A potent inhibitor of protein kinase C inhibits natural killer activity

Int. J. lmmunopharmac., Vol, 10, No. 3, pp. 211-216, 1988. Printed in Great Britain.

0192-0561/88 $3.00+ .00 International Society for lmmunopharmacology.

A POTENT INHIBITOR OF PROTEIN KINASE C INHIBITS NATURAL KILLER ACTIVITY MASAHIKO ITO, FUMINORI TANABE, AKIHIKO SATO, YOSHIYUKITAKAMI and SHIRO SHIGETA Department of Bacteriology, Fukushima Medical College, Fukushima, Japan

(Received 4 June 1987 and in final form 7 October 1987) Abstract - - A potent inhibitor of protein kinase C(PKC),l-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), dose-dependently inhibited natural killer (NK) activity in large granular lymphocytes (LGL) pretreated at 37°C for 30 min. However, neither N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (H-9), which inhibits more effectively cyclicnucleotide-dependent protein kinases than other kinases, nor N-(2-guanidinoethyl)-5-isoquinolinesulfonamidehydrochloride (HAl004), which was used as a control for H-7, reduced NK activity. The inhibitory effect of H-7 was not due to changes in effector cell viability or target cell binding. We also found that H-7 suppresses PKC activity in both the cytosol and membrane fractions of LGL. From these findings, PKC is considered to play an essential role in the lytic mechanism of NK cell-mediated cytolysis.

Natural killer (NK) cells are large granular lymphocytes (LGL) that contain azurophilic granules in the cytoplasm and exhibit cytolytic activity against tumor and virus-infected cells (Timonen, Ortaldo & Herberman, 1981). NK cells are considered to recognize and bind to the susceptible target cells, and then release the cytotoxic factors that bind to and lyse the target cells. Protein kinase C(PKC) was identified in 1979 by Takai, Kishimoto, Iwasa, Kawahara, Mori & Nishizuka (1979a). It requires calcium and phospholipid, and incorporates phosphate from ATP into the substrate. PKC is activated by diacylglycerol that is generated from inositol phospholipids (Takai, Kishimoto, Kikkawa, Mori & Nishizuka, 1979b) or by phorbol esters (Castagna, Takai, Kaibuchi, Sano, Kikkawa & Nishizuka, 1982). This enzyme is found in various organs and tissues and is considered to play an important role in various cell functions (Nishizuka, 1986). In 1984, Hidaka, Inagaki, Kawamoto & Sakai synthesized 1(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride(H-7) which selectively inhibits PKC. H-7 also inhibits superoxide formation in neutrophils stimulated by phorbol 12-myristate 13-acetate (Fujita, Takeshige & Minakami, 1986) and the differentiation of HL-60 cells induced by phorbol diester (Nishikawa, Uemura, Hidaka & Shirakawa, 1986). Recently, the release of NK cytotoxic factors has been suggested to result from protein phosphorylation

by PKC (Graves, Bramhall & Bonavida, 1986). We therefore examined whether the synthesized inhibitors of PKC affect NK cell-mediated cytolysis, by using isoquinolinesulfonamide derivatives, H-7, H-9 and HAl004(Hidaka et al., 1984).

EXPERIMENTAL PROCEDURES

Separation o f large granular lymphocytes (LGL) The heparinized peripheral blood from normal volunteers was placed on Lymphoprep (Nyegaard & Co., Oslo, Norway) and centrifuged at 400 g for 30 min at 4°C. The mononuclear cell layer was collected and washed three times in RPMI 1640 supplemented with 10 % heat-inactivated fetal calf serum (FCS, Boerhinger Mannheim, F.R.G.), 2 mM L-glutamine, 100 U/ml penicillin and 100 /ag/ml streptomycin. Non-adherent lymphocytes were obtained from mononuclear cells by adhesion to the plastic dish precoated with FCS in an incubator containing 5% CO2 for lh at 37°C, followed by passage through a nylon wool column. LGL were separated from nonadherent lymphocytes by centrifugation on discontinuous Percoll density gradients (Pharmacia Fine Chemicals, Uppsala, Sweden, 53, 48 and 43%) using the method of Katz, Zaytoun & Fauci (1982). The 43% Percoll fraction contained enriched natural killer (NK) activity and more than 70% of cells in this fraction had the morphological characteristics of

