PMA inhibits NK generation, cytotoxic activity and NK-1.1 expression

Int. J. Immunopharmac., Vol. 15, No. 1, pp. 1 1 - 1 7 , 1993. Printed in Great Britain.

0192-0561/93 $5.00 + .00 Pergamon Press Ltd. International Society for lmmunopharmacology.

PMA INHIBITS NK CELL GENERATION, CYTOTOXIC ACTIVITY AND NK-1.1 EXPRESSION EMIRA AYROLDI, LORENZA CANNARILE, GRAZIELLAMIGLIORATIand CARLO RICCARDI* Institute of Pharmacology, University of Perugia, Italy (Received 16 June 1992 and in final form 22 August 1992)

Abstract - - We investigated the role of protein kinase C activator phorbol 12-myristate 13-acetate (PMA) on IL-2-driven NK cell differentiation, by using an in vitro model previously set up by our laboratory. Bone marrow precursor cells, from mice treated with 5-fluorouracil (FUBM), when cultured with IL-2, generated mature NK cells. The biochemical system involved in this process has not yet been defined. We investigated the possible mechanism by analyzing the effect of PCK activator PMA on NK cell differentiation and lytic activity of mature NK cells. We now report that: (1) PMA inhibited the IL-2-induced NK cell differentiation and induced development of cells which lyse the NK-resistant target P815. (2) PMA inhibited the lytic ability of mature NK cells against NK-sensitivetarget YAC-1. We evaluated the effects of PMA using the expression of NK-associated antigen NK-1.1 and the ability to lyse YAC target as parameters of NK cell differentiation. PMA down-regulated both these parameters, reducing their expression during the differentiation process of NK cells and inducing down-modulation of these in mature NK cells. The results suggest that PKC regulatory control could be under the process of differentiation and activation of NK cells.

Natural Killer (NK) cells are a small population of lymphocytes that exhibit "natural" cytolytic activity against various targets with no restriction in relation to MHC (Koren & Herberman, 1983; Trinchieri, 1989). They express NK-I. 1 surface antigen. NK-I. 1 positive cells manifest lytic activity against NKsensitive tumor target cells whereas NK-I.I negative cells do not (Hackett, Tutt, Lipscomb, Bennett, Koo & Kumar, 1986; Sentman, Kumar, Koo & Bennett, 1989; Koo, Dumont, Tutt, Hackett & Kumar, 1986; Koo, Peppard & Hatzfeld, 1982). NK activity can be increased or decreased by a wide range of stimuli (Koren & Herberman, 1983; Trinchieri, 1989). Interleukin-2 (IL-2), not only boosts mouse, rat and human NK activity but is also able, in vitro, to induce development of the cytolytic effectors NK-I.1 ÷ and MAC-1 + from undifferentiated bone marrow precursors (Henney, Kuribayashi, Kern & Gillis, 1981; Domzig, Stadler & Herberman, 1983; Miyasaka, Darneli, Baron & Tabal, 1984; Migliorati, Moraca, Nicoletti & Riccardi, 1992). In an attempt to analyze the biochemical mechanism regulating these IL-2-induced effects, we

examined the role of PKC activator in both the differentiation and boosting of mature NK cells. Protein Kinase C (PKC) is a Ca +÷ and phospholipid dependent enzyme involved in membrane signal transduction. It has been reported that PKC activity is important for many leukocyte functions and previous studies have suggested its role in regulating NK activity (Goldfarb & Herberman, 1981; Chow, Norstedt, Fredholm & Jondal, 1988; Graves, Bramhall & Bonavida, 1986; Windebank, Abraham, Powis, Olsen, Barna & Leibson, 1988). Modulation of NK activity seems to be induced by the interaction of many stimuli with specific membrane receptors. The biological response is subsequently regulated by intracellular signals, activated by ligand-receptor interaction. However, the nature of postreceptor events leading to NK cell activation is still not well understood. Recently, it has been shown that interaction between NK effector and specific target induces phospholipase-C activation and production of a second messenger which might activate protein kinase C (Graves et aL, 1986). An increase in intracellular calcium has been correlated with the

*Author to whom correspondence should be addressed at: Prof. Carlo Riccardi, Istituto di Farmacologia Medica, via del Giochetto, 06100 Perugia, Italy. 11

E. AYROLD1et al.

12

early NK cell activation stage. It has been shown that PMA increases or decreases the spontaneous cytotoxicity of spleen cells (Graves et al., 1986; Windebank et al., 1988; Senik & Kolb, 1982; Keller, 1979). Several studies have given evidence suggesting that PKC is involved in activating mature NK cells (Goldfarb & Herberman, 1981; Chow et al., 1988; Graves et al., 1986; Windebank et al., 1988; Senik & Kolb, 1982; Keller, 1979), but the role of PKC in regulating NK cell differentiation remains unexplored. In this study we used bone marrow precursor cells and IL-2-generated mature NK cells and analyzed the effects of PMA on (1) cytolytic activity and (2) expression of NK-I.1.

