Protein Kinases in NK Cell Cytotoxicity N-Terminal Kinase 1 Mitogen

Differential Role of p38 and c-Jun N-Terminal Kinase 1 Mitogen-Activated Protein Kinases in NK Cell Cytotoxicity1

Rossana Trotta, Katia Fettucciari, Livio Azzoni, Bekele Abebe, Kristin A. Puorro, Laurence C. Eisenlohr, and Bice Perussia2

The serine-threonine mitogen-activated protein kinase (MAPK) family includes extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 kinases. In NK cells, spontaneous or Ab-mediated recognition of target cells leads to activation of an ERK-2 MAPK-dependent biochemical pathway(s) involved in the regulation of NK cell effector functions. Here we assessed the roles of p38 and JNK MAPK in NK cell-mediated cytotoxicity. Our data indicate that p38 is activated in primary human NK cells upon stimulation with immune complexes and interaction with NK-sensitive target cells. Fc␥RIIIA-induced granule exocytosis and both spontaneous and Ab-dependent cytotoxicity were reduced in a dose-dependent manner in cells pretreated with either of two speciﬁc inhibitors of this kinase. Target cell-induced IFN-␥ and Fc␥RIIIA-induced TNF-␣ mRNA accumulation was similarly affected under the same conditions. Lack of inhibition of NK cell cytotoxicity in cells overexpressing an inactive form of JNK1 indicates that this kinase, activated only upon Fc␥RIIIA ligation, does not play a signiﬁcant role in cytotoxicity. These data underscore the involvement of p38, but not JNK1, in the molecular mechanisms regulating NK cell cytotoxicity. The Journal of Immunology, 2000, 165: 1782–1789.

atural killer cells exert MHC-nonrestricted cytotoxicity, protein tyrosine phosphatases (4, 5, 12, 13). Biochemical pathways independently from prior sensitization, against a variety induced preferentially during ADCC or spontaneous cytotoxicity N of target cells, including virus-infected, transformed, have also been identified. For example, granule exocytosis-medi- and IgG Ab-coated cells (reviewed in Ref. 1). While receptors ated spontaneous cytotoxicity against the prototypic K562 target transducing activating signals to trigger spontaneous cytotoxicity cells depends on activation of protein kinase C, but not phospha- are just beginning to be defined (2), the well-characterized low tidylinositol 3-kinase, whereas the reverse is true for ADCC (14). affinity receptor for the Fc fragment of IgG (Fc␥RIIIA) is respon- Mitogen-activated protein kinases (MAPK) transduce signals sible for triggering Ab-dependent cell-mediated cytotoxicity that regulate cell growth and differentiation (15). They are serine- (ADCC)3 (reviewed in Ref. 3). Early biochemical events induced threonine kinases, the enzymatic activity of which is elicited upon upon cross-linking the receptors involved in binding IgG Ab- phosphorylation of threonine and tyrosine residues in a Thr-X-Tyr by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. coated or NK-sensitive target cells include protein tyrosine kinase motif in their regulatory domain (16). This is mediated by dual ϩ (PTK) activation and increased intracellular Ca2 concentration as specificity MAPK kinases, which also become activated, following a consequence of phospholipase C-␥1 and -␥2 activation (4–7). phosphorylation, under the same conditions. The MAPK family Intermediate molecules in the elicited signaling cascades have includes the extracellular signal-regulated kinases (ERK), the c- been reported to modulate NK cell cytotoxicity. The PTK Syk (8), Jun N-terminal kinase (JNK)/stress-activated protein kinase, and the adapter protein LAT (linker for activation of T cells) (9), and the p38 MAPK (p38). For most part, ERK are activated by mito- the Vav-Rac1 pathway (10, 11) play roles in both types of cyto- genic factors, while JNK and p38 are activated by stress-inducing toxicity, which are abolished upon PTK inhibition or activation of agents or proinflammatory cytokines. Although in most cell types each MAPK is activated by specific nonoverlapping kinases, http://classic.jimmunol.org named MEK, activation of both p38 and JNK by a single MEK4 kinase has also been reported (17). In different cell types the dif- Department of Microbiology and Immunology, Kimmel Cancer Center, Jefferson ferent MAPKs may act antagonistically or cooperate with each Medical College, Philadelphia, PA 19107 other to regulate different cell functions (15). Examples for this are Received for publication December 20, 1999. Accepted for publication May 26, 2000. the opposite effects of ERK (facilitating) and JNK (protecting) to The costs of publication of this article were defrayed in part by the payment of page control B cell receptor-induced apoptosis (18) and the requirement Downloaded from charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. for both kinases in TCR- and CD28-dependent T cell activation 1 This work was supported in part by U.S. Public Health Service Grants CA37155 and and IL-2 production (19). CA45284. K.F. was on leave of absence from the Department of Clinical Medicine, We and others have shown that ERK-2 activation occurs in NK Pathology, Pharmacology, Section of General Pathology and Immunology, University ␥ of Perugia (Perugia, Italy). cells upon target cell binding or Fc RIIIA stimulation, and that 2 Address correspondence and reprint requests to Dr. Bice Perussia, Jefferson Medical cytokine mRNA accumulation, spontaneous cytotoxicity, ADCC, College, Kimmel Cancer Center, BLSB 750, 233 South 10th Street, Philadelphia, PA and Fc␥R-induced degranulation depend at least in part on ERK-2 19107. E-mail address: [email protected] function (20–23). The possible role of the other MAPK family 3 Abbreviations used in this paper: ADCC, Ab-dependent cell-mediated cytotoxicity; members is unknown. The observation that p38 plays a role in BLT, sodium benzyloxycarbonyl-L-lysine thiobenzyl ester; EA, IgG-sensitized E; ERK, extracellular signal-regulated kinase; Fc␥R, receptor for the Fc fragment of actin reorganization leading to formation of filamentous actin in IgG; F-actin; filamentous actin; JNK, c-Jun N-terminal kinase; K-2, kinase 2; MAPK, endothelial cells upon platelet-derived growth factor (PDGF) (24) mitogen-activated protein kinase; MAPKAP kinase-2, MAPK-activated-protein kinase-2; PDGF, platelet-derived growth factor; PTK, protein tyrosine kinase; Vac, and vascular endothelial growth factor (25) stimulation suggests vaccinia; wt, wild type. the possibility that the same kinase may be activated to play a

