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The Journal of Immunology, 2008, 180, 2922 -2932
Copyright © 2008 by The American Association of Immunologists, Inc.
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KIR2DL4 Differentially Signals Downstream Functions in Human NK Cells through Distinct Structural Modules1

S. M. Shahjahan Miah, Tracey L. Hughes and Kerry S. Campbell2

Fox Chase Cancer Center, Division of Basic Science, Institute for Cancer Research, Philadelphia, PA 19111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
KIR2DL4 (2DL4) is a member of the killer cell Ig-like receptor (KIR) family in human NK cells. It can stimulate potent cytokine production and weak cytolytic activity in resting NK cells, but the mechanism for 2DL4-mediated signaling remains unclear. In this study we characterized the signaling pathways stimulated by 2DL4 engagement. In a human NK-like cell line, KHYG-1, cross-linking of 2DL4 activated MAPKs including JNK, ERK, and p38. Furthermore, 2DL4 cross-linking resulted in phosphorylation of I{kappa}B kinase β (IKKβ) and the phosphorylation and degradation of I{kappa}B{alpha}, which indicate activation of the classical NF-{kappa}B pathway. Engagement of 2DL4 was also shown to activate the transcription and translation of a variety of cytokine genes, including TNF-{alpha}, IFN-{gamma}, MIP1{alpha}, MIP1β, and IL-8. Pharmacological inhibitors of JNK, MEK1/2 and p38, blocked IFN-{gamma}, IL-8, and MIP1{alpha} production, suggesting that MAPKs are regulating 2DL4-mediated cytokine production in a nonredundant manner. Activation of both p38 and ERK appear to be upstream of the stimulation of NF-{kappa}B. Mutation of a transmembrane arginine in 2DL4 to glycine (R/G mutant) abrogated Fc{epsilon}RI-{gamma} association, as well as receptor-mediated cytolytic activity and calcium responses. Surprisingly, the R/G mutant still activated MAPKs and the NF-{kappa}B pathway and selectively stimulated the production of MIP1{alpha}, but not that of IFN-{gamma} or IL-8. In conclusion, we provide evidence that the activating functions of 2DL4 can be compartmentalized into two distinct structural modules: 1) through transmembrane association with Fc{epsilon}RI-{gamma}; and 2) through another receptor domain independent of the transmembrane arginine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human NK cells express killer cell Ig-like receptors (KIR),3 a family of NK cell receptors that recognize MHC class I molecules (1). KIRs are type I transmembrane glycoproteins with two (2D) or three (3D) Ig-like domains and cytoplasmic domains of varying lengths. KIRs with long (L) cytoplasmic domains (KIR2DL and KIR3DL) are mostly inhibitory receptors that contain two cytoplasmic ITIMs. Those KIRs with short (S) cytoplasmic domains (KIR2DS and KIR3DS) are activating receptors that lack ITIMs and associate with the adaptor DAP12 via a charged lysine residue in their transmembrane region.

KIR2DL4 (2DL4) is an evolutionarily conserved, framework member of the KIR gene family that is unique among KIRs in its genomic organization, protein structure, and function. Unlike other KIR family members, the 2DL4 gene is found in almost all KIR haplotypes (2, 3). 2DL4 is the only KIR that is transcribed in virtually all of the IL-2-cultured human NK cell clones that have been analyzed. Expression of the receptor on the surface of freshly isolated peripheral blood NK cells is restricted, however, to the minor CD56high subset and uterine NK cells, but IL-2 culture up-regulates surface expression on virtually all cells within 2 wk (4, 5, 6). Furthermore, common polymorphisms prevent surface expression altogether in a significant fraction of the human population (4, 5, 7). In contrast to other activating or inhibitory KIR family members that regulate NK cell cytotoxicity and cytokine production, 2DL4 activates potent cytokine production but weak cytotoxicity (4, 8). Surprisingly, 2DL4 exhibits structural characteristics of both activating and inhibitory KIR (6, 8, 9, 10, 11) by possessing both a charged transmembrane arginine residue and a single cytoplasmic ITIM. 2DL4 can physically interact with the transmembrane adaptor protein Fc{epsilon}RI-{gamma}, which is believed to be mediated through the transmembrane arginine residue to transduce activating signals (9). In contrast, the ITIM domain can recruit Src homology domain-containing tyrosine phosphatase 2 (SHP-2) and has the potential to mediate negative signals (11, 12). Little is known, however, regarding signal transduction pathways that are triggered to elicit functional responses by 2DL4.

The only known ligand of 2DL4 is soluble-HLA-G, which is produced by fetal-derived trophoblast cells (6, 13, 14). The signals that induce activation of uterine NK cells are still unknown, but 2DL4 interaction with HLA-G may play an important role in this process. During human pregnancy, 2DL4 is expressed on most decidual/placental, but not peripheral, NK cells (6). In both mice and humans, uterine NK cells have limited cytolytic potential but produce cytokines including IFN-{gamma} and TNF-{alpha} (15, 16). IFN-{gamma} produced by mouse uterine NK cells has been implicated in promoting the remodeling of the vasculature of the maternal arteries necessary for a normal pregnancy (17). Regulated surface expression, divergent structure, unique functional properties, and evolutionary conservation suggest distinct biological roles for 2DL4, possibly during pregnancy.

The aim of this study was to identify the signaling pathways that are specifically activated by the engagement of 2DL4. We have found that 2DL4 cross-linking activates calcium mobilization, several MAPKs, the NF-{kappa}B pathway, cytotoxicity, and production of IFN-{gamma}, IL-8, and MIP1{alpha}. Furthermore, the transmembrane arginine in 2DL4 mediates physical association with the transmembrane adaptor Fc{epsilon}RI-{gamma} to mediate the activating function of 2DL4. Mutation of the transmembrane arginine (R) to glycine (G), R/G, abrogates Fc{epsilon}RI-{gamma} association, receptor mediated IFN-{gamma} and IL-8 production, cytotoxicity, and calcium responses but, surprisingly, not MIP1{alpha} production. The R/G mutant also still activates MAPKs and the NF-{kappa}B members, indicating that the activating signals triggered by 2DL4 are not exclusively mediated through Fc{epsilon}RI-{gamma} association and suggesting that another structural domain of 2DL4 may provide an activating signal to the MAPK and NF-{kappa}B pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and culture

P815 (a mastocytoma cell line isolated from a DBA/2 mouse) and Phoenix-Ampho cells were cultured as described (12). The IL-2-dependent KHYG-1 cell line (JCRB0156; a human NK cell leukemia line obtained from Health Science Research Resources Bank, Japan Health Sciences Foundation, Osaka, Japan) (18) was cultured as previously described for NK-92 cells and passed every 4 days with fresh IL-2-containing medium (4, 12). Human NK cells were purified from a normal donor (homozygous for expression of full-length 2DL4) as described (4). Briefly, PBL were isolated from peripheral blood with Ficoll-Hypaque gradient centrifugation and NK cells were sorted on a FACSVantage SE flow cytometer (BD Biosciences). CD56+CD3– primary NK cells were cultured in RPMI 1640 with 5% autologous serum and 500 U/ml recombinant human IL-2 (generously provided by the Biological Resources Branch, National Cancer Institute, Frederick, MD). Volunteer blood donors were recruited by informed consent as approved by our Institutional Review Board.

