition of target cells. Effector: target cell ratio was 20:1. Target cells used were K562 (0) and JURKAT (0). Results from one typical experiment out of four. C, control.

Table 1. Effect of neomycin on NK cell binding % binding cells*

Effector cells Non-adherent lymphocytes (NK killing)

Target cells K562

Exp.

+Neomycin

-Neomycin

1 2

8-78 10-14

8-73 9-79

Effector and target cells were centrifuged together, with and without 16 mm neomycin at 100g. After 10 min incubation at 370, the cells were gently dispersed and scored as outlined in the Materials and Methods. Effector: target ratio is 1: 1. * Percentage effector cells binding to target cells.

ide (PI) hydrolysis was triggered only in NK cells after conjugating with susceptible target cells. In the presence of 16 mM neomycin, inositol phosphate production in effector cells was similar to control (effector cells only) after binding to JURKAT target cells (Fig. 2). Without neomycin there was a significant increase in total inositol phosphates (P

A central role for phosphoinositide hydrolysis in activating the lytic mechanism of human natural killer cells.

Using neomycin, which inhibits phosphoinositide breakdown, cytotoxicity mediated by natural killer (NK) cells was suppressed in a dose-dependent manne...
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