similar role in NK cells upon target cell binding and thus may be and 1 mM EDTA). Western blotting was performed according to our pub- involved in regulating granule exocytosis-mediated cytotoxicity. lished protocols (20), and Ab-reactive proteins were detected with HRP- Increased JunB mRNA and emergence of JunB:Fos heterodimers labeled sheep anti-rabbit Ig sera and enhanced chemiluminescence (Am- ersham, Arlington Heights, IL). Two methods were used to assess kinase with increased AP-1-activity during spontaneous cytotoxicity have activity: 1) expression (detected by Western blotting) of the enzymatically been reported in the human NKL cell line (26). Because ERK, but active phosphorylated forms of p38 and JNK, and 2) JNK1 and p38 kinase not JNK, is involved in the regulation of JunB transcription (27, assays, performed according to the protocols of Hibi and Rose (31, 32), 6 28), a possible differential role for distinct MAPK in cytotoxicity respectively. For these each kinase was immunoprecipitated from 5 ϫ 10 NK cell lysate equivalent using the specific Ab (1 ␮g) and protein A- may be envisaged. Sepharose (Pharmacia, Uppsala, Sweden). After four washes with lysis To define the role of non-ERK-2 MAPK in the regulation of NK buffer and two with 10 mM HEPES (pH 7.5), 25 mM MgCl2, 50 mM NaCl cell cytotoxicity, we investigated the involvement of p38 and JNK supplemented with 1 mM Na3VO4, 50 mM sodium fluoride, and 1 mM kinases. Our data indicate that both Fc␥RIIIA triggering and NK PMSF, the protein A-Sepharose beads were incubated (30 min, 30°C) with ␮ cell recognition of nonsensitized target cells generate signals lead- 30 l of reaction buffer and occasional tapping. The kinase buffer for the JNK1 assays was 20 mM HEPES (pH 7.5), 2 mM DTT, 20 mM ␤-glycerol ing to activation of p38, whereas only Fc␥RIIIA stimulation acti- ␮ phosphate, 20 mM MgCl2, 0.1 mM Na3VO4, 20 mM ATP, 10 Ci vates JNK1. Inhibition experiments indicate that only p38 plays a [␥-32P]ATP (sp. act., 4000 Ci/mmol; ICN, Costa Mesa, CA). GST-ATF2 role in ADCC and spontaneous cytotoxicity and exclude a role for (aa 1–96; Santa Cruz Biotechnology) and GST-c-Jun (aa 1–223) (31), each JNK1 in ADCC, demonstrating a differential role for p38 and 1 ␮g, were used as interchangeable substrates for JNK because both polypeptides have sequences specifically recognized by this kinase (33). JNK1 in regulating NK cell cytotoxic functions. Similar to ERK, The kinase buffer for the p38 kinase assays was 20 mM HEPES (pH 7.5), p38 is also involved in the regulation of target cell-induced cyto- ␤ 25 mM -glycerol phosphate, 25 mM MgCl2, 2 mM DTT, 0.1 mM ␮ ␥ 32 kine expression. Na3VO4, 20 mM ATP, 10 Ci [ - P]ATP. The substrate was GST-ATF2, as described above. After the reaction the kinases were eluted from the beads by heating and were analyzed in 10% SDS-PAGE (reducing condi- Materials and Methods tions). Western blots were performed to verify that equal amounts of pre- Cells lines and NK cell preparations cipitated MAPK were loaded per sample, and in vitro phosphorylation of The human monocytic THP-1, erythroleukemic K562, T lymphoid Jurkat the kinase substrates was detected after exposure of the filters to X-AR (clone J32), B lymphoblastoid RPMI-8866 and 721.221 cell lines were films (Eastman Kodak, Rochester, NY). MAPKAP kinase-2 assays were maintained in culture in RPMI 1640 medium (BioWhittaker, Walkersville, performed using a commercial kit following the manufacturer’s recom- MD) supplemented with 10% heat-inactivated FBS (Sigma, St. Louis, MO) mendations (MAPKAP kinase-2 IP-Kinase Assay Kit, Upstate Biotechnol- ogy, Lake Placid, NY; with a sheep serum reacting with both rabbit and hu- and 100 ␮g/ml L-glutamine (Life Technologies, Gaithersburg, MD). Homogeneous NK cell preparations were obtained from 10-day cocul- man MAPKAP kinase-2 for immunoprecipitation, and the KKLNRTLSVA tures of PBL from healthy individuals with 30-Gy irradiated RPMI-8866 peptide as a substrate). cells following negative selection using a mixture of anti-CD14, -CD3, and Vaccinia virus (Vac) recombinant preparations and NK cell -CD5 mAb and indirect anti-Ig rosetting as previously described (29). The ϩ ϩ Ϫ ϩ infection cell preparations contained Ͼ98% CD16 /CD56 /CD3 and Ͻ3% CD3 cells, as determined by indirect immunofluorescence (flow cytometry) us- To generate JNK1 recombinant Vac, cDNA fragments encoding the ing a panel of mAb. FLAG-tagged wild-type (wt) and dominant negative (APF) Ala183 and Phe185 JNK1 (33) were generated after BamHI and HindIII, or XbaI and Monoclonal and polyclonal Abs HindIII digestion, respectively. The blunt-ended cDNA were inserted into

