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The Journal of Immunology, 1999, 162: 4062-4068.
Copyright © 1999 by The American Association of Immunologists

Engagement of Natural Cytotoxicity Programs Regulates AP-1 Expression in the NKL Human NK Cell Line1

Karine Bernard*, Anna Cambiaggi{dagger}, Sophie Guia*, François Bertucci§, Samuel Granjeaud*, Rebecca Tagett*, Catherine N’Guyen*, Bertrand R. Jordan* and Eric Vivier2,*,{ddagger}

* Centre d’Immunologie, Institut National de la Santé et de la Recherche Médicale (INSERM)/Centre National de la Recherche Scientifique (CNRS) de Marseille-Luminy, Marseille, France; {dagger} Unité de Biologie Moléculaire du Gène, Institut Pasteur, Paris, France; {ddagger} Institut Universitaire de France; and § Laboratoire de Biologie des Tuneurs, Institut Paoli-Calmettes, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NK cell cytotoxicity is a fast and efficient mechanism of target cell lysis. Using transcription analysis, such as multiplex messenger assays, we show here that natural cytotoxicity exerted by the human NKL cell line correlates with mRNA accumulation of very early activator protein (AP)-1 transcription factor genes such as JunB, FosB and c-Fos. In addition, DNA-binding activities of Jun-Fos heterodimers were observed by electrophoretic mobility shift assays during the course of natural cytotoxicity. Interaction between immunoglobulin-like transcript-2/leukocyte Ig-like receptor 1 on NKL cells and HLA-B27 on target cells leads to an impairment of NKL natural cytotoxicity, which correlates with an absence of JunB, FosB, and c-Fos transcription, as well as an absence of their DNA-binding activity. Our studies thus indicate that, despite the rapidity of NK cell-mediated lysis, AP-1 transcription factor is activated during the early stage of NK cell cytolytic programs and that engagement of NK cell inhibitory receptors for MHC class I molecules impairs the very early activation of AP-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell cytotoxicity is a strategy used by the immune system to eliminate virus-infected cells and tumor-transformed cells (1). NK cells are cytotoxic lymphocytes that represent 5–14% of PBL and also constitute a subset of tissue-infiltrating lymphocytes. NK cells exert a cytolytic activity toward Ab-coated target cells (ADCC), as well as toward a spectrum of target cells that includes tumor cells, virus-infected cells, and allogeneic cells. NK cells also produce a variety of regulatory cytokines including IFN-{gamma}, TGF-ß1, TNF-{alpha}, IL-1ß, IL-10, granulocyte-CSF, granulocyte/macrophage-CSF, and CC-chemokines, such as RANTES, macrophage inflammatory protein (MIP)-1{alpha}, and MIP-1ß (2, 3, 4, 5, 6). Lysis of target cells and cytokine production by NK cells result from a balance between the action of inhibitory and activating receptors expressed at their surface.

Interaction between MHC class I molecules on target cells and MHC class I receptors expressed on NK cells can block NK cell cytolytic activity. The NK inhibitory receptors for MHC class I molecules are well characterized. They belong to the Ig superfamily (KIRs killer cell Ig-like receptors) and to the lectin-like superfamily (CD94/NKG2 heterodimers) in humans (7, 8, 9). Despite their structural heterogeneity, these receptors use a common inhibitory mechanism (10, 11, 12). Both the Ig-like and the lectin-like inhibitory receptors contain one or two immunoreceptor tyrosine-based inhibitory motifs (ITIMs)3 in their intracytoplasmic portion (11). Upon receptor engagement, the tyrosine residue in ITIMs is phosphorylated and recruits the tandem src homology domain 2 (SH2)-containing protein tyrosine phosphatases SHP1 and/or SHP2, which are responsible for the inhibition of NK cell activity (13, 14, 15, 16, 17).