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LGL. Leu l la positive cells comprised 65% of the cells in this LGL preparation as determined by flow cytometry (Graves et al., 1986). LGL were pretreated for 10, 30 or 60 min at 37°C with several protein kinase inhibitors: 1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride (H-7),N-(2a m i n o e t h y l ) - 5 - i s o q u i n o l i n e s u l f o n a m i d edihydrochloride(H-9) or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride(HA1004) (all reagents purchased from Seikagaku Kogyo Co., Tokyo, Japan) at various concentrations, washed two times, and then assayed for NK activity. N K assay Human erythroleukemia cell line, K562, and human T lymphoma cell line, Molt 4, were used as target cells. Ten million target cells were labeled with 100 taCi of Na25~CrO4 (Daiichi Isotope Laboratory, Tokyo) for lh at 37°C, and were washed three times in RPMI 1640 with 1007o FCS. LGL were suspended in 200 tal of RPMI 1640 with 1007oFCS in each well of microplates (Nunclon Delta, Nunc, Roskilde, Denmark), and were mixed with l x 104 labeled target cells at effector : target (E:T) ratios of 40:1, 20:1 or 10:1. The mixture was incubated at 37°C in a 507o CO2 incubator for 4h. After incubation, 100 tal of supernatant was collected and the radioactivity was counted. The 07o cytotoxicity was calculated by the following formula: ~ocytotoxicity Experimentalrelease-spontaneousrelease 100. - Maximumrelease-spontaneousrelease x The spontaneous release was the radioactivity of the culture medium containing ~Cr-labeled target ceils without effector cells. The maximum release was determined by incubating target with 1N NaOH. All experiments were performed in triplicate. Reagents Human native interferon-/3 (IFN-/3) was purchased from Toray Co. Ltd, Tokyo, and human recombinant interleukin 2(IL-2) was from Shionogi Pharmaceutical Co., Tokyo. Target binding assay The percentage of lymphocytes binding to target cells (TBC) was determined by a modification of the method of Katz et al. (1985). Briefly, 2 × 105 Percollpurified LGL were mixed with a five-fold excess of unlabeled K562 cells, and the mixture was centrifuged at 500 g for 2 min, incubated for 10 min at 37°C, and then stored on ice. The cell pellets were gently

resuspended and the number of TBC was determined under light microscopy by counting at least 200 LGL, K562 cells were easily differentiated from LGL by their size and considerable cytoplasmic granulation. Assay f o r protein kinase C To assay for protein kinase C (PKC), LGL(1 × 107 cells) were suspended in 2ml of 20mM Tris-HC1 at pH7.5, containing 2mM EDTA, 5mM EGTA, 0.SmM phenylmethylsulfonyl fluoride and 50mM 2mercaptoethanol, and disrupted by sonication for 10s three times at 0°C. The supernatant, obtained after centrifugation at 100,000 g for 60 min, was applied onto a DE52 (Whatman) column according to the method of Kikkawa, Takai, Minakuchi, Inohara & Nishizuka (1982). The column was washed with the same buffer and then the enzyme was eluted with 0.15M NaCl in the same buffer. The eluate was used as the cytosol fraction. The pellet in 2 ml of the same buffer was sonicated and treated with Triton X-100 at a final concentration of 0.1070 for 30 min at 0°C. The supernatant obtained after centrifugation at 12,000 rotations/min for 15 min was applied to a DE 52 column and the eluate was used as the membrane fraction. PKC was assayed according to the method of Kikkawa et aL (1982), by measuring the incorporation of 3:p into H1 histone from ), _32p ATP in the reaction mixture (0.25ml) containing 5 tamol of Tris-HC1 at pH 7.5, 1.25tamol of magnesium acetate, 50 tag of H1 histon (Sigma), 2.5 nmol of ),_32p ATP (ICN, 5 - 10 × 10 4 counts/ min/nmol), 125 nmol of CaCI:, 10 /ag of phosphatidylserine (Sigma), 0.2 tag of diolein (Sigma) and the enzyme preparation to be assayed. PKC activity was determined by subtracting the activity measured in the absence of phosphatidylserine and diolein from that measured in its presence. One unit of PKC was defined as that amount of enzyme that incorporated 1 nmol of phosphate from ATP into H1 histone per min.

RESULTS

Figure 1 shows the effects of various protein kinase inhibitors on NK activity of LGL. H-7, which is the most potent and selective PKC inhibitor among these reagents (Hidaka et al., 1984), inhibited NK activity against K562 cells(a) and Molt4 cells(b). A decline in NK activity was seen after a 10 min pretreatment and the degree of inhibition was almost maximal after a 30 min pretreatment. However,

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Fig. 1. Time course of the effect of H-7 and other protein kinase inhibitors on NK activity. Percoll-purified LGL(4 × 106) suspended in 2 ml of RPMI 1640 without FCS were pretreated at 37°C for various periods with H-7(©), H-9(A)and HA1004( • ) at a concentration of 10/~M. After washing, NK activity against K562(a) and Molt 4(b) cells was measured by a 4 h S~Cr-release assay at an E:T ratio of 20:1. The figure represents the mean _+ S.E. of three separate experiments. 100