Abs and reagents

Abs included anti-CD56-Cy7-PE and anti-CD3-FITC (BD Pharmingen), anti-FLAG (M2; Sigma-Aldrich), and rabbit anti-Fc{epsilon}RI-{gamma} sera (Upstate Biotechnology and Exalpha Biologicals). An anti-2DL4 mAb (53.1; mouse IgG1) and anti-NKp44 (3.43.13) were kindly provided by Dr. M. Colonna (Washington University, St. Louis, MO). Anti-phospho-p42/44 ERK (pThr202/pTyr204), anti-p42/44 ERK, anti-phospho-JNK (pThr183/pTyr185), anti-phospho-p38 MAPK (pThr180/pTyr182), anti-p38 MAPK, anti-phospho-IKK{alpha} (pSer180)/IKKβ (pSer181; where IKK is I{kappa}B kinase), anti-phospho p90 ribosomal S6 kinase (p90RSK), anti-phospho MEK1/2, anti-phospho I{kappa}B{alpha}, and anti-I{kappa}B{alpha} Abs were purchased from Cell Signaling Technology. Anti-JNK was from Upstate Biotechnology. HRP-conjugated affinity pure goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. Mouse mAb to GAPDH was from Chemicon International, Inc. Anti-tubulin Ab was purchased from Santa Cruz Biotechnology. Lipofectamine reagent, Lipofectamine Plus reagent, and Lipofectamine 2000 were purchased from Invitrogen. PMA and ionomycin were from FisherBiotech.

Retroviral cDNA constructs

Retroviral expression constructs for FLAG-tagged wild-type (WT) 2DL4 (FLAG-WT-2DL4) and Fc{epsilon}RI-{gamma} in the bicistronic pBMN-IRES-EGFP vector (kindly provided by G. Nolan, Stanford University, Stanford, CA) were previously described (12). The generation of stably transduced KHYG-1 and Ramos B cell lines was performed as previously described (4, 12, 19). Point mutation of 2DL4 at the transmembrane R to G was done with the QuikChange site-directed mutagenesis kit (Stratagene). Transduced cells were sorted in a FACSVantage SE (BD Biosciences) flow cytometer for expression of EGFP or surface FLAG-2DL4.

Immunoprecipitation and immunoblotting

KHYG-1 cells were cultured with fresh IL-2 every 4 days and used for experiments on day 2 of culture. Cells were stimulated with soluble anti-KIR2DL4 (53.1) or anti-FLAG (M2) for different time periods and cells were rapidly washed twice with ice-cold PBS and solubilized in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 10 mM EDTA, 1 mM NaF, 1 mM Na3VO4, and protease inhibitors (1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride and 2 µg each (per milliliter) of aprotinin, soybean trypsin inhibitor, and leupeptin)). Triton X-100 (1%) was used to prepare NK cell lysates and 1% digitonin for lysing Ramos cells was used for immunoprecipitation. The cell lysates were precleared by centrifugation and aliquots from the first centrifugation are referred to as total cell extracts. Supernatants were immunoprecipitated by incubation with 3 µg of each primary Ab prebound to protein G-agarose (Upstate Biotechnology). After incubation for 1 h at 4°C, the immunoprecipitates were washed five times with 0.1% digitonin lysis buffer. Immunoprecipitated proteins were eluted at 100°C for 5 min in 2x sample buffer, separated by SDS-PAGE, and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% milk in TBST (25 mM Tris (pH 8.0), 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. The blots were incubated with the indicated primary Abs for 1 h at 4°C. The membranes were washed four times with TBST and incubated with HRP-conjugated secondary Abs for 30 min at room temperature. After extensive washing with TBST, proteins were visualized by ECL (Millipore). For preparation of total cell extracts, cells were washed twice with PBS, lysed with 1% Triton X-100 lysis buffer, and directly analyzed by SDS-PAGE after the addition of sample buffer.

Redirected cytotoxicity assay

To specifically engage FLAG-tagged 2DL4, KHYG-1 cells were tested for redirected cytotoxicity against the Fc{gamma}RII/III+ P815 murine mastocytoma cell line in a 3–4 h 51Cr release assay in 200 µl of medium per well. The P815 target cells (2 x 106 cells) were prelabeled with 100 µCi 51Cr (5 mCi/ml; stock product 2030B, PerkinElmer Life Sciences) for 60–90 min and incubated with effector cells in V-bottom 96-well plates (12) (Costar). Spontaneous release and maximal release of 51Cr were determined by incubating in medium alone or 1% Triton X-100, respectively. Each assay condition was performed in triplicate. The percentage of specific lysis was determined as follows: [(mean cpm experimental release – mean cpm spontaneous release)/(mean cpm maximal release – mean cpm spontaneous release)] x 100. To engage specific receptors in the redirected assay, mAb (1 µg/ml) were mixed with P815 cells for 5 min before effector cell addition.

Intracellular Ca2+ measurement

The Ramos cell line transduced with Fc{epsilon}RI-{gamma} in combination with either FLAG-WT-2DL4 or FLAG-R/G-2DL4 (2 x 106/ml) were loaded for 30 min at 37°C with 3 µM Indo-1 acetoxymethyl ester (Sigma-Aldrich) and analyzed on a FACSVantage SE flow cytometer (BD Biosciences) as described (9). After baseline was acquired, 3 µg of anti-FLAG mAb and 3 µg of goat anti-mouse IgG F(ab')2 (Southern Biotechnology Associates) were sequentially added to 0.5 ml of cell suspension (1 x 106/ml). Positive control Ab was goat anti-human IgM (Southern Biotech). The mean 405/485 nm emission ratio was assessed over time using the FlowJo computer program (Tree Star).

