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The Journal of Immunology, 2007, 179, 3752 -3762
Copyright © 2007 by The American Association of Immunologists, Inc.
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Bcl10 Plays a Divergent Role in NK Cell-Mediated Cytotoxicity and Cytokine Generation1

Subramaniam Malarkannan2,*,{ddagger},§, Jeyarani Regunathan*, Haiyan Chu*, Snjezana Kutlesa*, Yuhong Chen{dagger}, Hu Zeng{dagger}, Renren Wen{dagger} and Demin Wang2,{dagger},{ddagger}

* Laboratory of Molecular Immunology, {dagger} Receptor Signaling Laboratory, Blood Research Institute, {ddagger} Department of Microbiology and Molecular Genetics, and § Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activating receptors such as NKG2D and Ly49D mediate a multitude of effector functions including cytotoxicity and cytokine generation in NK cells. However, specific signaling events that are responsible for the divergence of distinct effector functions have yet to be determined. In this study, we show that lack of caspase recruitment domain-containing protein Bcl10 significantly affected receptor-mediated cytokine and chemokine generation, but not cytotoxicity against tumor cells representing "missing-self" or "induced-self." Lack of Bcl10 completely abrogated the generation of GM-CSF and chemokines and it significantly reduced the generation of IFN-{gamma} (>75%) in NK cells. Commitment, development, and terminal maturation of NK cells were largely unaffected in the absence of Bcl10. Although IL-2-activated NK cells could mediate cytotoxicity to the full extent, the ability of the freshly isolated NK cells to mediate cytotoxicity was somewhat reduced. Therefore, we conclude that the Carma1-Bcl10-Malt1 signaling axis is critical for cytokine and chemokine generation, although it is dispensable for cytotoxic granule release depending on the activation state of NK cells. These results indicate that Bcl10 represents an exclusive "molecular switch" that links the upstream receptor-mediated signaling to cytokine and chemokine generations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Receptors NKG2D and Ly49D are two major activating molecules of NK cells (1, 2). NKG2D recognizes UL16-binding proteins 1–3 and MHC class I (MHC I)3 -related molecules (A and B) in humans (1, 3) and nonclassical MHC I molecules H60, Rae-1, and Mult-1 in mice (4, 5, 6). Expression of most of these ligands is "induced" in stressed, infected, and transformed cells (7, 8). Thus, NK cells provide surveillance against pathologically altered cells through NKG2D-mediated recognition of "induced-self." Ly49D, another major activating receptor of NK cells interacts with classical MHC class I, H2-Dd (9).

In recent years, tremendous progress has been made to understand the signaling pathways originating from NKG2D that regulate the functions of NK cells. NKG2D is expressed as two isoforms. Upon ligand binding, NKG2D uses Src family protein tyrosine kinases (PTK) to initiate at least two distinct signaling pathways (10, 11, 12, 13, 14). In one pathway, activated Src family PTK phosphorylates Tyr-Ile-Asn-Met (YINM) motif-bearing adaptor molecule DAP10, which in turn recruits PI3K (11, 12). DAP10 associates with the longer isoform of the NKG2D. In the second pathway, the Src family PTK phosphorylates the ITAM-containing adaptor molecule DAP12 that associates with the shorter isoform of NKG2D, and activates via Syk and Zap70 (12, 13, 14, 15, 16). Ultimately, further downstream signaling cascades lead to granule release for cytotoxicity and gene transcription for cytokine production. Attribution of each of these signaling components to NKG2D-mediated effector functions is only beginning to be defined. The absence of DAP10, DAP12, or its associated Syk/Zap70 (13, 14, 16), did not significantly affect NKG2D-mediated cytotoxicity, suggesting that DAP10-PI3K and DAP12-Syk/Zap70 pathways are redundant for mediating this effector function. However, lack of DAP12 or Syk/Zap70, but not DAP10, severely impaired NKG2D-mediated cytokine production (14, 15), indicating an exclusive role for DAP12-Syk/Zap70 pathway in cytokine generation. Ly49D associates with DAP12 adaptor protein and follows signaling pathway that are similar to that of NKG2D/DAP12 complexes (17). Despite all these progresses related to membrane proximal events, other key signaling molecules downstream of NKG2D and Ly49D that are uniquely responsible for distinct effector functions remain far from fully understood.