by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. mAb 3G8 (anti-CD16), B159.5 (anti-CD56), B36.1 (anti-CD5), OKT3 (anti- the NheI cloning site of the psC11 vector and introduced into Vac, WR CD3), and B52.1 (anti-CD14) have been previously described (29); the strain, by homologous recombination, as previously described (34). For infection, NK or Jurkat T cells were incubated with the indicated Vac anti-FLAG M2 mAb was obtained from Sigma. The polyclonal rabbit sera 7 anti-MAPK p38 and JNK1 were purchased from Santa Cruz Biotechnology recombinant (10–20 multiplicity of infection, 37°C, 1.5 h, 10 cell/ml, and ϫ 6 (Santa Cruz, CA); the polyclonal rabbit serum detecting enzymatically ac- an additional4hin2 10 /ml RPMI 1640 medium supplemented with tive, Thr183- and Tyr185-phosphorylated JNK1 and JNK2 was obtained 10% FBS). The cells were used immediately after washing. Expression of from Promega (Madison, WI); that anti-Thr180- and Tyr182-phosphorylated the wt or dominant negative JNK1 fusion recombinant proteins was con- active p38 was purchased from New England Biolabs (Beverly, MA); the ﬁrmed by Western blot with anti-JNK1 or anti-FLAG Ab, and the effect of anti-phospho-c-Jun mAb was obtained from Santa Cruz Biotechnology; the expression of APF JNK1 on c-Jun phosphorylation was determined by the anti-HSP90 Ab was purchased from Transduction Laboratories Western immunoblotting with anti-phospho c-Jun Ab. Cell lysates for the (Lexington, KY). latter were in 0.5 M NaCl. http://classic.jimmunol.org Cell stimulation Sodium benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT)-esterase release assay Cells were incubated (5 ϫ 106/ml; 37°C) for the indicated times with the different stimuli. These were: K562 and 721.221 cells (5:1, NK to target This was performed as described by Visonneau et al. (35) using as stimuli cell ratio), PMA (50 ng/ml) and ionomycin (1 ␮M; both from Sigma), plastic-immobilized mAb 3G8, B159.5 as a negative control, and PMA immune complexes (rabbit IgG-sensitized bovine erythrocytes (EA)), or E (50 ng/ml) and ionomycin (1 mM; both from Sigma) as the positive con- (negative control; 0.5% suspension) prepared as previously described (30), trol. Cell-free supernatants were collected after 4-h incubation at 37°C. The Downloaded from and biotin-labeled mAb 3G8 or B159.5 (both 20 ␮g/ml) with added strepta- percentage of released BLT esterase activity was calculated for each sam- vidin (50 ␮g/ml; Sigma). In the samples used for Western blotting, K562 ple according to the formula (S/S ϩ C) ϫ 100, where S is OD in the and 721.221 cells were ﬁxed (3 ϫ 106 cells/ml 1% paraformaldehyde, 30 supernatant, and C is that in the corresponding cell lysate. min on ice) and washed extensively before use. This treatment prevents Cytotoxicity assays possible activation of endogenous kinases and has no effect on target cell binding to NK cells or subsequent stimulation of early biochemical events K562, THP-1, and 721.221 cells, as indicated, were used as the target in and ERK activation in NK cells (23, 21) (data not shown). When target 3-h 51Cr release assays (30). For redirected ADCC, mAb 3G8 or B159.5 as cells were used, effector/target cell contact was facilitated by centrifugation the control (both supernatants, 1/4 predetermined optimal concentration) (600 rpm, 2 min) before incubation. When indicated, the p38 inhibitors was present throughout the assay with THP-1 cells. These were not lysed SB203580 and SB202190 (Calbiochem, San Diego, CA) were added to the in the presence of the control B159.5 mAb (not shown). A constant number effector cells at the indicated concentrations for1hat37°C before of target cells (5 or 10 ϫ 103/well, as indicated) and serial dilutions of stimulation. effector cells were used in triplicate. Spontaneous release from any target cell used was Ͻ10%; lytic units (36) were calculated at 40% cytotoxicity. Western blotting and kinase assays F-actin detection After stimulation the cells were lysed (108 cells/ml lysis buffer: 1% Non- idet P-40, 10 mM HEPES (pH 7.5), 0.15 M NaCl, 10% glycerol, 10 ␮g/ml After stimulation, NK cells were labeled with N-(7-nitrobenz-2-oxa-1,3-