RNase protection assay

Total cellular RNA was purified with the RNeasy mini kit (Qiagen). The production of cytokine mRNA was quantitatively measured by using the Multi-Probe RNase protection assay kit (BD Biosciences) (20). Briefly, with the RiboQuant in vitro transcription kit (BD Biosciences) biotin-labeled RNA probes were synthesized by using a Multi-Probe human cytokine template set (hCK-5; catalog no 556155, BD Biosciences). The synthesized probes were purified and then hybridized overnight at 56°C with 20 µg of total RNA purified from cells unstimulated or stimulated for 2 h by either 2DL4 cross-linking or PMA plus calcium ionophore stimulation. After the RNase treatment, the protected double-stranded RNAs were purified and separated on a urea gel and detected by ECL.

Cellular assays

For cytokine/chemokine assays, 2DL4-transduced KHYG-1 cells (2 x 105 cells/well) were incubated with plate-bound Abs in a 96-well plate. For 2DL4 stimulation, anti-FLAG Ab was preadsorbed at 3 µg/100 µl/well and anti-NKp44 Ab at 1 µg/100 µl/well. To analyze the effect of inhibitors, cells were preincubated with inhibitors for 30 min before stimulation with Abs and throughout the assay period. After 24 h of stimulation, supernatants were removed and tested for the presence of cytokines by ELISA. ELISA kits for IL-8 and MIP1{alpha} were purchased from Pierce Biotechnology and used according to the manufacturer’s instructions. Culture supernatants were also tested for the presence of IFN-{gamma} by ELISA according to the manufacturer’s instructions (BD Pharmingen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Mutation of the transmembrane arginine on 2DL4 abrogates Fc{epsilon}RI-{gamma} binding, cytotoxicity, and calcium responses

2DL4 is an unusual member of the KIR family because it contains both a transmembrane positive charge and a cytoplasmic ITIM, which are characteristic of activating and inhibitory receptors, respectively. The positively charged transmembrane arginine residue of 2DL4 is predicted to mediate the physical association with the transmembrane signaling adaptor protein Fc{epsilon}RI-{gamma}. The function of the cytoplasmic ITIM, which can recruit SHP-2, remains unknown.

To address the role of the transmembrane arginine in 2DL4 function, we mutated this residue to glycine (R/G mutation), which is found at the same position in all other KIR family members (Fig. 1A). Both WT and mutant constructs of 2DL4 were engineered with an amino-terminal FLAG epitope tag. Because the low level of Fc{epsilon}RI-{gamma} expression in NK cell lines limits the capacity to efficiently detect coimmunoprecipitation with 2DL4, we first retrovirally cotransduced the Ramos B cell line with Fc{epsilon}RI-{gamma} and either FLAG-WT-2DL4 or FLAG-R/G-2DL4 as previously described (12). Immunoprecipitation was performed with anti-FLAG mAb from cell lysates with digitonin buffer, and immunoblotting was conducted with anti-Fc{epsilon}RI-{gamma} Ab. The results indicate that loss of the positively charged arginine abrogates binding with Fc{epsilon}RI-{gamma} (Fig. 1B). We next tested whether the R/G mutation altered the receptor-mediated elevation in intracellular calcium concentration. Engagement of FLAG-WT-2DL4 receptor with anti-FLAG Ab induced a significant increase in intracellular calcium concentration. Calcium mobilization was completely absent, however, when FLAG-R/G-2DL4 was cross-linked with Ab. In contrast, stimulation with anti-IgM Ab induced a strong calcium mobilization response in both cell populations. These results indicate that the association of 2DL4 with Fc{epsilon}RI-{gamma} is mediated through the transmembrane arginine and that this residue is necessary for mediating the 2DL4-mediated calcium mobilization release (Fig. 1C).


Figure 1
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  		FIGURE 1. Transmembrane arginine in 2DL4 is essential for Fc{epsilon}RI-{gamma} association, calcium mobilization, and cytotoxicity responses. A, Construction of human (hu) WT-2DL4 and mutant (R/G; hu2DL4) cDNAs. The transmembrane (TM) arginine (R) was mutated to glycine (G) because this position is glycine in all other KIRs (examples of transmembrane sequences of inhibitory (hu3DL1) and activating (hu2DS4) KIRs are shown). Cyto, Cytoplasmic domain; D0 and D2, Ig-like domains. B, Association of Fc{epsilon}RI-{gamma} with 2DL4 in Ramos B cells. Ramos B cells were transduced with Fc{epsilon}RI-{gamma} in combination with either WT or R/G mutant of FLAG-2DL4. 2DL4 was immunoprecipitated (IP) with anti-FLAG Ab and immunoblotted with anti-Fc{epsilon}RI-{gamma} antiserum. C, Intracellular Ca2+ concentration was monitored in Indo-1-loaded Ramos B cells transduced with either WT or R/G mutant of 2DL4 in combination with Fc{epsilon}RI-{gamma}. Cells were stimulated with biotinylated anti-FLAG mAb, streptavidin cross-linker, and anti-human IgM Ab to stimulate the B cell Ag receptor as a positive control. Results are mean ratio of 405-/485-nm emission from a representative of three independent experiments. D, KHYG-1 cells (day 2 after IL-2) that had been transduced with either WT or R/G mutant of FLAG-2DL4 were stained for surface expression with anti-FLAG mAb (M2; dark lined histograms) plus PE-conjugated anti-{kappa} secondary by conventional FACS analysis. Gray line is secondary alone staining (upper panel). WT or R/G mutant FLAG-2DL4 transduced cells (day 2 after IL-2 culture) were assayed for Ab-redirected cytotoxicity against 51Cr-labeled Fc{gamma}R+ P815 target cells (lower panels) in the presence of anti-CD56 mAb (B159 used as a negative control; filled circles) or anti-FLAG mAb (M2; open circles) for 4 h. The results are mean of triplicate values from a representative of four experiments.