Bcl10 is an adaptor protein containing an N-terminal caspase recruitment domain and a C-terminal serine/threonine-rich domain. Bcl10 was originally identified in mucous-associated lymphoid tissue lymphomas (Malt) due to its chromosomal translocation (18, 19). Recent studies have demonstrated that Bcl10 is critical for AgR-induced nuclear translocation of NF-{kappa}B via ubiquitination-mediated activation of the I{kappa}B kinase (IKK) complex (20, 21). Generation of Bcl10-deficient (Bc110–/–) mice provided a valuable tool for defining the significance of this adaptor protein in AgR signaling (22, 23). In these mice, T cell development, TCR-induced proliferation/NF-{kappa}B activation/IL-2 production and up-regulation of activation markers were severely impaired (22). Bcl10 deficiency resulted in a dramatic reduction in follicular (FO), marginal zone (MZ), and peritoneal B1 B cells (23). In addition, Bcl10 deficiency impaired BCR-induced NF-{kappa}B activation, proliferation leading to a reduction in the basal levels of serum Igs and these mice failed to generate immune Ab responses to the T cell-dependent and -independent Ags.

In this study, we investigated the role of Bcl10 in NK cell development and functions (22, 23). We show that Bcl10 is highly expressed in both freshly isolated and IL-2-activated NK cells. Bcl10 deficiency does not affect the lineage commitment, development, and terminal maturation of NK cells. Bcl10–/– NK cells exhibited normal NKG2D- and Ly49D-mediated cytotoxicity compared with that of wild type (WT). In contrast, Bcl10–/– NK cells have severely impaired NKG2D-, Ly49D-, and NK1.1-mediated cytokine and chemokine generation. These data, for the first time, provide us important insights into the "molecular switches" responsible for the divergence of distinct immune effector functions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and cell lines

Bcl10–/– and WT mice which have been backcrossed to C57BL/6 for seven generations were as described (23). All mice used in this study were maintained in pathogen-free conditions at the Biological Resource Center (BRC) at the Medical College of Wisconsin (MCW; Milwaukee, WI) and were used between 6 and 12 wk of age. All the animal protocols used were approved by the BRC, MCW. EL4, EL4H60, RMA/S, CHO, and YAC-1 cells and their culture conditions were described (24, 25).

Flow cytometry

Cell preparations were stained with fluorescent-labeled mAbs as described (24). Hybridoma-secreting anti-NK1.1 (PK136) was obtained from American Type Culture Collection. Abs for NK1.1 (PK136), CD3{epsilon} (145-2C11), NKG2D (A10), NKG2A (16a11), CD11b (M1/70), CD43 (1B11), CD49b (DX5), CD51 (RMV-7), CD69 (H1.2F3), CD122 (5H4), and Ly49I (YLI-90) were obtained from eBioscience. Abs for Ly49A (A1), Ly49C/I (5E6), Ly49D (4E5), and Ly49G (4D11) were obtained from BD Biosciences. NK cells were stained in 1% FCS-PBS with appropriate Abs (24). Standard flow cytometry analysis was conducted in LSR-II using FACSDiva software (BD Biosciences).

Western blotting

Immunoblots of Bcl10 in T, B, and NK cell lysate (1 x 106) were performed as described (23). Forty micrograms of whole cell lysate were resolved using 8% SDS-PAGE gels and transferred to nitrocellulose membranes and probed with indicated Abs. Anti-mouse Bcl10 (Santa Cruz Biotechnology) and anti-actin (Boehringer Mannheim) were used and signals were detected using an ECL kit (GE Healthcare).

NK cell preparation

NK cells were purified as previously described (24). Briefly, single-cell suspensions from spleen and bone marrow (BM) were passed through nylon wool columns to deplete adherent populations consisting of B and macrophages. Nylon wool-nonadherent cells were cultured with 1000 U/ml IL-2 (National Cancer Institute-Biological Resources Branch-Preclinical Repository, Frederick, MD). Purity of the NK cultures was checked and preparations with >90% of NK1.1+ were used, where >75% of the cells were CD3 negative.