each aprotinin and leupeptin, 1 mM PMSF, 1 mM Na3VO4, 50 mM NaF, diazol-4-yl)phallacidin (Molecular Probes, Eugene, OR) and analyzed by 1784 p38 AND JNK1 IN NK CELL FUNCTIONS

flow cytometry as described by Salmon et al. (37). Briefly, 3.5 ϫ 105 and ionomycin as a control (Fig. 1A, left panels). Minimal levels cells/sample were fixed (10 min, 20°C) in 3.7% formaldehyde in PBS of active p38 and JNK1 were detected in NK cells before stimu- (Sigma), permeabilized, and labeled (20 min, 4°C) in PBS containing 0.5% lation or after 10-min incubation with control E. As with ERK1 saponin (Sigma), 0.2% FBS, 0.005% Tween-20, 0.01% NaN3, and N-(7- nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin-phallacidin (1 U/sample). Rel- and ERK2 (20, 22) (not shown), the levels of active p38 and JNK1 ative F-actin content is expressed as the ratio between the mean fluores- significantly increased to plateau by 5–10 min, started declining cence intensity of NDB staining in stimulated and nonstimulated control within 20 min (not shown), and returned to control levels by 1-h NK cells. stimulation with EA ( first and third panels). All samples expressed Northern blot analysis similar levels of total p38 and JNK1 (second and fourth panels). Active p38 and JNK1 were also detected upon stimulation with After 1-h incubation at 37°C with or without the p38 inhibitor SB202190 (50 ␮M), NK cells (5 ϫ 106/ml) were cultured with the indicated stimuli PMA/ionomycin. As expected, p38 and JNK1 immunoprecipitated for 1.5 h, and Northern blot analysis was performed as previously de- from EA-stimulated NK cells were able to phosphorylate the ex- scribed (38), with slight modifications. Briefly, total RNA was extracted ogenous substrates ATF2 and GST-c-Jun, respectively, in in vitro using TRIzol reagent (Life Technologies, Gaithersburg, MD), size frac- kinase assays (Fig. 1B). The same substrates were only minimally tionated in 1% agarose-formaldehyde gels, transferred to Hybond-nylon membranes (Amersham), and hybridized to cDNA probes specific for hu- phosphorylated by the kinases immunoprecipitated from E-stimu- man IFN-␥, TNF-␣, and TCR ␤-chain (detecting a nonfunctional, trun- lated cells. cated, 1.0-kb mRNA species in NK cells) for normalization. cDNA probes To determine whether binding of NK to sensitive target cells 32 were labeled with [␣- P]dCTP (spec. act., 3000 Ci/mmol; ICN) by nick leads to activation of the same kinases, the presence of active p38 translation (Roche, Indianapolis, IN) (20, 21). Hybridization was detected and JNK1 was analyzed in the lysates from NK cells stimulated and quantitated using a PhosphorImager (PhosphorImager SI, Molecular Dynamics, Sunnyvale, CA) with proprietary software (ImageQuant). with K562 or 721.221 target cells. Lysates from target and NK cells incubated separately for the same time as the experimental Results samples and mixed after lysis were used as the negative control; Fc␥RIIIA- and target cell-induced p38 and JNK1 kinase positive controls were lysates from NK cells stimulated with PMA/ activation in NK cells ionomycin. The levels of active p38 were significantly increased in To determine whether Fc␥RIIIA ligation induces p38 and JNK1 NK cells after interaction with K562 or 721.221 cells, whereas MAPK activation, Western blot analysis was performed with anti- those of active JNK1 were unchanged (Fig. 1A, right, first and active p38 or anti-active JNK Ab on lysates from NK cells non- third panels). Comparable levels of p38 and JNK1 were detected stimulated or stimulated with immune complexes (EA), E, or PMA in all samples (right, second and fourth panels). No active JNK1 was detected upon longer (1-h) stimulation of NK with the target cells (data not shown), and the immunoprecipitated JNK1 did not phosphorylate recombinant ATF2 used as the substrate in in vitro kinase assay (Fig. 1C).

Role of p38 in Fc␥RIIIA-induced granule exocytosis and by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. cytotoxicity The pyridinyl imidazoles SB203580 and SB202190 speciﬁcally inhibit p38, but not other MAPK activity (39), by occupying p38 ATP-binding sites (40). Low doses of SB203580 (1 ␮M) were sufﬁcient to inhibit by 50% the p38 activity in lysates of NK cells stimulated with PMA/ionomycin (not shown). The ability of SB203580 to inhibit the p38-dependent pathway in NK cells was assessed using as a readout the induced activation of MAPKAP

http://classic.jimmunol.org kinase-2 (K-2), a physiological substrate of p38 (40, 41) activated in NK cells upon stimulation with immune complexes or target cells (data not shown). Addition of 13 ␮M SB203580 to intact cells inhibited by 50% the PMA/ionomycin-induced MAPKAP FIGURE 1. p38 and JNK1 activation in NK cells upon Fc␥RIIIA stim- K-2 activity (Fig. 2, inset). Further inhibition was obtained at ulation and target cell binding. A, NK cells were incubated with the indi- higher doses. Concentrations of this inhibitor up to 50 ␮M did not cated stimuli under the conditions described in Materials and Methods