 
The human NK-like cell line KHYG-1 was retrovirally transduced with cDNA encoding either FLAG-WT-2DL4 or FLAG-R/G-2DL4. KHYG-1 can generate strong cytokine and cytolytic responses compared with other NK-like cell lines and, therefore, served as a good model cell line for our studies of 2DL4 function (18, 21). Both WT 2DL4 and R/G-2DL4 were expressed on the surface of KHYG-1 cells, but the R/G mutant was expressed at higher levels, presumably due to better membrane stability without the transmembrane charge (Fig. 1D, upper panels). In contrast to other NK cell activating receptors, 2DL4 induces only weak cytotoxicity (4, 8). As shown in Fig. 1D (lower panel), anti-FLAG Ab was used to engage FLAG-2DL4 receptors in a redirected cytotoxicity assay against P815 target cells. Our results indicate that, in comparison to WT-2DL4 receptor engagement, R/G-2DL4 completely lacked the capacity to stimulate receptor-mediated killing of target cells (Fig. 1D).

Cytokine and chemokine responses are differentially regulated by transmembrane R to G mutation of 2DL4

An earlier report showed that engagement of 2DL4 induces a unique profile of cytokine/chemokine gene transcription, including the up-regulation of an array of proinflammatory/proangiogenic cytokines, such as IL-6, IL-1β, TNF-{alpha}, and IL-23 (comprised of the IL-23{alpha} and IL-12β subunits), and chemokines, such as IL-8, MIP3{alpha}, MIP1{alpha}, MIP1{delta}, and MIP2β (22). Our unpublished gene array analysis comparing resting NK-92 cells with the same stimulation by cross-linking FLAG-WT-2DL4 with anti-FLAG mAb indicates a similar pattern of gene up-regulation (data not shown). We further tested the patterns of cytokine and chemokine gene transcription with an RNase protection assay in response to the cross-linking of 2DL4 in KHYG-1 cells. NK-92 and KHYG-1 cells lack the Fc receptor CD16, which eliminates concerns of Fc receptor-mediated signaling when engaging 2DL4 with anti-FLAG mAb. We found that 2DL4 engagement with anti-FLAG mAb up-regulates mRNA for MIP1{alpha}, MIP1β, IL-8, TGF-β3, and IFN-{gamma} (Fig. 2 and data not shown). Surprisingly, however, the R/G-2DL4 mutation differentially affected chemokine gene transcription, because IL-8 gene transcription was lost but transcription of MIP1{alpha} and MIP1β was still robustly stimulated upon cross-linking of the mutant receptor (Fig. 2, compare lanes 2 and 5). Alternatively, control stimulation by PMA and ionomycin resulted in production of all cytokines by both transduced cell lines (Fig. 2, lanes 3 and 6). The transcript expression level of the control housekeeping genes (L32 and GAPDH) were comparable among all cells. In summary, our data indicate that despite a lack of association with Fc{epsilon}RI-{gamma}, the R/G mutant of 2DL4 still induces MIP1{alpha} and MIP1β gene transcription, indicating Fc{epsilon}RI-{gamma}-independent regulation of these chemokine genes by 2DL4 receptor.


Figure 2
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  		FIGURE 2. Analysis of 2DL4-stimulated cytokine gene transcription by RNase protection assay. KHYG-1 cells transduced to express either FLAG-WT-2DL4 or FLAG-R/G-2DL4 were cultured with IL-2 and, at day 2, cells (10 x 106) were either unstimulated (–) or stimulated with (+) soluble anti-FLAG (M2; 10 µg/ml) or PMA (P; 10 nM) and ionomycin (I; 1 µM) for 2 h. Total RNA was extracted and hybridized with the biotin-labeled RNA probes of a human cytokines template set (Ltn, Lymphotactin). After RNase treatment, the protected double-stranded RNA was purified, separated by urea gel, and detected by chemiluminescence. The results are representative of two independent experiments.

 
We next tested whether activation of 2DL4 receptor by Ab cross-linking stimulates the secretion of these cytokines and chemokines into the culture supernatant. After stimulation of FLAG-2DL4-transduced KHYG-1 cells with plate-bound anti-FLAG Ab, we harvested culture supernatants at 24 h to detect the release of IFN-{gamma}, IL-8, and MIP1{alpha} by ELISA. As predicted, activation of WT-2DL4 receptor stimulated potent release of IFN-{gamma}, IL-8, and MIP1{alpha} (Fig. 3A). Mutation of transmembrane arginine-abrogated IFN-{gamma} secretion and suppressed IL-8 production by ~70%, indicating that the activating signals triggering their production are primarily driven by association with the transmembrane adaptor Fc{epsilon}RI-{gamma}. In contrast, MIP1{alpha} secretion was only modestly affected by the mutation (30%). As positive control, stimulation with anti-NKp44 strongly induced secretion of IFN-{gamma}, IL-8, and MIP1{alpha} in KHYG-1 cells expressing either form of 2DL4, indicating the lack of a general defect in the cells expressing the mutant receptor. Taken together, our data indicate that activation signals stimulated by 2DL4 are not completely dependent on physical association with Fc{epsilon}RI-{gamma} through the transmembrane arginine residue.


Figure 3
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  		FIGURE 3. Cytokine and chemokine secretion by KHYG-1 cells after cross-linking of WT or R/G mutant of 2DL4 and impacts of MAPK inhibitors. A, KHYG-1 cells transduced with either FLAG-WT or FLAG-R/G mutant form of 2DL4 were cultured either with or without (NS) plate-bound anti-FLAG mAb (M2) or anti-NKp44 (3.43.13). A, Culture supernatants were harvested after 24 h of stimulation and supernatants were analyzed by ELISA for secretion of IFN-{gamma} (upper panel), IL-8 (middle panel), and MIP1{alpha} (lower panel). The secretion of IFN-{gamma} is expressed as the percentage of total release upon engagement of WT-2DL4, because amounts of secretion varied between experiments (upper panel). The results are mean ± SD of triplicate determinations from three experiments for IFN-{gamma}, a single determination of nine experiments for IL-8, and a single determination of four experiments for MIP1{alpha}. B, Dose responses of MAPK inhibitors on 2DL4-mediated IL-8 production. Cells were pretreated with different doses of MAPK inhibitors of JNK (SP600125; 10, 30, and 50 µM), p38 (SB 202190; 1, 10, and 20 µM), or MEK (U0126; 1, 10, and 20 µM) before stimulation with anti-FLAG Ab (M2). IL-8 was detected in culture supernatant after 24 h of stimulation. The results are mean ± SD of two independent experiments with triplicate determinations. C, MAPK inhibitors block 2DL4-mediated cytokine production. KHYG-1 cells transduced with FLAG-WT-2DL4 were pretreated with MAPK inhibitors at different doses for 30 min before stimulation with anti-FLAG Ab (M2). Culture supernatants were harvested at 24 h after stimulation. Cytokine and chemokine secretion in culture supernatants were analyzed by ELISA. IFN-{gamma} (upper panel) and MIP1{alpha} secretion (lower panel) were assayed in supernatants from cells treated with inhibitors of JNK (SP600125), p38 (SB 202190), or MEK (U0126) at doses of 10 µM. The results are mean ± SD of two independent experiments with duplicate wells.