Cytotoxicity assays and quantification of perforin release

NK-mediated cytotoxicity was quantified using 51Cr-labeled target cells (27) at varied E:T ratios. Percent-specific lysis was calculated using amounts of absolute, spontaneous, and experimental 51Cr release from target cells. Loss of perforin was quantified through intracellular staining using anti-perforin Ab (eBioOMAK-D). Briefly, IL-2-cultured NK cells were incubated with EL4 or EL4H60 cells at an 20:1 E:T ratio for 4 h at 37°C. After 4 h, cells were Fc-blocked, stained for NK1.1, fixed, permeabilized, and quantified for intracellular perforin using FITC-conjugated anti-perforin mAb through flow cytometry. Staining cells with anti-NK1.1 Ab excluded the target cells from analyses, which are negative for NK1.1.

Secretion and quantification of cytokines/chemokines through ELISA and flow cytometry

IL-2-cultured, Fc-blocked NK cells were activated with titrated concentrations of plate-bound anti-NKG2D (A10), anti-Ly49D (4E5), or anti-NK1.1 (PK136) mAbs. Standard curves generated using recombinant cytokines were used to calculate the concentrations of IFN-{gamma} and GM-CSF in the culture supernatants using ELISA kits (eBioscience). IFN-{gamma}, GM-CSF, MIP-1{alpha}, MIP-1beta, and RANTES were quantified using the Bioplex kit (Bio-Rad). Intracellular IFN-{gamma} was quantified using established methodologies (28). Briefly, NK cells were activated with 10 µg/ml plate-bound anti-NKG2D (A10) mAb for 16 h. Cells were then Fc-blocked, stained for NK1.1, fixed, permeabilized, and quantified for intracellular IFN-{gamma} using PE-conjugated anti-IFN-{gamma} mAb through flow cytometry. For pharmacological inhibitor assays, NK cells were incubated for 1 h with varying concentrations of inhibitors for NF{kappa}B (BAY 11-7085; Calbiochem) or protein kinase C (PKC; GF 109203X; Biomol), washed, and added anti-NKG2D or anti-Ly49D Ab-coated plates. After 18 h, culture supernatants were collected to quantify the indicated cytokines using the Bioplex kit (Bio-Rad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Bcl10 is highly expressed in murine NK cells

Bcl10 is expressed in T and B cells (18, 19, 23) and its deletion impairs the development and functions of these immune cells (22, 23). To assess its potential roles, we first examined the expression levels of Bcl10 in NK cells. Fresh T, B, and NK cells were purified from mouse spleen by cell sorting and cell lysates were analyzed by Western blotting, which demonstrated that Bcl10 was amply expressed in fresh NK cells (Fig. 1A). Expression levels of Bcl10 in NK cells were much higher than in T cells but similar to that of B cells (Fig. 1A). Bcl10 protein was also readily detectable in IL-2-activated NK cells derived from the WT (Fig. 1B). As expected, Bcl10 was absent in the Bcl10–/– NK cells. These data demonstrate that Bcl10 is abundantly expressed in murine NK cells.


Figure 1
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  		FIGURE 1. Lack of Bcl10 does not affect the commitment and maturation of NK cells. A, Expression of Bcl10 protein was quantified by Western blot in fresh B, T, and NK cells derived from WT mice. Blot is reprobed with anti-actin mAb as loading control. B, Expression of Bcl10 in the IL-2-activated NK cells derived from WT and Bcl10–/– mice as analyzed by Western blot. C, Single-cell suspensions prepared from the indicated organs of WT and Bcl10–/– mice were stained for NK1.1 and CD3{epsilon}. T (CD3+NK1.1–), NKT (CD3+NK1.1+), and NK (CD3–NK1.1+) cells are gated as shown. Histograms representing the percentages of T, NKT, and NK cells in total lymphocyte population are shown in the rightmost panels. Data from five WT and Bcl10–/– mice each were used. Values of p were calculated using "two-tail"-two sample Student’s t tests.