Downloaded from affect activation of ERK and JNK MAPK (data not shown). Both (iono, ionomycin). Western blot analysis (10% SDS-PAGE, reducing con- Fc␥RIIIA- and PMA/ionomycin-induced BLT esterase secretion ditions) was performed on the lysates (106 cells/samples) with active anti- p38 and anti-p38 Ab (top panels) sequentially on the same filter, and with were inhibited in a dose-dependent manner in NK cells preincu- active anti-JNK and JNK1 Ab (bottom panels) sequentially on a duplicate bated with SB203580 (Fig. 2) or SB202190 (data not shown). filter. Right panels, NK and paraformaldehyde-treated K562 or 721.221 The involvement of p38 in ADCC and spontaneous cytotoxicity cells were incubated separately (Ϫ) or together (ϩ) for 10 min. The lysates was assessed analyzing the effect of its pharmacologic inhibition in from NK and target cells incubated separately (i.e., not stimulated) were 51Cr release assays (Fig. 3). The levels of redirected ADCC against mixed after lysis and used as controls. B, p38 (top) and JNK1 (bottom)in THP-1/3G8 (left panels) and of spontaneous cytotoxicity against vitro kinase assays were performed on p38 and JNK1 immunoprecipitated K562 and 721.221 target cells (middle and right panels) mediated from cells stimulated with control erythrocytes (E) or IgG-coated E (EA) by NK cells pretreated with SB203580 or SB202190 (top and bot- for 10 min. ATF2 and GST-c-Jun fusion proteins were used as substrates tom, respectively) were significantly lower than those mediated by (see Materials and Methods). C, In vitro kinase assay was performed, using ATF2 recombinant protein as substrate, on JNK1 immunoprecipitated from control nontreated cells. Based on calculation of lytic units at 40% ␮ the same lysates as in A, right panels (top). JNK1 immunoprecipitated from cytotoxicity in the two experiments reported, 50 M SB203580 or each sample was detected with anti-JNK1 Ab (bottom). This experiment is SB202190 inhibited ADCC by 77 and 84%, respectively, sponta- representative of three performed with similar results. neous cytotoxicity against K562 by 90 and 91%, and spontaneous The Journal of Immunology 1785

Table I. Actin polymerization upon Fc␥RIIIA stimulation

Stimulusb

Inhibitor a Anti-CD56 Anti-CD16 PMA/ionomycin

None 1.1 Ϯ 0.1c 1.4 Ϯ 0.1* 1.5 Ϯ 0.2* SB203580 1.0 Ϯ 0.1 1.5 Ϯ 0.3* 1.4 Ϯ 0.2* SB202190 0.9 Ϯ 0.2 1.3 Ϯ 0.2* 1.4 Ϯ 0.3*

a NK cells were incubated (3.5 ϫ 106/ml, 1 h, 37°C) in medium without (none) or with the indicated inhibitors, 50 ␮M. b Biotin-labeled anti-CD56 (B159.5) and anti-CD16 (3G8) mAb, 20 ␮g/ml, with added streptavidin, 50 ␮g/ml; PMA, 5 ng/ml; ionomycin, 1 mM (see Materials and Methods). c Relative F-actin content was determined in the cells by ﬂow cytometry after NBD-phallacidin incorporation. Values are the ratio between mean ﬂuorescence intensity in stimulated and nonstimulated cells (mean Ϯ SD, n ϭ 5, 10,000 cells sample analyzed). -p Ͻ 0.01 (two-tailed, Student t test), CD16 or PMA/ionomycin vs CD56 stim ,ء ulation, under any condition; inhibitor-treated vs control nontreated cells, nonsignif- icant.

kinases, regulates it, F-actin content was analyzed in NK cells FIGURE 2. Effect of p38 inhibition on Fc␥RIIIA-induced granule exo- treated with inhibitors of p38 (SB203580, or SB202190; Table I) cytosis. NK cells were incubated (1 h, 37°C) in medium with the indicated and/or of MEK (PD098059; not shown) and stimulated for 5 min concentrations of SB203580. The anti-CD16 mAb 3G8, anti-CD56 mAb with anti-Fc␥RIIIA mAb (Table I). Similar to the changes reported B159.5, and PMA/ionomycin, as indicated, were used as stimuli in a 4-h for granulocytes (37) in similar experimental conditions, the F- BLT esterase release assay (see Materials and Methods). This experiment is representative of three performed with similar results. Inset, After incu- actin levels detectable in NK cells were significantly increased bation (40 min, 37°C) in medium with the indicated concentrations of upon PMA/ionomycin or CD16, but not CD56, stimulation. The SB203580, NK cells were incubated (10 min, 37OC) without (Ϫ) or with levels of CD16-induced actin polymerization did not change in (ϩ) PMA/ionomycin, and a kinase assay was performed on the MAPKAP cells pretreated with any of the inhibitors, alone or in combination K-2 immunoprecipitated from the cell lysates using KKLNRTLSVA pep- (not shown). tide as a substrate (see Materials and Methods). This experiment is representative of two performed with similar results. Role of JNK in ADCC and spontaneous cytotoxicity As detected in Western blotting with an Ab recognizing specifi- cytotoxicity against 721.221 target cells by 64 and 71%. In the cally Ser63-phosphorylated c-Jun (Fig. 4), expression of the same conditions, cell viability, expression of Fc␥RIIIA, LFA-1, FLAG-tagged, Vac-encoded, recombinant inactive APF mutant by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. CD18, and CD11b, and formation of conjugates with any of the (33), but not of the wt JNK, correlated with lack of c-Jun phos- target cells used were not affected (not shown). phorylation induced by PMA-ionomycin stimulation in J32 cells and by EA in NK cells. This confirms that JNK1 activity is sig- Role of p38 in actin rearrangement nificantly reduced in the APF-inactive mutant-expressing cells, in- To determine whether Fc␥RIIIA ligation mediates induction of dicating that overexpression of APF inhibits the enzymatic activity actin polymerization and, if so, whether p38, alone or with ERK of endogenous JNK on its natural substrate (c-Jun) in intact cells. http://classic.jimmunol.org