 
Role of MAPKs in stimulation of cytokine and chemokine production by 2DL4 engagement

The MAPKs represent a large family of protein kinases that regulate diverse intracellular signal transduction cascades controlling cellular proliferation, differentiation, and apoptosis. The three major families of mammalian MAPKs are ERK, JNK, and p38. Activated MAPKs translocate to the nucleus where they phosphorylate and activate AP-1 family transcription factors (i.e.; Fos, c-Jun). It has been previously reported that 2DL4-mediated IFN-{gamma} production can be blocked by an inhibitor of p38 MAPK (11). Therefore, we tested the effects of JNK, MEK1/2 (the kinase immediately upstream of ERK), and p38 MAPK inhibitors on 2DL4-mediated cytokine and chemokine production. We found that, in addition to p38 inhibitor, inhibitors of both JNK and MEK1/2 also blocked production of IFN-{gamma} (Fig. 3, B and C).

Surprisingly, we found that IL-8 production and MIP1{alpha} production were also suppressed by JNK, p38, and MEK1/2 inhibitors, indicating that all three classes of MAPKs are critical and nonredundant signaling elements for 2DL4-mediated cytokine and chemokine production. The effects of inhibitors were observed at doses that were commonly used by others (Fig. 3, B and C) (11, 23, 24, 25). We also tested the viability of cells after treatment with doses of the inhibitors used in our study and found no impact on the viability or apoptosis of KHYG-1 cells as assayed by annexin V and propidium iodide staining (data not shown).

Activation of MAPKs after stimulation of 2DL4 in the human NK cell line KHYG-1

Because MAPK inhibitors suppress 2DL4-mediated cytokine and chemokine production, we next tested whether 2DL4 can also activate these MAPK signaling cascades in KHYG-1 cells expressing WT or R/G mutant forms of 2DL4. Our results show that cross-linking of 2DL4 stimulates a rapid and transient phosphorylation of ERK1/2 within 2 min (Fig. 4A). ERK was similarly activated in primary NK cells from a donor that is homozygous for full-length 2DL4 receptor, which is expressed on the surface of IL-2-cultured NK cells (Fig. 5, A and B).


Figure 4
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  		FIGURE 4. Activation of the ERK MAPK cascade upon 2DL4 cross-linking. A, KHYG-1 cells (2 days after IL-2 culture) transduced with either WT-2DL4 (upper panel) or R/G-2DL4 (lower panel) were stimulated with anti-FLAG (M2) Ab (10 µg/ml) for the indicated times or PMA and ionomycin (P/I) for 10 min as a positive control. Cells were then lysed and aliquots of precleared cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-ERK (pERK) or total ERK. The results are representative of at least three independent experiments. B, Activation of both upstream and downstream kinases in the ERK signaling cascade after cross-linking of 2DL4. KHYG-1 cells (day 2 after IL-2 culture) transduced with either WT-2DL4 or R/G-2DL4 were stimulated with anti-FLAG (M2) Ab (10 µg/ml) for the indicated times or PMA and ionomycin (P/I) for 10 min as a positive control. Cells were then lysed and aliquots were separated by SDS-PAGE and immunoblotted with anti-phospho-MEK1/2 (upper panel), anti-phospho-p90RSK (middle panel), or anti-tubulin (lower panel). The results are representative of at least three independent experiments. C, Selective suppression of ERK by the MEK inhibitor. KHYG-1 cells transduced with WT-2DL4 were pretreated (bars above) or not with inhibitors of MEK (U0126), p38 (SB202190), or JNK (SP600125) for 30 min before stimulation with anti-FLAG Ab (10 µg/ml). Cells were processed as described above and lysates were immunoblotted with anti-phospho ERK. Reprobing with anti-GAPDH demonstrated equivalent loading of lanes (data not shown). Results are representative of three experiments.

 

Figure 5
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  		FIGURE 5. Activation of MAPKs after cross-linking of 2DL4 receptor in human primary NK cells. Primary NK cells (CD3–CD56+) were sorted from peripheral blood and cultured with IL-2 for 10 days to up-regulate 2DL4 surface expression. A, Cells (day 2 after IL-2) were stained for surface expression with anti-KIR2DL4 (53.1; gray lined histograms) plus PE-conjugated anti-{kappa} secondary by conventional FACS analysis. The faint dark line corresponds to secondary alone staining. B, Cells (day 2 after IL-2 culture) were then stimulated with anti-2DL4 (53.1) Ab at 10 µg/ml for the indicated times and cell lysates were prepared, separated on SDS-PAGE, and immunoblotted with anti-phospho-ERK (pERK), anti-phospho p38, anti-phospho-JNK (pJNK), or anti-GAPDH, as indicated.

 
ERK is regulated by the activation of MEK1/2, which is the upstream MAPK kinase, and a physiological substrate of ERK is p90RSK, a serine/threonine kinase family. Therefore, we tested the effect of 2DL4 stimulation on the activity of both of these kinase families. Our results demonstrated that the engagement of WT-2DL4 stimulated a very rapid phosphorylation of MEK1/2 (Fig. 4B) that terminates within 2 min, thereby preceding the kinetics of ERK activation. The rapid reversal of MEK phosphorylation suggests recruitment/activation of an inhibitory phosphatase to shut down activity. Our data further showed that p90RSK is also activated according to a similar time course as ERK upon cross-linking WT-2DL4. Taken together, we can conclude that engagement of 2DL4 leads to rapid and transient activation of the MEK-ERK-p90S6K kinase pathway.

Next, we tested the effect of the transmembrane R/G mutant of 2DL4 on the activation of the ERK cascade in KHYG-1 cells transduced with R/G-2DL4. Surprisingly, cross-linking of the mutant 2DL4 follows similar kinetics and intensity of stimulating phosphorylation of MEK1/2, ERK1/2, and p90RSK as those induced by WT-2DL4 receptor (Fig. 4, A and B). This indicates that the activation signal from 2DL4 to MAPK activation is independent of the transmembrane arginine, suggesting that the signal is generated through either the cytoplasmic domain or another domain of 2DL4.