EXPERIMENTAL PROCEDURES

A n tib odies

Monoclonal antibodies (mAbs) directed against Thy-I and CD8 were purchased from New England Nuclear (Boston, MA, U.S.A.). NK-1.1 supernatant was semipurified by ammonium sulfate precipitation. Proteinic title was 17 mg/ml. PK136, a hybridoma producing NK-I.1, was purchased from the American Type Culture Collection (Rockville, MD, U.S.A.). Phenotypic characterization

Expression of NK cell markers was measured by indirect immunofluorescence staining and flow cytometric analysis on a FACS 440 (Becton Dickinson, Mountain View, CA, U.S.A.). The first reagents were mouse NK-I.1, Thy-1 and CD8. The second reagent was a mouse mAb labeled with fluorescein isothiocyanate (FITC). All antibodies were titered to determine the optimal dilution.

Mice

C h r o m i u m release cyotoxicity assay

Inbred C57BL/6 male mice 6 - 8 weeks old were obtained from Charles River (Calco, Italy).

Various concentrations of effector cells were incubated with 5 × 103 / well 5~Cr-labelled cells for 4 h at 37°C in round-bottomed, 96-well plastic microtiter plates as previously described (Herberman et al., 1974). The percentage of specific lysis was calculated as follows:

Tumors

YAC-1 and P815 cells were maintained as previously described (Riccardi, Vose & Herberman, 1983) and used as targets for lytic activity.

Cytotoxicity=

test counts/min-autologous counts/min x 100, total counts/min

Factors

Recombinant human IL-2 (rlL-2) was provided by Cetus (Emeryville, CA, U.S.A.); phorbol 12-myristate 13-acetate (PMA) and 4-phorbol 12, 13-Didecanoate (4aPDD) were purchased from Sigma (Gallarate, Milan, Italy).

Culture conditions

Femoral BM cells from mice treated with 5-fluorouracil (5-FU), 150 mg/kg i.v. 1 day before cell harvesting were cultured in round-bottomed 96-well plates (Sterilin U.K.) at a final volume of 200/A/well (1 x 105 cells/well) with RPMI 1640 medium supplemented with 10% fetal calf serum (Flow Laboratories, McLean, VA, U.S.A.), 2-mercaptoethanol (2 × 10 5 M), 1% gentamycin, 10 U/ml penicillin and 100 mg/ml streptomycin (Ayroldi, Cannarile & Riccardi, 1991) with IL-2 and/or PMA. Cells were cultured for 7 days at 37°C in 5% COz, harvested and used as effectors in the cytotoxicity assay or for evaluating phenotypic markers.

where test counts/min is the mean counts/min released in the presence of effector cells, autologous control counts/rain is the mean counts/min released by target cells incubated with unlabeled cells in place of effector cells and total counts/min is the amount of radioactive chromium incorporated into target cells. For simplicity, standard errors, usually less than 5% of mean values, are omitted from the table and figures. Assessment o f proliferation

Proliferation of BM cells was assessed by incorporation of 3H-thymidine (3H-TdR). 1 × 105 FUBM cells were supplemented with IL-2 and/or PMA into 96-well round bottomed plates at a final volume of 200/A/well. Cells were cultured for 6 days at 37°C in 5% CO2, pulsed for 24 h with 1 /aCi of 3H-TdR per well, harvested with MashII harvester and the radioactivity incorporated measured by liquid scintillation counting.

PMA Inhibits NK Cell Generation

13

CPM (Thousands)

%LYSIS

a5 /1

80 70

30

60 25 50 20 40 15 30 10

20

5 ¸

10

0 0

0.01

5

25

0

50

Fig. 1. Effect of PMA on IL-2-dependent development of NK cells from BM progenitors. BM cells from mice pretreated 1 day before with 5-FU were cultured for 7 days with IL-2 (100 U/ml) and serial doses of PMA. The cytotoxicity was measured in 4 h 5tCr-release assay. The results are expressed as average % of lysis of triplicate cultures. The S.E. were always less than 5% of the mean. E:T=25:l.