FIGURE 3. Effect of p38 inhibition on Fc␥RIIIA-dependent and spontaneous cytotoxicity. NK cells from two separate donors were incubated (1 h, 37°C) without (f)or Downloaded from with (Œ and F) the indicated concentrations of SB203580 (top) or SB202190 (bottom). Fc␥RIIIA-redirected lysis (THP-1 target cells in the presence of anti-CD16 mAb 3G8, THP-1/3G8) and spontaneous cytotoxicity against K562 and 721.221 cells were tested in a 3-h 51Cr release assay. Target cells were 5 ϫ 103/well. The x-axis shows the E:T cell ratio; the y-axis shows the percent speciﬁc 51Cr release. Each experiment is representative of two performed with similar results. 1786 p38 AND JNK1 IN NK CELL FUNCTIONS

FIGURE 6. Effect of p38 inhibition on Fc␥RIIIA- and K562-induced IFN-␥ mRNA accumulation. NK cells (5 ϫ 106/sample) were incubated (1 h, 37°C) in medium with or without 50 ␮M SB202190. After an addi- FIGURE 4. Effect of overexpression of a dominant negative JNK1 on tional 1.5-h incubation with EA (or E as control) and K562 (106/sample), induced c-Jun phosphorylation. Jurkat cells, clone J32 (left panels), and total RNA was extracted, and Northern blot analysis was performed using NK cells (right panels) were not infected (none) or were infected with Vac IFN-␥ (top panel), and TCR␤ (bottom panel) cDNA probes sequentially on recombinants encoding FLAG-tagged wt or kinase-inactive JNK1 (APF). the same filter. This experiment is representative of two performed with The cells were left unstimulated (none) or were stimulated with PMA- similar results. ionomycin (Jurkat) or EA (NK cells; see Materials and Methods). Western blot analysis was performed on the lysates using anti-phospho c-Jun Ab (top panel), anti-FLAG (middle panel), and anti-HSP90 (bottom panel) sequentially on the same filters to assay for c-Jun phosphorylation, Vac- encoded protein expression, and total amount of protein loaded per lane. recombinant Vac encoding APF or wt JNK1 and stimulated with EA. In cell expressing the kinase inactive or the wt form of JNK1 the levels of active JNK1 were, respectively, significantly lower or ADCC and spontaneous cytotoxicity were tested in NK cells ex- higher than those in noninfected cells (Fig. 5A, right panel). Ad- pressing wt JNK1 kinase or its inactive mutant (APF; Fig. 5). ditionally, CD16-redirected and spontaneous cytotoxicity were not Expression of the exogenous proteins was confirmed by Western inhibited in APF-JNK1-expressing NK cells or in NK cells in- blot using an anti-JNK1 or an anti-FLAG Ab (Fig. 5A, left panels). fected with wt (Fig. 5B) or empty Vac (not shown). Similar results An ϳ46-kDa band corresponding to endogenous JNK1 was de- were obtained using the human NK cell line NKL infected with the tected in all lysates analyzed, while a slower migrating band, cor- JNK1 recombinant viruses (not shown). responding to FLAG-tagged APF or wt JNK1, was detected only in the lysates from cells infected with the wt or the mutant JNK1 ␥ Vac recombinant. Similar levels of wt and APF JNK1 were ex- Role of p38 in Fc RIIIA- and target cell-induced cytokine pressed in the Vac-infected cells. To confirm that overexpression mRNA accumulation of the kinase-inactive form of JNK1 results in inhibition of en- To determine whether p38 plays a role in regulating cytokine ex- dogenous JNK1 activation, the expression of active JNK1 was pression induced by Fc␥RIIIA ligation or target cell binding, we analyzed by Western blot in NK cells noninfected or infected with analyzed the effects of the p38 inhibitor SB202190 on IFN-␥ (Fig. by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 5. Effect of overexpression of a dominant negative JNK1 on Fc␥RIIIA-dependent and spontaneous cytotoxicity. A, Left panels, Western blot analysis performed with anti-FLAG and anti-JNK1 Ab on the 1% Nonidet P-40 lysates from NK cells not infected (none) or infected with Vac recombinants encoding FLAG-tagged wt or dominant negative JNK1 (APF). The lysates were from the same cells as those used for cytotoxicity in B. Right panel, Western blot analysis performed with anti-active JNK on the lysates of cells not infected (E, none) or infected with Vac recombinants encoding FLAG-tagged wt or dominant negative JNK1 (APF) and stimulated with E or EA. B, NK cells were left uninfected (E, none) or were infected with Vac recombinants encoding FLAG-tagged wt (f)or kinase inactive JNK1 (Œ, APF). Redirected cytotoxicity using THP-1 in the presence of anti-CD16 3G8 mAb and spontaneous cytotoxicity against K562 target cells were measured in a 3-h 51Cr release (104 target cells/well). This experiment is representative of three performed with similar results. The Journal of Immunology 1787