We also tested the effect of 2DL4 cross-linking on the activation of the other major MAPKs, JNK and p38. We found that cross-linking of 2DL4 on KHYG-1 cells also activated the rapid and transient phosphorylation of p38 (Fig. 6A). Furthermore, Ab cross-linking stimulated phosphorylation of JNK in KHYG-1 cells, but the induction kinetics were delayed (first evident at 5 min) and sustained to at least 30 min (Fig. 6B). We also found that cross-linking of 2DL4 on primary NK cells activates p38 and JNK (Fig. 5) in a similar fashion. Similar to the ERK pathway, both p38 and JNK were also stimulated with similar kinetics by engagement of the R/G mutant of 2DL4 in KHYG-1 cells. We also observed nearly equivalent stimulation of ERK, p38, and JNK by Ab engagement of WT or R/G forms of 2DL4 when expressed in the transformed human kidney cell line 293T (data not shown), which further demonstrates that the signaling is Fc{epsilon}RI-{gamma}-independent in a cell lacking the adaptor. Taken together, our data indicate that 2DL4 activates all three MAPK pathways and that the activation is independent of the 2DL4 transmembrane arginine residue and the association of Fc{epsilon}RI-{gamma}.


Figure 6
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  		FIGURE 6. JNK and p38 are activated by 2DL4 cross-linking. A and B, WT-2DL4 and R/G-2DL4-transduced KHYG-1 cells were stimulated for the indicated times with anti-FLAG Ab or PMA and ionomycin (P/I, 10 min) and cell lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-p38 and anti-p38 (A) or anti-phospho-JNK and anti-JNK Abs (B). The results are representative of at least three independent experiments. C, Selective suppression of p38 by the p38 inhibitor. KHYG-1 cells transduced with WT-2DL4 were pretreated (bars above lower blots) or not with inhibitors of MEK (U0126), p38 (SB202190), or JNK (SP600125) for 30 min before stimulation with anti-FLAG Ab (10 µg/ml). Cells were processed as described above and lysates were immunoblotted with anti-phospho-p38. Reprobing with anti-GAPDH demonstrated equivalent loading of lanes. Results are representative of three experiments. D, Suppression of JNK by the MAPK inhibitors. KHYG-1 cells transduced with WT-2DL4 were pretreated (bars above lower blots) or not with inhibitors of MEK (U0126), p38 (SB202190), or JNK (SP600125) for 30 min before stimulation with anti-FLAG Ab (10 µg/ml). Cells were processed as described above and lysates were immunoblotted with anti-phospho-JNK. Reprobing with anti-GAPDH demonstrated equivalent loading of lanes (data not shown). Results are representative of two experiments.

 
We further tested the specificity of our MAPK inhibitors in KHYG-1 cells at the doses used in Fig. 3. We found that each MAPK pathway inhibitor blocked 2DL4-mediated activation of its target kinase at doses that inhibit cytokine production (Figs. 4C and 6, C and D). The p38 (SB202190) and JNK (SP600125) inhibitors did not cross-inhibit the 2DL4-mediated activation of ERK (Fig. 4C). Although the MEK inhibitor (U0126) had no effect on p38 phosphorylation, the JNK inhibitor reproducibly increased the activation of p38 (Fig. 6C). Interestingly, the p38 inhibitor had a significant inhibitory effect on the activation of JNK, while the MEK inhibitor had a slight but less consistent inhibitory impact on JNK (Fig. 6D). Importantly, we have observed this inhibition of 2DL4-stimulated JNK activation by two separate inhibitors of p38 (SB202190 and ML3403 (EMD Biosciences); Fig. 6D and data not shown). Although we are unaware of any other evidence that p38 is upstream of JNK, we conclude that the late phase JNK activation is dependent upon prior early p38 activation in the 2DL4 signaling pathway. The enhanced p38 activity in the presence of the JNK inhibitor further indicates cross talk between these MAPKs in the signaling response to 2DL4 engagement.

Activation of the NF{kappa}B pathway after cross-linking of 2DL4

NF-{kappa}B proteins comprise a family of transcription factors that are induced by stimuli such as proinflammatory cytokines, bacterial toxins (e.g., LPS and exotoxin B), viruses/viral products (e.g., HIV-1, human T cell leukemia virus I, hepatitis B virus, EBV, and herpes simplex) and proapoptotic and necrotic stimuli (oxygen free radicals, UV light, and gamma irradiation). NF-{kappa}B is involved in regulating many aspects of cellular activity, growth, stress, injury, and apoptosis and especially in pathways of the immune response (26). In addition, these transcription factors are persistently active in a number of disease states, including cancer, arthritis, chronic inflammation, asthma, neurodegenerative diseases, and heart disease (27, 28, 29).

The activity of NF-{kappa}B is primarily regulated by interaction with inhibitory I{kappa}B proteins in the cytosol. After receptor stimulation, I{kappa}B is rapidly phosphorylated, ubiquitylated, and degraded to release active NF-{kappa}B, which enters the nucleus and activates gene transcription. Therefore, a key step for controlling NF-{kappa}B activity is the regulation of the I{kappa}B-NF-{kappa}B interaction. A serine-specific IKK phosphorylates two specific serines near the N terminus of I{kappa}B{alpha}, which targets I{kappa}B{alpha} for ubiquitylation.

We cross-linked WT-2DL4 with anti-FLAG mAb for different lengths of time in KHYG-1 cells and found a transient increase in the phosphorylation of IKK{alpha}β, whereas the total levels of IKKβ were comparable in all lanes (Fig. 7A). We also found that the phosphorylation of I{kappa}B{alpha} was increased after stimulation of WT-2DL4 receptor (Fig. 7B, upper panel), which correlated with the degradation of total I{kappa}B{alpha} protein within the first 10 min (Fig. 7B, middle panel). We subsequently probed the membrane with anti-GAPDH Ab (Fig. 7B, bottom panel) and found that all lanes contain equal amounts of this loading control. Analogous to the impacts through WT-2DL4, cross-linking of the R/G mutant of 2DL4 also stimulated the phosphorylation of IKK{alpha}β and I{kappa}B{alpha} and induced degradation of I{kappa}B{alpha} protein (Fig. 7). Consistent with the MAPK results, our data indicate that the engagement of 2DL4 stimulates the NF-{kappa}B pathway in NK cells independently of the transmembrane arginine residue.