Knowledge of NK cell surface receptors involved in the activation of natural cytotoxicity is emerging. It is likely that an array of triggering receptors, including p46, p44, DNAM-1, B7 ligands as well as CD2, ß2-integrins, NKRP-1, and CD44 are involved in natural cytotoxicity, depending upon NK cell activation state and upon the availability of the relevant ligand on target cells (18, 19, 20, 21, 22). Moreover, subsets of NK cells express at their surface activating isoforms of killer cell Ig-like receptor and CD94/NKG2 heterodimers (7, 23). These activating molecules lack ITIMs in their intracellular portion and present a charged amino acid in their transmembrane portion (11, 24). These receptors are noncovalently associated with an ITAM-bearing polypeptide, KARAP/DAP-12, expressed as homodimers and responsible for transducing activating signals upon receptor triggering (25, 26, 27, 28, 29). Consistent with the association of CD3{zeta}, Fc{epsilon}RI{gamma}, or KARAP/DAP-12 with a variety of activating receptors, including p46 and p44 (18, 19), it has been shown that the early phase of natural cytotoxicity requires protein tyrosine kinase (PTK) activation (30). However, the precise cascade of biochemical events involved in natural cytotoxicity is not yet elucidated.

To dissect the mechanisms involved in natural cytotoxicity, we compared the transcription patterns of the human NK cell line NKL when interacting with the sensitive target cell line C1R or with resistant C1R-HLA-B27 (C1R-B27) transfectants. In this complex biological system, the large number of genes that are involved, together with the fact that different genes become critical at different times, requires experimental techniques able to examine the expression levels of many genes simultaneously. Array hybridization methods provide this possibility; we used the flexible "multiplex messenger assay" (MMA), in which genes are represented by IMAGE cDNA inserts arrayed on nylon membranes and hybridized with complex radioactive probes prepared from relatively small amounts of total RNA (31, 32, 33). We observed that members of the AP-1 transcription factor complex are produced during early stages of natural cytotoxicity and that engagement of MHC class I inhibitory receptors impairs the transcription of AP-1 genes and DNA binding of Jun-Fos heterodimers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, chemicals, and mAbs

The following cell lines have been described previously: the IL-2-dependent human NK cell line NKL (34), the human B-LCL cell line C1R, and its stable HLA-B27 transfectant (C1R-B27) (35). The mitogen-activated protein kinase (MAPK) inhibitor PD098059 was purchased from New England Biolabs (Beverly, MA). The transcription inhibitor actinomycin D (ActD) was purchased from Sigma (St. Louis, MO).

Cytotoxicity assays

The cytolytic activity of NKL cells was assessed against C1R and C1R-B27 cell lines. Briefly, 5 x 10351Cr-labeled target cells were added to serial dilutions of NKL cells at the initiation of a 51Cr-release assay. After the indicated period of time, 100 µl of supernatant was collected, and radioactivity was measured in a gamma counter for the determination of 51Cr release and percentage of specific lysis.

For the experiments with ActD and PD098059, NKL were preincubated for 3 h (ActD) and 1 h (PD098059) at 37°C. Cell viability was assessed by trypan blue exclusion.

Selection of cDNA clones corresponding to known genes

The complete set of genes and the corresponding IMAGE cDNA clones used in these experiments are displayed at our Web site (http://tagc.univ-mrs.fr/). cDNA clones corresponding to the 3' portion of their mRNA were identified using the EST database (dbEST release 091495) from the National Center for Biotechnology Information (Bethesda, MD) via the World Wide Web (http://www.ncbi.nlm.nih.gov) and obtained through the Integrated Molecular Analysis of Genomes and Their Expression (IMAGE) Consortium. The clones to be used were then selected from libraries constructed by B. Soares according to the following criteria: pT7T3D (with a modified polylinker) cloning vector, Escherichia coli DH10B host bacteria, an insert size of approximately 1 kb, and no detectable repeat sequences. All clones were provided by the Human Genome Mapping Project Resource Centre (Hinxton, U.K.). Their identities were checked by vector-PCR amplification of the inserts with T7 and T3 primers and comparison with the size recorded in dbEST; only clones displaying a single PCR product of the expected size were used. Some clones were further checked by 5' tag sequencing. Four negative controls were included: three poly(A) sequences (50, 60, and 90 bp long) and the cloning vector pT7T3D without insert. A plant gene, the Arabidopsis thaliana cytochrome c554 gene, was used to normalize different hybridizations. Membranes on which vector-PCR amplification products had been spotted were used. For this set of genes, the cDNA to be arrayed were first amplified in three 96-well microtiter plates using a vector-PCR amplification with T7 and T3 primers (T7 primer, 5'-ATTATGCTGAGTGATATCCCT-3'; T3 primer, 5'-TCCCTTTAGTGAGGGTTAAT-3'), the 4th plate containing, in all wells, the PCR-products of the A. thaliana cytochrome c554 gene.