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Fig. 2. D o s e - effect of H-7 and other protein kinase inhibitors on NK activity. Percoll-purified LGL(4 × 10 6) suspended in 2 ml of RPMI 1640 without FCS were pretreated with H-7(C)), H-9(A) and HA1004( • ) at various concentrations for 30 min at 37 °C. After washing, NK activity against K562(a) and Molt4(b) cells was measured by a 4 h 5'Cr-release assay at an E: T ratio of 20:1. The figure represents the mean _+ S.E. of three separate experiments. neither H-9, which inhibits m o r e effectively cyclic n u c l e o t i d e - d e p e n d e n t protein kinases t h a n o t h e r kinases, n o r HA1004, which is the weakest P K C i n h i b i t o r a m o n g t h e m a n d was used as a c o n t r o l for H-7, affected N K activity. As s h o w n in Fig. 2, H-7 i n h i b i t e d N K activity in a d o s e - d e p e n d e n t m a n n e r . M o r e o v e r , the i n h i b i t i o n o f NK activity was o b s e r v e d at various E : T ratios (Fig. 3). This i n h i b i t o r y effect o f H-7 was n o t a cytotoxic effect o n L G L , because the viability o f L G L p r e t r e a t e d with H-7 for 30 m i n a n d f u r t h e r i n c u b a t e d for 4 h at the c o n c e n t r a t i o n s used was m o r e t h a n 95°7o by t r y p a n blue exclusion assay. T h e O7o cytotoxicity against the

target cells pretreated with H-7 was not different f r o m t h a t against the u n t r e a t e d target cells. Since NK activity is stimulated by I F N a n d IL-2 ( H e r b e r m a n , O r t a l d o & B o n n a r d , 1979, Suzuki, H a n d a , Itoh & K u m a g a i , 1983), the effect o f H-7 o n stimulated NK cells was next examined. As s h o w n in T a b l e l, H-7 reduced the stimulated N K activity to the level of u n s t i m u l a t e d N K activity at a c o n c e n t r a t i o n o f 10 /aM. Twenty/~M H-7 was m o r e i n h i b i t o r y a n d did not alter cell viability during a 4 h NK assay. W e next e x a m i n e d w h e t h e r H-7 suppresses the b i n d i n g o f L G L to target cells. As s h o w n in T a b l e 2, H-7 significantly i n h i b i t e d NK activity, b u t it did n o t

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Fig. 3. Effect of H-7 and other protein kinase inhibitors on NK activity at various E : T ratios. Percoll-purified LGL (4× 106) suspended in 2 ml of RPMI 1640 without FCS were pretreated with medium alone (IS]), H-7(O), H-9(A) and HA10IM( • ) at a concentration of 10 taM for 30 min at 37 °C. After washing, NK activity against K562(a) and Molt4(b) cells was measured at various E : T ratios. The figure represents the mean _+ S.E. of three separate experiments.

Table 1. Effect of H-7 on stimulated NK activity by IFN-/3 and IL-2

H-7 concentration (/aM)

Medium

0 10 20

47.5 ± 1.5 20.6 ± 2.8 16.8 ± 3.2

NK activity (07o) of LGL pretreated with IFN-/3 (1000 U/ml)

IL-2 (100 U/ml)

70.2 _+ 2.5 43.1 ± 1.9 22.5 _+ 2.8

68.4 _ 3.0 45.5 ± 2.4 21.8 ± 1.6

Percoll-purified LGL were pretreated with 1FN-B or IL-2 for 1 h and were then treated with H-7 for 30 min at 37°C. After washing, NK activity against K562 cells was measured at an E: T ratio of 20:1. The data represent the mean _+ S.E. of three separate experiments.

Table 2. Effects of H-7 and other reagents on binding of LGL to target cells and PKC activity in LGL Pretreatment* (10 taM) Medium H-7 H-9 HA1004

o70 TBC* 14.4 14.8 15.0 15.0

_+ 1.1 _+ 0.5 ± 2.5 ___ 3.5

070 cytotoxicity* 49.6 22.4 48.4 50.2

± 2.6 ± 4.0" _+ 1.9 _+ 1.5

PKC activity (°70 of control) ~ Cytosol Membrane 100 55.8 _+ 4.5 97.1 ± 3.5 97.5 ± 3.8

100 32.4 _+ 2.4 99.8 + 8.7 102.9 ___8.8

* Percoll-purified LGL were pretreated with various reagents (10/aM) for 30 min at 37°C. * Target binding assay was performed using K562 cells. * The °70 cytotoxicity against K562 cells at an E : T ratio of 20:1. LGL (1 × 107) pretreated with various reagents (10/aM) were assayed for PKC activity. The data are expressed as the o7o of PKC activity found in LGL pretreated with each reagent compared with that found in LGL pretreated with medium alone. The control specific activities in cytosol and membrane fractions are 7.8 _+ 0.3 and 0.4 + 0.1 (unit/mg protein), respectively. II Significant, P < 0.01. The data represent the mean _+ S.E. of three separate experiments.