Figure 7
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  		FIGURE 7. 2DL4 cross-linking activates the NF-{kappa}B pathway. A and B, WT-2DL4- or R/G-2DL4-transduced KHYG-1 cells (day 2 after IL-2 culture) were stimulated with anti-FLAG (M2; 10 µg/ml) Ab or PMA and ionomycin (P/I, 10 min) for the indicated times. Cell lysates were prepared with 1% Triton lysis buffer and precleared cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-IKK{alpha}β (pIKK{alpha}β) and anti-IKKβ (A) or anti-phospho-I{kappa}B{alpha} (pI{kappa}B{alpha}), total I{kappa}B{alpha}, and GAPDH (B). The results are representative of at least three independent experiments. C, Suppression of IKK{alpha}β by the MEK and p38 inhibitors. KHYG-1 cells transduced with WT-2DL4 were pretreated (bars above) or not with inhibitors of MEK (U0126), p38 (SB202190), or JNK (SP600125) for 30 min before stimulation with anti-FLAG Ab (10 µg/ml). Cells were processed as described above and lysates were immunoblotted with anti-phospho IKK{alpha}β. Reprobing with anti-GAPDH demonstrated equivalent loading of lanes. Results are representative of three experiments.

 
Finally, we tested whether the inhibitors of MAPK pathways impacted on the NF-{kappa}B pathway, because there are many reports in the literature that MAPKs can control NF-{kappa}B activation (30, 31, 32, 33). We found that MEK and p38 MAPK inhibitors effectively blocked the phosphorylation of IKK{alpha}β upon 2DL4 cross-linking, indicating that ERK and p38 MAPKs are indeed upstream activators of the NF-{kappa}B pathway in response to 2DL4 cross-linking. In contrast, JNK inhibitor did not affect early phosphorylation of IKK{alpha}β (Fig. 7C). The abrogation of IKK{alpha}β phosphorylation by inhibitors of MEK and p38, but not JNK, is consistent with the early and transient activation kinetics of ERK, p38, and IKK{alpha}β followed by later activation of JNK. Therefore, our data indicate that 2DL4 stimulates the phosphorylation of ERK and p38, which are upstream of the phosphorylation of the NF-{kappa}B regulatory kinase IKK.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we expressed and analyzed the function of FLAG epitope-tagged 2DL4 in the human NK-like cell line KHYG-1 by retroviral transduction. Our goal was to identify the signaling intermediates triggered by 2DL4 and establish the signaling role of the associated transmembrane adaptor Fc{epsilon}RI-{gamma} in mediating biological responses. We found that 2DL4 activates calcium mobilization, all three MAPKs, and the NF-{kappa}B pathway, which ultimately leads to potent cytokine and chemokine responses but weak cytotoxicity. The activation of ERK and p38 MAPKs was very early and transient in response to 2DL4 cross-linking, while JNK activation was delayed and sustained for at least 30 min. Pharmacological inhibitors demonstrated that the activities of all three MAPK families are necessary and function nonredundantly in 2DL4-mediated production of IFN-{gamma}, IL-8, and MIP1{alpha}. This is the first report providing evidence that the cross-linking of 2DL4 activates all three MAPKs and NF-{kappa}B in NK cells and that all of these MAPKs contribute to 2DL4-mediated cytokine production. Furthermore, our studies with pharmacological inhibitors suggest that the p38 and MEK/ERK pathways are upstream of the NF-{kappa}B pathway and that p38 activation may be a prerequisite for subsequent JNK activation.

Interestingly, our data also indicate that activation of the MAPKs and the NF-{kappa}B pathways, as well as the production of the MIP1{alpha} chemokine, are independent of receptor association with the transmembrane adaptor protein Fc{epsilon}RI-{gamma} through a positively charged transmembrane arginine residue. In contrast, our results clearly demonstrate that the transmembrane R/G mutation disrupts Fc{epsilon}RI-{gamma} association, calcium mobilization, cytotoxicity, and IL-8 or IFN-{gamma} responses. Therefore, these latter functional responses are dependent upon Fc{epsilon}RI-{gamma}, while the former responses are independent of association with the transmembrane adaptor protein.

To our knowledge, this is the first report of the involvement of JNK in IFN-{gamma} responses by NK cells. Previously, JNK was shown to be dispensable for natural cytotoxicity and Ab-dependent cellular cytoxicity responses by NK cells (23). Although defects in IFN-{gamma} production have been described in JNK1–/– T cells (34), at least one previous report failed to show inhibition of CD16- and IL-12-stimulated IFN-{gamma} production in NK cells pretreated with a JNK inhibitor (24). These findings indicate that IL-12 and CD16 may be triggering a JNK-independent pathway that is distinct from that of 2DL4. Furthermore, Snyder et al. previously reported that KIR2DS2 can stimulate IFN-{gamma} production and parallel JNK activation in the absence of association with its cognate transmembrane adaptor protein, DAP12 (35). That report, however, attributed the signaling to association with an alternative adaptor, because the signaling was lost when the basic transmembrane residue in KIR2DS2 was mutated. Therefore, our findings highlight several unique attributes of 2DL4 structure and function when compared with other NK cell activating receptors.

Our data support previous evidence that 2DL4 signaling to downstream events may be initiated directly through its cytoplasmic domain. Recently, Rajagopalan et al. reported that transfection of 2DL4 into the 293T fibroblast cell line results in constitutive production of IL-8, which is dependent on the cytoplasmic domain of the receptor (22). That result suggests that 293T cells express a ligand to engage the receptor. Although another domain of 2DL4 may still physically link the receptor to an alternate signaling adaptor, the cytoplasmic domain stands out as the likely source for signal initiation in our studies as well. It is currently unclear how the 2DL4 cytoplasmic domain might mediate activation signaling, because it contains an ITIM domain that exhibits potent inhibitory capacity in isolation through selective recruitment of the SHP-2 protein tyrosine phosphatase (11, 36). SHP-2, however, has been shown to facilitate activation signaling in a number of receptors, which is thought to be due to it serving as an adaptor to initiate downstream signaling pathways, especially the ERK cascade (37, 38, 39, 40). Faure and Long showed previously that mutation of adjacent RY residues in the 2DL4 transmembrane domain (to GT) generates a receptor with a slight inhibitory function (11). In our experiments, however, mutating only the arginine did not create an inhibitory receptor. The basis for the difference is currently unclear, but could involve the transmembrane tyrosine.