Calculation o f lytic units D o s e - r e s p o n s e curves were obtained by plotting the percentage of specific 5'Cr released as a function of the effector-to-target ratios (3 - 4 for each curve). The best fit curve for these functions was logarithmic (from Hewlett - Packard calculator HP-97, program Standard Pac, 01-03) in accordance with previous reports. A lytic unit (LU) was defined as the number of effector cells, extrapolated from the d o s e response curve, required to achieve percent specific target cell lysis. The a m o u n t o f L U / c u l t u r e was calculated by dividing the total number of cells recovered at the end of the culture by the number of cells corresponding to 1 LU.

Statistical analyses Statistical comparisons were performed Student's t-test, **P<0.01.

0.01

5

25

50

PMA (ng/ml)

PMA (ng/ml)

using

RESULTS

Effects o f P M A on IL-2 induced development o f N K cells f r o m bone marrow precursors We have previously shown that IL-2 induces, from bone marrow cells of mice treated with 5-fluorouracil (FUBM), the development of NK-I.1 ÷ and MAC-1 + cells lysing NK-sensitive target YAC-1, but

Fig. 2. Effect of PMA on FUBM cell proliferation. FUBM cells were cultured with IL-2 (100 U/ml) and increased doses of PMA for 7 days. One day before harvesting, the cells were pulsed with 1 t~Ci/well of thymidine and the incorporation assessed as described in Experimental Procedures. not NK-resistant target P815 (Migliorati et al., 1992). To determine whether activation of PKC could regulate this phenomenon, F U B M cells were cultured for 7 days with IL-2 in the presence or absence of several concentrations of P M A . As shown in Fig. l, P M A inhibited the cytotoxicity against YAC-target, in a dose-dependent manner. The cell recovery was increased, suggesting a P M A mitogenic activity on F U B M cells (not shown). To test this last hypothesis F U B M cells, cultured with IL-2 a n d / o r P M A , were pulsed with thymidine I day before harvesting. Figure 2 shows the results of a representative experiment demonstrating that P M A increased the proliferation of F U B M cells in a dose-dependent manner. The cells cultured with P M A alone did not survive to the end o f incubation time (not shown). Because P M A was such a potent inhibitor of NK cytotoxicity, it was important to test its ability to affect surface antigen NK-I.1, another important marker of NK cell differentiation (Koo et al., 1986). Therefore, F U B M cells cultured with IL-2 or IL-2 + P M A for 7 days, were analyzed by flow cytometry analysis, for some surface antigens. The results shown in Fig. 3 demonstrate that IL-2 induces the development of NK-I.1 positive cells from F U B M precursors which were demonstrated as being negative for this marker (Migliorati et al., 1992). While P M A did not affect the expression of CD8 and Thy-1 antigens, used as controls, it greatly reduced the number of cells bearing NK-associated antigen NK-I.1.

E. AYROLD1 et al.

14

THY 1.2

MK1.1

]

CD8

lh-2

I.I I

.....

.... i~

MK 1.1

I h-2

I

I

i~ t

THY 1.2

i - ' - " - ' ~ ~ ~ %

CD8

~ 92.1

~ PMA

12.4

3.5

]

l'%,\

~

I

I

Fig. 3. Flow cytometric analysis of effector cells. F U B M cells cultured for 7 days with IL-2 (100 U / m l ) or IL-2 (100 U / m l ) + P M A (5 ng/ml) were analyzed with a panel of m A b s by flow cytometry as described in Experimental Procedures. Log fluorescence intensity (x-axis) versus cell n u m b e r (y-axis) is depicted. Numbers indicate the % of positive cells. The background (cell stained with the secondary antibody only) falls left of the horizontal line. Table 1. Effect of P M A and 4 a P D D on NK and LAK activity

Table 2. Effect of P M A on IL-2-generated mature NK cells % Lysis +

% NK-11 ~ cells

% Lysist Treatment IL-2 IL-2+ PMA IL-2+4aPDD IL-2 + P M A *

% Cell recovery 29 114 25 22

YAC 17.5 7.5** 16.8 0.6

P815 3.1 16.8 0.8 1.6

F U B M cells were cultured for 7 days with IL-2 (100 U / m l ) or IL-2 (100 U / m l ) + P M A (5 ng/ml) or IL-2 (100 U / m l ) + 4 PDD, P M A analogue, inactive on PKC. The cytotoxicity was measured in a 4 h 5~Cr release assay against the target NK-sensitive YAC-1 and the target NK-insensirive P815. Standard errors were routinely less than 5% of mean cytotoxicity. * P M A , added on day 6 of culturing. **P<0.01. t E : T = 2 5 : 1. Because inhibition of NK differentiation was c o u p l e d to a n i n c r e a s e d u p t a k e o f t h y m i d i n e it w a s i m p o r t a n t to e s t a b l i s h t h e b i o l o g i c a l c h a r a c t e r i s t i c s o f t h e p r o l i f e r a t i n g cells. T h e r e f o r e , F U B M cells c u l t u r e d w i t h I L - 2 o r I L - 2 + P M A , were t e s t e d against the target NK-sensible YAC- 1 and against the t a r g e t N K - r e s i s t a n t P 8 1 5 cells in a 4 h C h r o m i u m release assay. As previously observed PMA inhibited