Design of the experiment for quantitative differential screening and preparation of MMA membranes

Membranes were spotted using a commercial robotic device (PBA) and a 96-pin tool depositing approximately 50 nl of solution per spot (0.6 pmol/µl). Before gridding, Hybond-N+ 8 x 12-cm membranes (Amersham Pharmacia Biotech, Uppsala, Sweden) were positioned on Whatman 3 MM paper saturated with denaturing solution (1.5 M NaCl, 0.5 M NaOH). Following gridding, membranes were transferred to a sheet of dry Whatman 3 MM paper and air dried for 2 h. Membranes were then processed though the following stages: 5 min on a sheet of 1.5 M NaCl, 0.5 M NaOH-saturated Whatman 3 MM paper; and 1 min on each of 3 sheets of 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2), 1 mM EDTA-saturated Whatman 3 MM paper. Membranes were then transferred to a sheet of Whatman 3 MM paper and air dried for 2 h. Finally, a UV treatment (254 mµ, 70 mJ/cm2) was performed to cross-link DNA to the membrane.

Oligonucleotide probe labeling and vector hybridization conditions

In spite of adjustment of DNA concentrations, the exact amount of DNA on the membrane does vary somewhat from spot to spot and from membrane to membrane and must be measured to obtain meaningful results. An oligonucleotide corresponding to a sequence present in all amplified segments was used for this normalization, with the following sequence: 5'-GGGAATTTGGCCCTCGAGGCCAA-3'. One µg of oligonucleotide was labeled with ({gamma}-33P)ATP using standard methods (36). Membranes were prehybridized for 3 h at 42°C in 6x standard sodium citrate (SSC), 5x Denhardt’s reagent, 1% SDS, and 100 µg/ml sonicated salmon sperm DNA and then hybridized with labeled oligonucleotide (at 105 cpm/ml) and unlabeled oligonucleotide (at 100 ng/ml) overnight. Filters were washed twice in 2x SSC, 0.1% SDS for 15 min at room temperature and then exposed with phosphor screens.

Preparation and labeling of complex probes from total RNA and hybridization conditions

Total RNA was isolated from cell lines using the "Trizol" reagent (Life Technologies Cergy-Pontoise, France). Complex probes were prepared from total RNA (5 µg) as previously described with an excess of oligo(dT) (25) to saturate the poly(A) tails and ensure that the reverse transcribed product does not contain long poly(T) sequences (31). A constant amount of RNA (1 ng) transcribed in vitro from cytochrome c554 DNA was added to the total RNA before 33P complex probe preparation to standardize hybridization intensities between different experiments. Filters were prehybridized and hybridized for 20 and 40 h respectively in 10 ml of hybridization mix. After hybridization, filters were washed in 0.1x SSC, and 0.1% SDS twice for 2 h at 68°C and then exposed to phosphor screens for 5 days. Before complex probe hybridization, membranes were hybridized with oligonucleotide probe, quantified, then gently stripped as previously described (31). Membranes were normally used for two such cycles.

Detection and quantification of hybridization signals

Detection and quantification of hybridization signals and normalization of hybridization intensities were done as described previously (37) except for the "vertical normalization" that was performed to allow comparison of different complex probe hybridizations, dividing for each clone the "horizontally normalized intensity" by the mean of the 192 intensities of the control clone c554.

Northern blot analysis and quantification

Total cellular RNA (10 µg) extracted from cells grown in culture were size fractionated in 1.2% agarose gels containing 3.7% formaldehyde and blotted to positively charged nylon membranes (Sure Blot hybridization membranes from Oncor). Prehybridization and hybridization were performed according to the manufacturer’s instructions, with cDNA probes specific for human junB, c-jun, and GAPDH. cDNA were labeled by random priming (38) and added to the hybridization mix at 106 cpm/ml. Northern blots were exposed to an imaging plate.