A number of studies have linked MAPKs to triggering the production of IFN-{gamma}, IL-8, and MIP1{alpha}. NK cells produce IFN-{gamma} following activation through several receptors, and costimulation with cytokines such as IL-2 and especially IL-12 can synergistically enhance these cytokine responses (41, 42). The production of IFN-{gamma} by cross-linking the activating Ly49D receptor on murine NK cells was previously shown to also be partially blocked by an ERK pathway inhibitor but significantly reduced by combined inhibitors of the p38 and ERK pathways, further demonstrating the importance of MAPKs in the production of IFN-{gamma} by NK cells (43). A role for ERK in the production of IFN-{gamma} by NK cells has also previously been established in SHIP-1-knockout mice (24). Another report using a pharmacological inhibitor also showed the potential role of p38 in controlling 2DL4-mediated production of IFN-{gamma} (11). Cross-linking of many NK cell receptors, including CD28, NKG2D, NKp30, NKp46, NKG2C/CD94, and 2B4, leads to the phosphorylation of both ERK2 and JNK1, although they use different proximal signaling modules (44). Consistent with our results, Mainiero et al. previously showed that p38 is required for integrin-stimulated IL-8 production in human NK cells (45). In contrast, another study showed that LPS induced production of IL-8, MIP1{alpha}, and MIP1β from syncytiotrophoblast cells and coordinated the activation of ERK1/2, but not that of p38 or JNK1/2. Inhibition of the ERK pathway led to a reduction in the secretion of MIP1β and IL-8 in that report, suggesting that their production is at least partly dependent on ERK1/2 activation (46).

The transcription factor NF-{kappa}B has been shown to regulate IFN-{gamma} production in NK cells through IL-12 and IL-18 receptors (47, 48, 49, 50). Moreover, NF-{kappa}B is involved in many aspects of cell growth, differentiation, and proliferation by promoting transcription of certain growth and transcription factors (e.g., c-Myc, Ras, and p53). In our study, we have also found that 2DL4 receptor activates NF-{kappa}B pathways within 1–2 min. The NF-{kappa}B family members have been previously shown to differentially regulate NK cell proliferation and production of IFN-{gamma} (51). c-Rel-deficient NK cells demonstrated reduced IFN-{gamma} production in response to stimulation with numerous cytokines, whereas p50–/– NK cells exhibited a significant increase in IFN-{gamma} production (51). Consistent with this result, p50 homodimers can act as inhibitors of gene transcription and are constitutively associated with the IFN-{gamma} promoter in NK cells (52), which exists in a constitutively open configuration that would allow for rapid production of the cytokine (53). In addition, it has been shown that mutation of the NF-{kappa}B binding site in the IL-8 promoter abrogates transcription of the gene, indicating an important role for NF-{kappa}B in the IL-8 production response (54). MIP1{alpha} has also been shown to be a target gene for NF-{kappa}B regulation (55). Consistent with these previous observations, our results indicate that 2DL4 cross-linking activates the classical NF-{kappa}B pathway, which is important for controlling IL-8 and MIP1{alpha} production. The intronic enhancer region (C3) of the IFN-{gamma} gene binds both the NF-{kappa}B protein c-Rel and NFAT, which is activated by calcium mobilization (53, 56, 57, 58). Similarly, it has also been shown in mast cells that IL-8 gene expression and protein secretion are induced by the combined stimulation of an increase in intracellular Ca2+ and the activation of NF-{kappa}B, which are dependent on the activation of MAPKs (30). In fact, the lack of Fc{epsilon}RI-{gamma}-mediated Ca+2 mobilization may contribute significantly to the lack of IFN-{gamma} and IL-8 production upon engagement of the R/G mutant of 2DL4. Furthermore, we also believe the lack of Ca+2 signaling by the R/G mutant abolishes the cytotoxicity response, because elevation in cytosolic Ca+2 is a major requirement for granule release during NK cell cytotoxic responses (59).

Combined activation of NF-{kappa}B and AP-1 in human airway smooth muscle cells has been shown to be dependent on MAPKs by using inhibitors (31). It has also been shown that p38 controls transcriptional regulation of the gene expression of COX-2 and also posttranscriptional regulation involving the transcript stability of TNF-{alpha} (60, 61). Furthermore, p38 has also been shown to be necessary for the transactivation of NF-{kappa}B in response to TLR ligands, microorganisms, and cytokines including TNF-{alpha} (32). Consistent with these findings, we found coordinate activation of ERK, p38, and NF-{kappa}B after 2DL4 cross-linking and inhibitors of MEK or p38 blocked phosphorylation of IKK{alpha}β, indicating that the ERK and p38 are upstream regulators of the NF-{kappa}B pathway.

In conclusion, we have characterized some of the key downstream signaling events triggered by 2DL4. Cross-linking 2DL4 leads to the production of IFN-{gamma} and IL-8, cytotoxicity, and Ca+2 mobilization responses, which are dependent on transmembrane arginine-mediated association with the transmembrane adaptor Fc{epsilon}RI-{gamma}. In contrast, 2DL4 can also stimulate the activation of MAPKs and NF-{kappa}B and secretion of MIP1{alpha}, which are independent of the transmembrane arginine. The results indicate that 2DL4 transduces activating signals into NK cells through two distinct structural modules.


   Acknowledgments
 
We thank Drs. Glenn Rall and Xiangxi (Mike) Xu for constructive criticism and advice. We also thank Drs. Marco Colonna and Garry Nolan for reagents, the Biological Resources Branch of the National Cancer Institute for rIL-2, and the DNA synthesis, DNA sequencing, flow cytometry, and cell culture facilities at Fox Chase Cancer Center for reagents and technical support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant CA100226 (to K.S.C) and partially by Centers of Research Excellence Grant CA06927 from the National Institutes of Health. This work was also supported in part by an appropriation from the Commonwealth of Pennsylvania. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. Back

2 Address correspondence and reprint requests to Dr. Kerry S. Campbell, Fox Chase Cancer Center, Institute for Cancer Research, 333 Cottman Avenue, Philadelphia, PA 19111. E-mail address: kerry.campbell{at}fccc.edu Back

3 Abbreviations used in this paper: KIR, killer cell Ig-like receptor; 2DL4, KIR2DL4 (KIR with two Ig-like domains and a long cytoplasmic domain); IKK, I{kappa}B kinase; p90RSK, p90 ribosomal S6 kinase; R/G, transmembrane arginine to glycine mutation; WT, wild type. Back

Received for publication August 4, 2007. Accepted for publication December 25, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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