Treatment Medium IL-2 PMA IL-2+PMA

30 min

4 h

8

7

8 3.5 8

9.7 7.1 8.9

30 min

4 h

42 42 25* 31

50 65 52 48

F U B M cells incubated for 7 days with IL-2 (100 U/ml), were washed and recultured with IL-2 (100 U / m l ) a n d / o r P M A (5 ng/ml) or medium control for 30 min or 4 h. The cells were then analyzed for NK-I.1 expression and cytotoxic activity as described in Experimental Procedures. The cytotoxicity of 1L-2-generated NK cells before reculturing was 15.1 at the E : T = 25 : 1, the % NK-I1 ~ cells was 56. *P<0.01. +E : T = 25 : 1.

t h e c y t o l y t i c activity a g a i n s t Y A C cells, b u t a l s o induced lysis o f NK-insensitive target P815 ( T a b l e 1). T h i s e f f e c t w a s d e t e r m i n e d b y a c t i v a t i o n o f P K C , as d e m o n s t r a t e d b y t h e f a c t t h a t t h e P M A a n a l o g u e 4 - p h o r b o l 12, 1 3 - d i d e c a n o a t e ( 4 a P D D ) u n a b l e to active t h e P K C , d i d n o t a f f e c t t h e d e v e l o p m e n t o f cells l y s i n g Y A C t a r g e t ( T a b l e 1).

PMA Inhibits NK Cell Generation Effect o f P M A on IL-2-generated mature N K cells Different results were obtained when P M A was added on day 6 of culturing. Table 1 shows that P M A added 1 day before cell harvesting, when the cells are already mature effectors (Migliorati, Cannarile, Herberman, Bartocci, Stanley & Riccardi, 1987), induced inhibition of lysis against the YAC target, but did not trigger cytotoxicity against the target NK-resistant P815. P M A induced a stronger inhibition of cytolytic activity in mature cells than precursor cells (Table I). Because significant inhibition of cytotoxicity was observed 24 h after addition of PMA, we investigated whether inhibitory effects of PMA on mature NK cells could occur in a shorter time and in culture conditions where the cells were deprived of their conditioned medium. Therefore, IL-2generated effector cells, were washed, recultured with IL-2 a n d / o r P M A for 0.5 or 4 h and then assayed for lytic activity and N K - I . I expression. As seen in the representative experiment in Table 2 the cytotoxicity and NK-I.1 expression were slightly decreased after incubation with control medium. In spite of this, when the cells were recultured with P M A alone for 0.5 h, cytotoxicity and NK-I.1 expression were further decreased, with respect to the control medium group. In contrast, no down modulation of cytolytic activity or NK-I.1 expression was observed, 4 h after incubation with P M A thus suggesting that the inhibitory effects of P M A were spontaneously reversed. These data indicate that P M A induced inhibitory effects on precursors and mature NK cells and suggests a correlation between NK-1.1 expression and cytolytic activity of NK cells.

DISCUSSION The results of this study demonstrate that PKC may regulate the differentiation process of NK cells from BM precursors and may modulate the cytotoxicity in mature NK cells. The development of NK cells from undifferentiated BM precursors is a strictly IL-2dependent process (Migliorati et al., 1987). BM precursors in the presence of IL-2, acquired the NK-associated antigens NK-I.1, MAC-1 and cytolytic activity only against the target NK-sensitive YAC-1 (Migliorati et al., 1992). The biochemical mechanism involved in the IL-2-driven differentiation of NK cells remains practically unexplored.