RT-PCR analysis

Total RNA was isolated from cell lines using the "Trizol" reagent (Life Technologies). Five micrograms of total RNA were reverse transcribed using oligo(dT) primers and Superscript reverse transcriptase according to the manufacturer’s instructions. Three percent of cDNA was then subjected to 25–30 cycles of PCR in a Perkin-Elmer thermocyler 9600 using human specific primers pairs (Table IGo). Three microliters of diluted first-strand cDNA were mixed with 2 µl 10x PCR buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 1 µl 50 mM MgCl2, 1 µl each of 50 pM sense and antisense primers, 2 µl 10 mM dNTP, and 1 unit Taq DNA polymerase (Life Technologies) in a final 20-µl reaction volume. Amplification conditions were 94°C for 5 min plus 25–30 cycles with 94°C for 40 s, annealing temperature (Table IGo) for 40 s, 72°C for 40 s, and a final extension of 72°C for 10 min. Fifty percent of each PCR reaction was electrophoresed on 2% agarose gels.


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  		Table I. Primers used for the analysis of c-Fos, FosB, Fra-1, Fra-2, JunB, and ß-actin transcripts1

 
Preparation of nuclear extracts and EMSA

Nuclear extracts from NKL incubated with C1R or C1R-B27 for 0 h, 1 h, 2 h, or 4 h were prepared as described (39). Protein concentrations in the extracts were determined by the Bradford assay (Bio-Rad protein assay; Bio-Rad, Richmond, CA). Three micrograms of nuclear extract were used for EMSA of AP-1 oligonucleotides. The consensus AP-1 oligonucleotide 5'-CGCTTGATGACTCAGCCGGAA-3' was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Binding assays were conducted by incubating the labeled DNA (50,000 cpm.) with 3 µg of nuclear proteins and 1 µg of poly(dI-C) in a buffer containing 10 mM HEPES, 50 mM KCL, 0.1 mM EDTA, 5 mM MgCl2, 10% glycerol, 1 mM DTT, and 0.1% Nonidet P-40. After 30 min on ice, the reaction mixtures were loaded onto a 4% polyacrylamide gel in 0.25x TBE buffer and electrophoresed. Gels were dried and exposed to a Fuji RX film overnight. For competition, Abs or a 50-fold molar excess of unlabeled oligonucleotide was added to the reaction mixture 30 min before the addition of labeled DNA. Anti-Fos (reactive with c-Fos, FosB, Fra-1, and Fra-2), anti-Jun (reactive with c-Jun, JunB, and JunD), anti-c-Jun, anti-JunB, and anti-JunD were purchased from Santa Cruz Biotechnology.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MMA reveals an increase of JunB and CD69 mRNA during the initial phase of natural cytotoxicity

NKL cells are able to efficiently lyse the B cell line C1R, whereas transfection of C1R cells with HLA-B27 allele confers C1R-B27 cells protection from NKL-mediated lysis (40, 41). The inhibition of NKL cytolytic activity against C1R-B27 target cells is due to the expression of the ITIM-bearing molecule ILT-2 at the surface of NKL cells (40). NKL cytotoxicity is a rapid phenomenon, since target cell lysis is already detectable after 1 h of contact between effector and target cells and increases with time (data not shown). We used this in vitro model to determine whether NK-mediated cytolytic activity is correlated with gene transcription.

To identify the genes whose transcription is involved in natural cytotoxicity, an MMA profiling experiment was performed on a set of 120 human known genes. Complex probes were prepared from total RNA of NKL cells incubated with C1R for 0 h and 2 h, and NKL cells incubated with C1R-B27 for 2 h (Fig. 1GoB). Control vector hybridizations were performed as described in Materials and Methods (Fig. 1GoA). Reproducible changes in expression levels were observed for two genes, JunB and CD69, that both showed a 100–200% increase when NKL were in the presence of C1R for 2 h (Fig. 1GoC). In both cases, this increase did not occur when NKL were confronted with the resistant C1R-B27 target cells. Expression levels for other genes included in the set, e.g., other costimulatory molecules CD2 and CD11a or other early genes such as myb and myc, showed either no alteration or only marginal, irreproducible changes and were not investigated further. Since CD69 is a well-known marker of NK cell activation, the accumulation of CD69 mRNA during the course of natural cytotoxicity was used as a positive control in our analysis (42). We focused our attention on the variations observed for the JunB transcription factor and on its potential involvement in NK cell cytotoxicity.



