Many NK cell receptors activate ERK2 and JNK1 to trigger microtubule organizing center and granule polarization and cytotoxicity

Abstract

Natural killer (NK) cells are components of the innate immune system that recognize and kill tumor or virus-infected target cells through specific NK activating receptor/ligand interactions. Lymphocyte function-associated antigen (LFA)-1 and its ligand ICAM-1 are also required to initiate conjugation and actin cytoskeletal remodeling. The NK activating receptors, many of which are expressed on a single NK cell, signal the polarization of the microtubule organizing center (MTOC) together with cytolytic granules to the synapse with target cells. After ligation of any one of these receptors, Src family kinases initiate activation of two signal pathways, the phosphoinositide-3 kinase --> ERK2 and the phospholipase Cgamma --> JNK1 pathways. Both are required for polarization of the MTOC and cytolytic granules, a prerequisite for killing the targets. Crosslinking of CD28, NKG2D, NKp30, NKp46, NKG2C/CD94, or 2B4 leads to the phosphorylation of both ERK2 and JNK1, although they use different proximal signaling modules. Thus, many, if not all, activating receptors stimulate these two distal pathways, independent of the proximal signaling module used. By contrast, CD2, DNAM-1, and beta(1)-integrin crosslinking do not activate either pathway; they may be costimulatory molecules or have another function in the synapse.

Fig. 1.

Distinct pairs of NK receptors are required for killing different target cells by the NK cell lines. YTS, NKL, and NK92 tumor cell lines were used to kill 721.221 and K562 targets in a ⁵¹Cr-release cytotoxicity assay. Specific mAb (10 μg/ml) were incubated with the NK cells before mixing with the targets. The means and SDs of the percent lysis at effector:target ratio (E:T) = 10:1 are shown after three or four independent experiments. ND, not determined; ∗, the mAb used in this experiment is specified separately.

Fig. 2.

NK activating receptors are required for the polarization of the MTOC and cytolytic granules, whereas LFA-1 mediates conjugate formation. (A) YTS cells were conjugated with 721.221 cells in the presence of control mIgG or anti-CD28 mAb. The conjugates were fixed and permeabilized for intracellular staining of F-actin (green), microtubules (MT, red), and granules (blue) simultaneously. (B) The percentage of conjugates showing F-actin polymerization in the NKIS and MTOC and granule polarization to the NKIS (means and SDs). At least 50 conjugates images were examined in each case. The FACS-based conjugation assay was used with YTS-GFP cells and 721.221-RFP cells preincubated with control mIgG, anti-LFA-1 mAb, or anti-CD28 mAb. Results from six independent conjugation assays were averaged. (C) Similar imaging experiments were done with NK92 cells conjugated with K562 targets in the presence of either control mIgG, anti-LFA-1 mAb, or anti-NKG2D mAb. (D) The results of observing >50 images are shown with SDs. ∗, Percent F-actin polymerization and MTOC and granule polarization in the presence of anti-LFA-1 mAb are the values obtained in examining the small number of conjugates formed under these conditions.

Fig. 3.

LFA-1 cross-linking initiates F-actin polymerization, whereas CD28, NKG2D, or 2B4 cross-linking polarize the MTOC. (A–F) YTS cells (A and B) and NKL cells (C–F) were coated with biotinylated anti-CD18 (the β₂ subunit of LFA-1) mAb (A and C), anti-CD28 mAb (B), anti-NKG2D mAb (D), anti-2B4 mAb (E), or anti-class I ME1 mAb as control (F) and then mixed with streptavidin-coated polystyrene beads. After 0.5–2 h at 37°C, the cells were fixed and permeabilized for the intracellular staining of F-actin (green) and MT (red). (D) The percentage of conjugates showing F-actin polymerization at the bead-cell contact site and of MTOC polarization toward the bead-cell contact site (arrows) are shown. P values were calculated after examining >50 images for each experiment. (G) Quantitation and statistical analysis.

Fig. 4.

Many NK activating receptors are coupled to the ERK2 and JNK1 phosphorylation pathways. LFA-1 and CD28 on YTS cells (A); NKG2D, NKp30, and NKp46 on NK92 cells (B); and DNAM-1, 2B4, CD2, NKG2D, NKG2C/CD94, and β₁ integrin on NKL cells (C) were cross-linked (×) for the indicated times. Cells treated with control mIgG were used as negative controls, and phorbol 12-myristate 13-acetate/ionomycin (PMA/ION) stimulation was used as a positive control. The total cell lysate was loaded into SDS/PAGE gels for Western blot analysis. Anti-phosphERK, anti-phosphoJNK, anti-panERK, and anti-panJNK rabbit polyclonal antibodies were used to detect the pattern of phosphorylation as a function of time. The second batch of polyclonal anti-phosphoJNK antibody obtained from Cell Signaling used in C did not detect phosphoJNK as strongly as that used in A and B. The results are representative of at least three independent experiments.

Fig. 5.

NKG2D cross-linking on NKL cells activates a bifurcated signaling pathway to phosphorylate ERK and JNK. Biotinylated anti-NKG2D mAb was used to cross-link the activating receptor on NKL cells by adding streptavidin for 10 min. Experiments were done in the presence of DMSO as control, 200 nM Wortmannin (WM, inhibitor of PI3K), 2 μM PP2 (inhibitor of SFK), and 1 μM U73122 (U731, inhibitor of phospholipase Cγ), all prepared in DMSO. After cross-linking, the NKL cells were immediately fixed and permeabilized for intracellular staining of phospho-ERK or phospho-JNK with Alexa647 nm conjugated-specific mAb. One representative result from three independent flow cytometry analyses is shown. The gray line indicates control mIgG staining, the green line shows the specific mAb staining in resting cells, the red line represents the specific staining after NKG2D cross-linking in the presence of DMSO, and the blue line is the result of NKG2D cross-linking in the presence of the indicated inhibitor.

Fig. 6.

Schematic representation showing that many NK activating receptors employ the same SFK-dependent PI3K → ERK2 pathway and phospholipase Cγ → JNK1 pathway to polarize the MTOC and cytolytic granules during NK cell cytotoxicity, independent of the proximal signaling modules.