6) mRNA accumulation. In two separate experiments, no signifi- the inhibitors or on inhibition of ERK or other biochemical path- cant variations in basal cytokine mRNA levels were observed in ways upstream of MAPK. Specifically, 1) cell viability, conjugate SB202190-pretreated cells, whereas EA- and K562- induced formation, and expression of several adhesion molecules involved IFN-␥ mRNA accumulation were inhibited by 86 and 83% and by in spontaneous cytotoxicity or ADCC are not modified in the in- 71 and 74%, respectively, compared with those in nontreated cells. hibitor-treated cells; 2) the same treatment does not affect PMA/ In the same experiments EA-induced TNF-␣ mRNA accumulation ionomycin-induced ERK activation (not shown); and 3) both in- was inhibited by 64 and 79%, whereas the levels of target cell- hibitors used, which specifically bind the ATP-binding site of the induced TNF-␣ mRNA were too low to allow meaningful com- p38 kinase, do not affect the activity of the closely related ERK parison (not shown). and JNK or other serine-threonine kinases, such as c-Raf, p90 S6kinase, and P70 S6 kinase (41). The possibility that in our pre- vious report using a MEK inhibitor to prevent ERK activation (21), Discussion p38 inhibition was responsible for the almost complete inhibition of both ADCC and spontaneous cytotoxicity can be discounted We report that phosphorylation and activation of the p38 MAPK based on the observation that p38, but not ERK, phosphorylation occur in human primary NK cells upon Fc␥RIIIA ligation and (and thus activation) is maintained in cells treated with a MEK target cell binding, and that, like ERK-2 (21, 23), it plays a role in inhibitor (not shown). cytotoxicity and cytokine expression, whereas JNK, activated ex- Similar to what we (21) and others (23) previously reported for clusively upon Fc␥RIIIA ligation, does not play a role in either the ERK pathway, our findings indicate that the inhibition of ADCC or spontaneous cytotoxicity. These observations serve to ADCC following p38 inactivation depends on a regulatory effect establish that all MAPK family members can be activated upon of this kinase on NK cell degranulation. Our data suggest that both ligand binding to Fc␥RIIIA and, among the MAPK family, iden- ERK- and p38-mediated signals, although necessary, are not suf- tify p38 and ERK as key molecules in the biochemical pathway(s) ficient alone to activate Fc␥RIIIA-mediated degranulation and the that regulates activation of two of the NK cell functions, namely lytic process. As discussed above, sequential and/or interdepen- cytotoxicity and cytokine production. dent activation of the two kinases is unlikely, and their roles do not All MAPK family members have been previously shown to be appear to be redundant. Whether the two kinases phosphorylate activated in murine macrophages (32) upon Fc␥R cross-linking. distinct substrates, or both kinases are needed to phosphorylate a However, the identity of the Fc␥R type responsible for this effect single substrate remains to be determined. Among specific p38 was not definitively established. Using NK cells, we extend those substrates, MAPKAP K-2, activated both upon Fc␥RIIIA stimu- data to report that Fc␥RIIIA is, in itself, capable of this effect. lation and target cell binding (data not shown), may represent a Upon ligand binding, other immune receptors structurally similar biochemical mediator common to the two types of cytotoxicity. ␥ to Fc RIIIA, e.g., the B cell Ag receptor, also transduce signals Direct detection of perforin- and granzyme B-containing intra- resulting in activation of the same three kinases (42). cellular granules has indicated the ERK2 dependence of ␥ We previously reported that Fc RIIIA-dependent ERK-2 acti- Fc␥RIIIA-induced granule migration along cytoskeletal structures vation plays a role in cytokine production, ADCC, and spontane- by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. in NK cells (23). Here we show that, as previously reported for ␥ ous cytotoxicity in NK cells (20, 21). The kinetics of Fc RIIIA- Fc␥RIIIB and Fc␥RIIA in neutrophils (37), Fc␥RIIIA stimulation induced activation and inactivation (dephosphorylation) of p38 induces actin polymerization in NK cells. Activation of p38 is and JNK in NK cells are very similar, if not identical, to those of required for PDGF-induced cell motility responses such as cell ERK, and it is likely that a specific phosphatase(s) induced via migration and actin reorganization (24) and mediates the vascular ␥ Fc RIIIA stimulation controls activation of all MAPK. The most endothelial growth factor-induced ERK-independent actin reorga- likely candidate for this is the dual specificity protein MKP-1, nization in endothelial cells (25) and the TGF-␤1-induced actin which dephosphorylates ERK-2, JNK, and p38 MAPK in PMA- polymerization in neutrophils (44). However, we obtained no ev- stimulated U937 cells (43). idence of a role for p38 (this manuscript), ERK (21), or the two http://classic.jimmunol.org Unlike immune complexes, tumor target cells binding to NK kinases combined (not shown) in target cell-, Fc␥RIIIA-, or PMA/ cells induce activation of p38, but not JNK1 kinase. One or more ionomycin-induced actin polymerization in NK cells. In cytotoxic activating receptors may be sensitive to fixation, as used here. T cells degranulation is regulated by the motor protein kinesin, and However, neither ERK (21, 23) nor p38 activation is prevented several kinesin-associated proteins have been identified, the state under these conditions. This indicates that at least one of the target of phosphorylation of which affects the extent of kinesin motor surface molecules triggering spontaneous cytotoxicity is still ca- activity and subsequent granule release (45, 46). Kinesin and/or Downloaded from pable of responding to fixed cells to induce early biochemical kinesin-associated proteins might be among the direct or indirect events and activation of at least two MAPK family members, mak- targets of the Ser/Thr kinase phosphorylation cascade induced by ing it unlikely that lack of JNK activation depends on lack of NK p38 or ERK activation during NK cell cytotoxicity. cell activation by the fixed target cell. Thus, we favor the hypoth- Our data indicate that both p38 and ERK MAPK activation reg- esis that, unlike Fc␥RIIIA, the receptor(s) triggering spontaneous ulate at least in part the immune complex-induced IFN-␥ and cytotoxicity transduces signals leading specifically to the activa- TNF-␣ mRNA accumulation and the target cell- induced IFN-␥ tion of ERK and p38, but not JNK, similar to the PDGF receptor mRNA accumulation in human NK cells. These data add to a (24) or the TCR (19). Whatever the reason for the lack of JNK recent report indicating a role for p38 MAPK in integrin-triggered activation upon target cell binding, our data support a nonredun- IL-8 production by human NK cells (47). Fc␥RIIIA stimulation dant role of individual MAPK members in NK cells. induces AP-1-dependent transcription of the cytokines tested (48), We have used pharmacologic inhibitors to determine the nec- and both ERK and p38 phosphorylate and regulate the activity of essary role of p38 in ADCC and spontaneous cytotoxicity and in this and other transcription factors (49, 50). Thus, in this case, cytokine expression, similar to ERK2. Several lines of evidence cytokine regulation may occur at least in part at the transcriptional support that the observed inhibition does not depend on toxicity of level. However, post-transcriptional regulation of the expression of 1788 p38 AND JNK1 IN NK CELL FUNCTIONS