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  		FIGURE 1. Hybridization of MMA filters using NKL complex probes. MMA membranes hybridized with the vector oligonucleotide probe (A) and with a complex probe made from 5 µg total RNA of NKL cells incubated with C1R cells during 2 h (B). Each colony has been spotted in duplicate on a single nylon membrane from the four 96-well plates as indicated in the enlargement (A). With the complex probes, signals corresponding to JunB, CD69, and negative or positive controls (poly(A), vector, and cytochrome c554) are indicated. C, Quantification of the hybridization signal obtained on JunB, CD69, and the cytochrome c554 control. The intensities corresponding to the average values of duplicate spots are corrected by vector hybridization and cytochrome c554 intensities and are expressed in percentage of abundance relative to cytochrome c554 added in the complex probe at 1% concentration with respect to poly(A)+ RNA (assuming that poly(A)+ RNA represents 2% of total RNA).

 
Transcripts for JunB, c-Jun, c-Fos, and FosB are up-regulated during natural cytotoxicity

The JunB protein associates with various Fos family members to form functional AP-1 transcription factors (43). mRNA rates of members of Jun and Fos families were thus assessed by Northern blotting for c-Jun and JunB genes, and by RT-PCR for c-Fos, FosB, Fra1, and Fra2 genes. The differential expression of JunB previously detected by MMA was confirmed (Fig. 2GoA). We also detected a small increase of c-Jun mRNA levels during activation of natural cytotoxicity (Fig. 2GoA), whereas the rate of JunD mRNA is unchanged in all the conditions tested (data not shown). Among members of Fos family, only c-Fos and FosB mRNA accumulated during lysis of target cells (Fig. 2GoB). In all cases, mRNA rates found for NKL incubated with C1R-B27 target cells remained at the level found with NKL alone. These results indicate that the increases observed when NKL cells interact with C1R targets are specifically correlated with natural cytotoxicity programs and are abrogated by the engagement of inhibitory MHC class I receptors. To insure that the modulation of AP-1 factors did occur in the effector cells, NKL cells were pretreated for 3 h with ActD, an inhibitor of transcription. Results in Fig. 2GoC show that ActD pretreatment severely impairs the accumulation of JunB, c-Fos, and FosB transcripts detected when NKL cells encounter the sensitive C1R cell line. In addition, actinomycin pretreatment of NKL cells inhibits their natural cytotoxicity toward C1R (Fig. 2GoD). By contrast, actinomycin pretreatment of C1R cells does not alter their sensitivity toward NKL-induced natural cytotoxicity (Fig. 2GoD). Together these results indicate that the regulation of AP-1 transcription factors occurs in NKL cells.



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  		FIGURE 2. Transcription analysis of Jun and Fos family members. A, Analysis of JunB and c-Jun mRNA expression by Northern blotting. The gel was loaded with 10 µg of total RNA from NKL incubated with C1R (a lanes) or C1R-B27 (b lanes) after 0 h (0), 1 h (1a and 1b), 2 h (2a and 2b) or 4 h (4a and 4b). The blot was successively hybridized with the JunB, c-Jun, and GAPDH (used as a control) probes. B, RT-PCR analysis of Fos family members mRNA levels. NKL cells were incubated with C1R (a lanes) or C1R-B27 (b lanes) after 0 h (0), 1 h (1a and 1b), 2 h (2a and 2b), or 4 h (4a and 4b). cDNA was prepared from total RNA and subjected to 30 cycles of PCR amplification with sense and antisense primers designed for Fos family members c-Fos, FosB, Fra-1, and Fra-2 at the annealing temperature indicated in Table IGo. ß-actin was used as a positive control of RT-PCR. C, NKL cells were pretreated for 3 h at 37°C in the presence or absence of ActD (20 µg/ml), washed, and then incubated for 1 h with C1R target cells. RT-PCR analyses of JunB, c-Fos, and FosB mRNA levels were performed as described in Fig. 2GoB. ß-actin was used as a positive control of RT-PCR. D, Cytolytic activity of NKL effector cells against the sensitive target cell line C1R was performed over a classical 4-h 51Cr-release assay (circle). NKL cells or C1R cells were pretreated for 3 h at 37°C in the presence or absence of ActD (20 µg/ml), washed, and then incubated for 4 h with C1R (square) or NKL (triangle) cells, respectively.