 
NK cell development and maturation in the absence of Bcl10 is largely unaffected

Bcl10 expression is critical for the normal development of T and B cells (22, 23). Because NK cells express significant amounts Bcl10, we reasoned that Bcl10 might play a role in the development of NK cells. To address this, we examined the population of NK (CD3–NK1.1+) cells derived from BM, spleen, liver, and thymus of WT and Bcl10–/– mice. The total number of lymphocytes were comparable among the organs analyzed as previously reported (23). Cells were divided into T (CD3+NK1.1–), NKT (CD3+NK1.1+) and NK (CD3–NK1.1+) cells based on their staining patterns. Total number of CD3+NK1.1– T cells did not differ between the Bcl10–/– and WT mice in any of the organs tested. However, CD3+NK1.1+ NKT cells were increased in the BM and decreased in the spleen, liver, and thymuses of Bcl10–/– mice. The percentages of CD3–NK1.1+ cells in BM that represented NK population were slightly reduced in Bcl10–/– compared with that of WT mice (Fig. 1C, right panels). This indicates that the commitment to NK lineage and the maturation of these effector cells in the BM is largely unaffected in the absence of Bcl10. Similarly, spleen-derived CD3–NK1.1+ cell numbers were also reduced while the percentages of these cells were increased in the liver in Bcl10–/– mice. Reasons for these reductions are currently not understood. Thymuses from these animals did not yield statistically significant numbers of CD3–NK1.1+ cells.

NK cell development occurs in the BM. Recent studies have demonstrated that the process of NK cell development can be defined into distinct stages based on the expression pattern of different cell surface markers (29). Expression of CD122, which is the beta-chain of IL-2 and IL-15 receptors, marks the commitment of progenitor cells into NK lineage in the BM and it is expressed throughout NK cell development. NKG2D and NK1.1 are the earliest known NK cell-specific markers (30). CD11b (Mac-1) and CD43, which are critical regulators of lymphocyte migration and homing, are up-regulated in the final stages maturation, while CD51 ({alpha}v) expression declines (29). Acquisition of distinct Ly49 by the developing CD3–NK1.1+ cells is a most important developmental step in which NK cells become exclusive subsets and "calibrate" their activation potentials based on the inhibitory Ly49R(s) they express. Expression of Ly49Rs also signifies the terminal maturation of the NK cells.

To determine whether the lack of Bcl10 could affect the development of NK cells, we analyzed the expression of these markers on fresh NK cells derived from BM and spleen (Fig. 2, A and B). Gated CD3–NK1.1+ NK cells were analyzed for the expression of maturation and activation receptors such as CD122, NKG2D, and NKG2A or integrins and developmental markers such as CD11b, CD43, CD49b, and CD51. Results demonstrate that the expression of most of these markers was comparable between the Bcl10–/– and WT. One interesting exception is the levels of the inhibitory receptor NKG2A (BM, WT 31.2 ± 2.1; Bcl10–/– 36.5 ± 0.4; spleen, WT 25.6 ± 2.6; Bcl10–/– 35.7 ± 2.0), which appears to be increased in the Bcl10–/– mice. Additionally, the levels of CD51 ({alpha}v) were decreased in the Bcl10–/–-derived fresh NK cells (BM, WT 38.9 ± 1.9; Bcl10–/– 24.3 ± 2.2; spleen, WT 20.0 ± 1.0; Bcl10–/– 11.2 ± 2.0). The molecular mechanism and the significance of the increase in NKG2A and the reduction in CD51 expression are not clearly understood at present. Irrespective of these differences, the population of the matured NK cells (CD3–NK1.1+CD11b+ or CD3–NK1.1+CD43+) was comparable in the BM and spleen between Bcl10–/– and WT mice (Fig. 2, A and B). Recent studies have demonstrated that the acquisition and expression of different Ly49Rs marks the terminal differentiation and maturation of NK cells (29, 31). Thus, the expression levels of Ly49Rs can be an indicator of the maturation status of the developing NK cells (29). Our earlier studies have indicated that the lack of phospholipase C {gamma}2 (PLC{gamma}2) significantly reduced the ability of maturing NK cells to express Ly49Rs (32). Moreover, Ly49R expression has been strongly correlated to the functional abilities of mature NK cells (33, 34). Therefore, we analyzed the expression of these developmental markers on IL-2-activated NK cells. Among different Ly49Rs, expression of Ly49A was consistently increased in Bcl10–/–-derived NK cells compared with that of WT. However, expression of all the inhibitory Ly49 (I, C, and G) including Ly49A or the expression of activating Ly49D were unaffected (Fig. 2C) in IL-2-activated NK cells in the absence of Bcl10. Based on these observations, we conclude that the lack of Bcl10 does not seem to overtly affect the commitment, early development, and the terminal maturation of NK cells.