several cytokines has been reported for p38 (39, 51), and the lev- 16. Su, B., and M. Karin. 1996. Mitogen-activated protein kinase cascades and reg- ␥ ulation of gene expression. Curr. Opin. Immunol. 8:402. el(s) at which this kinase regulates Fc RIIIA- and/or target cell 17. Derijard, B., J. Raingeaud, T. Barrett, I. H. Wu, J. Han, R. J. Ulevitch, and induced IFN-␥ and TNF-␣ mRNA remains to be determined. The R. J. Davis. 1995. Independent human MAP-kinase signal transduction pathways possibility that JNK1 may also be involved (at least in cytokine defined by MEK and MKK isoforms. Science 267:682. 18. Sakata, N., H. R. Patel, N. Terada, A. Aruffo, G. L. Johnson, and E. W. Gelfand. expression induced upon Fc␥RIIIA stimulation) is not excluded by 1995. Selective activation of c-Jun kinase mitogen-activated protein kinase by our data and unfortunately cannot be tested at present. No specific CD40 on human B cells. J. Biol. Chem. 270:30823. 19. Su, B., E. Jacinto, M. Hibi, T. Kallunki, M. Karin, and Y. Ben-Neriah. 1994. JNK inhibitors of this kinase are available, and the use of cells infected is involved in signal integration during costimulation of T lymphocytes. Cell with Vac-encoding inactive kinase is inappropriate to study non- 77:727. immediate events that require host cell RNA transcription and pro- 20. Trotta, R., P. Kanakaraj, and B. Perussia. 1996. Fc␥R-dependent MAP kinase activation in leukocytes: a common signal transduction event necessary for ex- tein translation, both known to be subverted by the vaccinia virus. pression of TNF-␣ and early activation genes. J. Exp. Med. 184:1027. Collectively, our data serve to establish that only p38 and ERK, 21. Trotta, R., C. Puorro, M. Paroli, L. Azzoni, B. Abebe, L. C. Eisenlohr, and B. Perussia. 1998. Dependence of both spontaneous and antibody-dependent, among MAPK, play a role in NK cell cytotoxicity and cytokine granule exocytosis-mediated, NK cell cytotoxicity on extracellular signal-regu- production induced upon target cell recognition. They also open lated kinases. J. Immunol. 152:6648. the way to future studies to define the mechanism(s) through which 22. Milella, M., A. Gismondi, P. Roncaioli, L. Bisogno, G. Palmieri, L. Frati, M. G. Cifone, and A. Santoni. 1997. CD16 cross-linking induces both secretory p38 regulates NK cell lytic functions, the common or specific and extracellular signal-regulated kinase (ERK)-dependent cytosolic phos- MAPK substrates involved, and the possible role of JNK1 specif- pholypase-A2 (PLA2) activity in human natural killer cells: involvement of ERK, but not PLA2, in CD16-triggered granule exocytosis. J. Immunol. 158:3148. ically in functions other than cytotoxicity. 23. Wei, S., A. M. Gamero, J. H. Liu, A. A. Daulton, N. I. Valkov, J. A. 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