 
Accumulation of specific AP-1 complexes correlates with the course of natural cytotoxicity

Further analysis of the involvement of AP-1 transcription factors in natural cytotoxicity was investigated by measuring the DNA binding activity of AP-1 polypeptides present in protein extracts prepared from NKL cells alone as compared with protein extracts prepared from NKL cells incubated with C1R or C1R-B27 targets. EMSA revealed that protein extracts from NKL cells alone or from NKL cells incubated with the resistant C1R-B27 targets bound the AP-1 oligonucleotide and displayed by EMSA a retarded band representative of the basal AP-1 level in unstimulated NKL cells (Fig. 3GoA, lanes 0a, 1b, 2b, and 4b). In contrast, protein extracts prepared from NKL cells incubated with C1R targets bind the AP-1 oligonucleotide with two different electrophoretic mobility bands (Fig. 3GoA, lanes 1a, 2a, and 4a). The appearance of the upper band correlates with the course of natural cytotoxicity, since this low-mobility band appears at 1 h of incubation with target cells and increases until 4 h.



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  		FIGURE 3. Gel shift analysis of AP-1 factors in NKL cells incubated with C1R or C1R-B27 targets. Nuclear extracts were obtained from NKL incubated with C1R (a lanes) or C1R-B27 (b lanes) after 0 h (0), 1 h (1a, 1b), 2 h (2a, 2b), or 4 h (4a, 4b). The presence of AP-1 complexes in these nuclear extracts was analyzed by EMSA. For competition analysis, the binding reactions were performed in the presence of AP-1 oligonucleotide alone (A) and in the presence of specific antisera against Fos family members (anti-Fos) (B), Jun family members (anti-Jun) (C), JunB (D), c-Jun (E), and JunD (F). A bracket indicates the "upper" and the "lower" complex of AP-1. Arrow indicates the supershift Fos band.

 
To identify the components of these two AP-1 complexes, we performed competition experiments with antisera against the Fos and Jun protein families. The AP-1 upper band supershifted after treatment with antiserum directed against multiple members of the Fos families (Fig. 3GoB) and disappeared or was strongly reduced after treatment with antiserum directed against multiple members of Jun families (Fig. 3GoC). These results demonstrate that the upper AP-1 band mostly contains Jun:Fos heterodimers. In contrast, the intensity of the lower AP-1 band was reduced by anti-Jun Abs and unaffected by anti-Fos Abs, indicating that the lower AP-1 complex primarily consists of Jun-Jun homodimers. To assay for the relative contribution of JunB, c-Jun, and JunD to AP-1 complexes in unstimulated or activated NKL cells, the AP-1 electrophoretic mobility shift was assayed in the presence of specific anti-JunB, c-Jun, and JunD antisera. Anti-JunB antiserum altered the majority of the lower and upper complex (Fig. 3GoD). However, anti-c-Jun or anti-JunD Abs did not significantly reduce the number of these complexes (Fig. 3GoE and 3F). Taken together, these results indicate that the lower complex is mainly composed of JunB homodimers whereas the upper complex is mainly composed of JunB:Fos heterodimers.

Based on these results, we used a pharmacological approach to further analyze the involvement of AP-1 transcription factors in NKL natural cytotoxicity. NKL were pretreated with the MEK inhibitor PD098059, which interferes with the MAPK-dependent transcription of AP-1 complexes. As shown in Fig. 4Go, a decrease of NKL natural cytotoxicity was observed upon treatment with the MAPK inhibitor PD098059. Therefore, the pharmacological approach is in agreement with our MMA findings, as well as with previous pharmacological studies (44), and supports the involvement of the MEK-dependent AP-1 pathway in NKL cytotoxicity programs.



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  		FIGURE 4. Effect of PD098059 on NKL-mediated cytotoxicity. NKL cells were pretreated for 1 h at 37°C in the presence (squares) or absence (circles) of PD098059 (100 µM) and then incubated for 4 h with 51Cr-labeled C1R (filled symbols) and C1R-B27 (open symbols) target cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although a variety of cell surface receptors have been shown to be involved in natural cytotoxicity, the pathways that initiate and propagate NK cell activation programs remain to be unveiled. We report here that the natural cytotoxicity programs of NKL cells are correlated with the activation of AP-1 transcription factors, consistent with the increase in AP-1 DNA-binding activity observed in human NK clones during natural cytotoxicity (45).