Figure 2
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  		FIGURE 2. Expression of early NK cell developmental markers is normal in Bcl10–/– mice. Bone marrow (A) and spleen (B) cells were stained with anti-CD3{epsilon} and anti-NK1.1 mAbs and the gated CD3–NK1.1+ population was analyzed for the expression of NKG2D, NKG2A CD11b, CD43, CD49b, CD51, CD122, and Ly49A receptors. Percentages of positive NK cells for each receptor are shown. Gates used to calculate percent-positive cells are shown in the leftmost panels (Neg). Data presented were representative of four independent experiments. C, Percent-positive cells expressing Ly49 receptors were analyzed in the WT and Bcl10–/– mice-derived, IL-2-activated NK cells. Data presented were an average of seven Ly49 expressions in seven mice in each group.

 
NKG2D-mediated recognition of induced-self is reduced in fresh NK but unaffected in IL-2-cultured Bcl10–/– NK cells

Bcl10 expression is critical for AgR-mediated signaling in T and B cells (22, 23). Although our data demonstrate that the Bcl10 deficiency did not affect NK cell development, we speculated that Bcl10 might play a role in NKG2D-mediated signaling. NKG2D is ubiquitously expressed on NK cells and the activation through NKG2D results in cytotoxicity against ligand-expressing target cells (1). Notably, the levels of NKG2D expression on Bcl10–/– NK cells were normal compared with the WT (Fig. 2). Earlier studies from us and others have shown that ectopic or stress-induced expression of H60 on tumor cells renders them susceptible to NKG2D-mediated cytotoxicity (5, 24, 35). To assess the ability of Bcl10–/– NK cells in mediating cytotoxicity through the NKG2D, we used EL4 cell lines stably expressing H60 (EL4H60) in 51Cr-release assays (24). Parental EL4 cells were used as negative controls. Fresh NK cells were isolated, tested for purity, and used in cytotoxicity assays. Absence of Bcl10 led to a reduction in NKG2D-mediated cytotoxicity (Fig. 3A). Freshly isolated NK cells are known to express only NKG2D/DAP10 complexes (12). However, IL-2-activated NK cells express both NKG2D/DAP10 and NKG2D/DAP12 complexes (36). Therefore, we also tested the cultured NK cells in cytotoxicity assays. Compared with the freshly isolated NK cells, IL-2-activated Bcl10–/– NK cells exhibited normal abilities to lyse EL4H60 similar to WT cells (Fig. 3B). Another tumor cell, YAC-1 that intrinsically expresses H60 was used to further test the cytotoxic ability of Bcl10–/– NK cells (Fig. 3B). Similar to that of EL4H60, YAC-1 was also lysed equally well by the Bcl10–/– NK cells. The defect in the ability of fresh NK cells to recognize induced-self is unexpected. It can be explained either by a yet undefined defect in the development and maturation of NK cells or the granule release in fresh NK cells is in part dependent on the Bcl10-mediated pathway. Irrespective of this defect in the fresh NK cells, the NKG2D-mediated recognition of induced-self by the IL-2-cultured NK cells occur independent of Bcl10-mediated signaling events.


Figure 3
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  		FIGURE 3. "Induced-self" and "missing-self" recognitions of NK cells are unaffected in the absence of Bcl10. A, Fresh NK cells were isolated from spleens of WT and Bcl10–/– mice using DX5-coated magnetic beads. NK cell preparations (>75%) of comparable purity between WT and Bcl10–/– mice were used to test their ability to mediate cytotoxicity of indicated target cells. B, IL-2-activated NK cells were tested against 51Cr-labeled EL4, EL4H60, YAC-1 (H60+), (C) Ly49D ligand (H2-Dd)-expressing CHO cells, or (D) MHC class I-negative RMA/S cells or at the indicated E:T ratios. Six to eight mice were used for each category. {circ} and •, The NK cells from individual WT and Bcl10–/– animals, respectively.

 
Ly49D-mediated recognition of tumor cells is unaffected in Bcl10–/– IL-2-activated NK cells

Among many members of Ly49 family, Ly49D belongs to a unique subset of type II lectin that associates with adaptor protein DAP12 (17). Thus, Ly49D functions as an activating receptor in murine NK cells. The only known natural ligand for Ly49D is the MHC I H2-Dd (27). Therefore, we used CHO cells that express a hamster homolog of murine MHC I H2-Dd (37). Similar to that of EL4H60, CHO cells were also efficiently lysed by the Bcl10–/– NK cells (Fig. 3C). These results indicate that Ly49D/DAP12-mediated cytotoxicity, similar to that of NKG2D/DAP12 complexes, does not require Bcl10.