Inhibitor of Protein Kinase C Inhibits NK Activity affect the percentage of TBC as compared with the control. Therefore, the inhibition of NK activity by H-7 was not due to the suppression of binding of NK cells to target cells. To examine whether H-7 inhibits PKC activity in NK cells, PKC activity in Percoll-purified LGL pretreated with these inhibitors at 37°C for 30 min was measured (Table 2). H-7 inhibited PKC activity in both the cytosol and membrane fractions of LGL, whereas H9 and HAl004 did not. These findings suggested that the suppression of PKC activity in NK cells by H-7 is related to the inhibition of NK activity. DISCUSSION H-7 is the most potent inhibitor of PKC among the reagents used in our experiments and also inhibits cyclic nucleotide-dependent protein kinases, whereas H9 inhibits cyclic nucleotide-dependent protein kinases more markedly than other kinases (Hidaka et al., 1984). However, the inhibitory effect of H-7 on NK activity is not due to the inhibition of cyclic nucleotide-dependent protein kinases, since H-9 did not reduce NK activity. In addition, H-7 did not decrease the viability of LGL at the concentrations used during the 4 h NK assay. Graves et al. (1986) recently demonstrated that both phorbol ester and calcium ionophore, which are known to activate PKC, are required for release of natural killer cytotoxic factors. Thus, they suggested that the release of natural killer cytotoxic factors results from protein phosphorylation by PKC. Jondal, Patarroyo & Broliden (1986) showed that anti-estrogenic drugs, tamoxifen and clomiphene,

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which inhibit PKC, suppress NK activity. However, they did not examine whether these drugs inhibited PKC activity in the effector cells. In the present study, H-7 significantly inhibited NK activity of LGL in a dose-dependent manner without affecting cell viability of NK cells. The effect of H-7 was observed after a 10 min pretreatment and the inhibition was almost maximal after a 30 min pretreatment. H-7 (10 /aM) also reduced the NK activity stimulated by IFN and IL-2, to the level of unstimulated NK cells. Since H-7 did not inhibit the binding ability of NK cells to target cells, H-7 probably affects the lytic step of NK cell-mediated cytolysis. We also demonstrated that H-7 suppresses PKC activity in both the cytosol and membrane fractions of LGL. We assume that H-7 penetrates the cell membrane at an early stage and directly binds to PKC, thereby blocking its activity. Although we do not know whether the PKC in the cytosol or that in the membrane is involved in NK cell-mediated cytolysis, the suppression of PKC activity in NK cells may be responsible for the inhibition of NK activity. NK activity is suppressed by serine protease inhibitors (Goldfarb, Timonen & Herberman, 1982) and lipoxygenation inhibitors (Seaman,1983) which are considered to block the release of arachidonic acid from membrane phospholipids. Neither of these inhibitors reduces the binding capacity of NK cells to target cells, and these agents were suggested to affect the lytic mechanism of NK cell-mediated cytolysis. The findings of Graves et al. (1986) and our findings indicate that PKC plays an important role in NK cellmediated cytolysis, although the correlation between PKC, serine proteases and lipoxygenase pathway remains to be resolved.

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CASTAGNA,M., TAKAI,Y., KAIBUCHI,K., SANO,K., KIKKAWA,U. ~; NISHIZUKA,Y. (1982). Direct activation of calciumactivated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J. bioL Chem., 257, 7847 - 7351. FUJITA, I., TAKESHIGE,K. & MINAKAMI. S. (1986). Inhibition of neutrophil superoxide formation by 1-(5isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C. Biochem. Pharmac., 35, 4555 - 4562. GOLDFARB.R. H., TIMONEN,T. ~; HERBERMAN.R. B. (1982). The role of neutral serine proteases in the mechanism of tumor cell lysis by natural killer cells. In N K Cells and Other Natural Effector Cells, pp. 931 - 938. Academic Press, New York. GRAVES, S. S., BRAMHALL,J. & BONAVIDA,B. (1986). Studies on the lethal hit stage of natural killer cell-mediated cytotoxicity. I. Both phorbol ester and ionophore are required for release of natural killer cytotoxic factors (NKCF), suggesting a role for the protein kinase C activity. J. Immun., 137, 1977- 1983. HERBERMAN,R. B., ORTALDO,J. R. 8£ BONNARD,G. D. (1979). Augmentation by interferon of human natural and antibody-dependent cell-mediated cytotoxicity. Nature, 277, 221 -223.

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