15

Phosphorylation of protein kinase C is involved in several mammalian cell signal transductions (Bensussan, Tourvieille, Chen, Kuang, Dousset & Sasportes, 1985; Abrams, Bray & Brahmi, 1983) and it has been suggested that the IL-2 response in T-cells is mediated, at least in part, by PKC activation (Farrar & Anderson, 1985; Evans & Farrar, 1987). We investigated the effects of the PKC activator P M A on IL-2-mediated differentiation of NK cells by monitoring cytolytic activity and expression of NK-I.1, both of which are characteristic only of mature NK cells. PMA, the potent tumor promoter, consistently inhibited both NK-specific cytotoxicity and NK-1.1 expression, but triggered lysis against the target P815 (Table 1) and induced proliferation of FUBM cells (Fig. 2). To test whether the inhibitory effects of P M A on NK precursors could be due to activation of PKC, we examined whether 4aPDD, PMA analog inactive on enzyme, could mimic the effects of PMA. We found that 4aPDD did not affect the development of NK cells from FUBM precursors. The development of LAK activity required the continuous presence of P M A during the culturing or the pretreatment of BM precursor cells with P M A (not shown). Although the P M A added on day 6, when the cells are mature effectors, completely inhibited NK activity it did not induce lysis against P815. These data suggest a different effect of PKC activation on precursors and mature NK cells. The level of inhibition of cytolytic activity induced by PMA was stronger in mature NK cells than precursor cells and was almost eliminated in mature effectors (Table I). A number of experimental systems has shown that PKC is involved in the expression of basal or IL-2induced cytotoxicity in mature NK cells. Abrams et al. demonstrated that P M A inhibits human NK cell mediated cytotoxicity when the compound is present in the cytotoxicity assay or when the effector cells are pretreated with P M A (Abrams et al., 1983). Goldfarb and Herberman showed that P M A inhibited both mouse and human NK cells (Goldfarb and Herberman, 1981). Keller demonstrated inhibition of NK cell activity by TPA in the rat (Keller, 1979). However many reports have shown that TPA enhanced NK killing (Windebank et al., 1988; Senik & Kolb, 1982) and that both the basal and IL-2-induced cytolytic activities (NK and LAK) of CD3- LGL are inhibited by H7 the inhibitor of PKC (Ortaldo, Young & Varesio, 1989). Differences in experimental procedures related to the presence of contaminating cells, or to the presence of P M A in the chromium release assay have

16

E. AYROLDI el al.

been i n v o k e d to a c c o u n t for the discrepancy in the data. W e report t h a t P M A eliminated cytolysis w h e n a d d e d one day before cell harvesting a n d d o w n m o d u l a t e d cytolytic ability a n d the N K - I . I expression o f m a t u r e N K cells after 0.5 h t r e a t m e n t . A f t e r 4 h neither m a r k e r was affected by P M A t r e a t m e n t . These results are particularly interesting because, in a d d i t i o n to the role o f P K C in m o d u l a t i n g cytotoxicity, which could be supposed o n the basis o f previous studies (Trinchieri, O ' B r i e n , Shade & Perussia, 1984; Leibson, M i d t h u m , W i n d e b a n k & A b r a h a m , 1990) they imply a direct role for P K C in NK-I.1 m o d u l a t i o n a n d establish a c o r r e l a t i o n between the expression o f this m a r k e r a n d the level o f cytotoxicity (Table 2). O u r d a t a suggest t h a t P M A has a potentially relevant d o u b l e role o n F U B M cells: (1) o n precursor cells by inducing the d e v e l o p m e n t of a cell p o p u l a t i o n whose lineage is different f r o m that o f NK; (2) o n effector cells by inhibiting lytic ability against the YAC-sensitive target. The different regulatory effects o f P K C activation o n precursor a n d m a t u r e effector cells are not yet

fully u n d e r s t o o d . H o w e v e r , we would suggest that activation o f P K C , inducing p h o s p h o r y l a t i o n of different proteic p a t t e r n s in precursor a n d effector cells, induces different biologic effects. The i n h i b i t o r y effect of P M A o n m a t u r e NK cells, could be due to d o w n regulation of some surface receptor a n d / o r signal t r a n s d u c t i o n system involved in regulating cytotoxic activity. O n the other h a n d , the i n h i b i t o r y effects o n the d i f f e r e n t i a t i o n o f NK precursor cells could be a consequence o f the mitogenic effect of P M A + IL-2 o n a different lineage. This last conclusion is s u p p o r t e d by the fact that IL-2 + P M A not only induced cellular proliferation but also lytic activity against NKresistent target P815. It is particularly interesting that two interacting signals ( P M A a n d IL-2) induced the d e v e l o p m e n t of a lineage different f r o m that induced by a single signal (IL-2 alone). Studies are in progress in o u r l a b o r a t o r y to analyze the intracellular events responsible for the P M A biological effects. Acknowledgement - - This work was supported by AIRC,

PF ACRO, CNR, Italy.

REFERENCES

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