AP-1 transcription factors consist of a complex combination of various members of the Fos (c-Fos, FosB, Fra1, Fra2) and Jun (c-Jun, JunB, and JunD) families of proteins (43). Each of these proteins contains a leucine zipper domain that allows the association with other members of the Fos-Jun family. Although homodimers of Jun, but not of Fos, bind to TPA-responsive elements (TRE), the DNA-binding affinity and the transcriptional activity is notably higher for Jun:Fos heterodimers. AP-1 transcriptional activity is therefore regulated by 1) the regulation of Fos and Jun gene expression, 2) the combination of proteins that form AP-1 homo- or heterodimers, and 3) the posttranslational modification of both preexisting and newly synthesized Jun and Fos protein, which occurs mainly by phosphorylation. Our results show that, in the absence of target cells or in the presence of HLA Class I+ target cells resistant to natural cytotoxicity, NKL cells express Jun:Jun homodimers. By contrast, in the presence of target cells sensitive to natural cytotoxicity, Jun:Fos heterodimers are detected in addition to Jun:Jun homodimers. Therefore, engagement of natural cytotoxicity programs correlates with the formation of Jun:Fos heterodimers.

The transcriptional regulation of Fos and Jun genes depends upon the activation of MAPK-dependent pathways (46). Two categories of MAPK, the extracellular regulatory kinases (ERK) and the c-Jun N-terminal kinase (JNK), are involved in c-Fos transcription (47), whereas c-Jun transcription depends only upon JNK (48). PD098059 was initially identified to inhibit the activity of MEK1, which acts upstream of ERKs, but not the activity of SEK, a MAPK kinase homologue that participates to JNK activation. In addition, PD098059 does not alter the function of Raf, which acts upstream of MEK1 (49, 50). Using PD098059, we show that activation of MAPK is necessary for NKL natural cytotoxicity toward C1R cells (Fig. 4Go). Our data are consistent with the inhibition induced by PD098059 on natural cytotoxicity exerted by another human IL-2 dependent NK cell line, YT, as well as by freshly isolated NK cells toward Raji or K562 cells (51). Similarly, CD16-induction of c-Fos mRNA in NK cells (44), as well as rADCC, are inhibited by PD098059 treatment (data not shown). These results underscore the role of MAPK as signal-transducing molecules that govern NK cell activation programs. The correlation between the inhibitory effect of PD098059, as well as mRNA accumulation, and the DNA-binding activity of AP-1 factors strongly supports the involvement of AP-1 complexes in natural cytotoxicity programs. Further, the increase in JunB mRNA and the emergence of JunB:Fos heterodimers during natural cytotoxicity are consistent with the involvement of ERKs (52), but not JNK (53), in the regulation of JunB gene transcription. The correlation between the extinction of cytotoxicity and the absence of JunB, c-Fos, and FosB gene up-regulation observed when NKL encounter C1R-B27 cells is further exemplified by the absence of JunB, c-Fos, and FosB expression in anergic CD4+ T cells (54). Taken together, our results suggest that AP-1 transcription factors play a role in NK cell cytotoxicity in addition to their broad involvement in the regulation of lymphocytes, monocytes, and mast cell activation programs (55).


   Acknowledgments
 
We thank Brigitte Granel and Pascale André for their collaboration in the initial phases of this work; Nathalie Auphan, Katia Simon, and Sylvie Guerder for their expertise in AP-1; Michael Starkey (HGMP Resource Center) for provision of some membranes used in this work; and Corinne Béziers La Fosse (CIML) for graphics.


   Footnotes
 
1 This work was supported by institutional grants from INSERM/CNRS, Ministère de l’Enseignement Supérieur et de la Recherche, and specific grants from Association pour la Recherche contre le Cancer (E.V. and B.R.J.), Ligue Nationale contre le Cancer (E.V. and B.R.J.), Association Française contre les Myopathies (B.R.J.), and from the Training and Mobility of Researcher Programme (A.C). Back

2 Address correspondence and reprint requests to Dr. Eric Vivier, Centre d’Immunologie INSERM/CNRS de Marseille-Luminy, Case 906, 13288 Marseille cedex 09, France. E-mail address: Back

3 Abbreviations used in this paper: ITIM, immunoreceptor tyrosine-based inhibition motif; EMSA, electrophoretic mobility shift assay; MMA, multiplex messenger assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ActD, actinomycin D; AP-1, activator protein-1; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAP kinase kinase; ERK, extracellular regulatory kinase; JNK, c-Jun N-terminal kinase. Back

Received for publication August 19, 1998. Accepted for publication January 4, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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