Although the ability to mediate cytotoxicity against different target cells was not affected, we hypothesized that Bcl10 may still play a role in the generation and release of cytotoxic granules in NK cells. To test this, we quantified the levels of perforin in NK cells before and after NKG2D-mediated activation. IL-2-activated NK cells from WT or Bcl10–/– mice were incubated with EL4 or EL4H60 for 4 h at a 20:1 E:T ratio. Percentages of perforin-positive and -negative NK cells in gated NK1.1+ populations were quantified. This indicated that Bcl10 does not affect the generation of perforin. To quantify NKG2D-mediated release of perforin, we incubated NK cells with EL4H60 cells. Bcl10–/– NK cells released considerable amounts of perforin demonstrating that Bcl10 does not play a major role in the signaling requirements for the generation and release of cytotoxic granules (data not shown). These observations are consistent with our cytotoxicity data.

Bcl10 is dispensable for missing-self recognition by IL-2-activated NK cells

Cells that lack or have reduced expression of self MHC class I molecules are susceptible to NK cell-mediated cytotoxicity (38). Although it is well-characterized that lack of engagement by the inhibitory Ly49Rs to respective MHC I relieves the NK cells from inhibition, the signaling events that positively regulate this cytotoxicity are not fully understood. To test whether Bcl10 plays a role in missing-self recognition, we measured the cytotoxic potential of IL-2-activated NK cells against RMA/S tumor cell, which expresses significantly lower levels of MHC I. Fig. 3D demonstrates that the Bcl10–/– NK cells lysed RMA/S cells as efficiently as that of WT cells. NK cells from both WT and Bcl10–/– mice fail to mediate cytotoxicity against the MHC-sufficient, parental RMA cells (data not shown). Thus, we conclude that Bcl10 is not required for the missing-self recognition by NK cells.

Bcl10 deficiency impairs NKG2D, Ly49D, and NK1.1-mediated cytokine and chemokine generation

As part of their innate immune response, NK cells, via NKG2D-mediated activation, generate substantial quantities of cytokines such as IFN-{gamma}, GM-CSF, and chemokines MIP-1{alpha}, MIP-1beta, and RANTES (39). Therefore, we analyzed the role of Bcl10 in the generation of cytokines and chemokines. IL-2-activated NK cells cultured with titrated concentrations of plate-bound anti-NKG2D (A10), anti-Ly49D (4E5), or anti-NK1.1 (PK136) mAbs. Supernatants were collected and the levels of IFN-{gamma}, GM-CSF, MIP-1{alpha}, MIP-1beta, and RANTES were measured by ELISA. WT NK cells produced large amounts of IFN-{gamma} and GM-CSF with plate-bound mAbs in a dose-dependent manner (Fig. 4, A and B). In contrast, Bcl10–/– NK cells were severely impaired in their ability to produce either IFN-{gamma} (Fig. 4A) or GM-CSF (Fig. 4B). Similar to these results, Bcl10–/– NK cells also generated significantly lesser amounts of chemokines, MIP-1{alpha}, MIP-1beta, and RANTES compared with the WT cells (Fig. 4C). These observations strongly imply that Bcl10 plays a critical role in receptor-mediated cytokine and chemokine generations. Although the signaling pathway used by NK1.1 complex is yet to be defined, Ly49D is shown to recruit DAP12 molecule for signal transductions (17). Thus, Bcl10 plays a critical regulator of signaling events that are initiated via NKG2D/DAP12, Ly49D/DAP12 or NK1.1 complexes for the generation of cytokines and chemokines.


Figure 4
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  		FIGURE 4. Ability of NK cells to generate cytokines or chemokines is severely impaired in the absence of Bcl10. Generation of (A) IFN-{gamma} and (B) GM-CSF in IL-2-activated NK cells from Bcl10–/– and WT mice in response to anti-NKG2D, anti-Ly49D, or anti-NK1.1 mAbs-mediated activations. {circ} and •, NK cells from individual WT and Bcl10–/– mouse. Three to seven mice were used for each group. Cross bars in the middle of SDs indicate average levels of cytokines. C, Average MIP-1{alpha}, MIP-1beta, and RANTES in response to anti-NKG2D mAb are shown with SDs. Open and filled histograms stand for WT and Bcl10–/– NK cells. Three of WT and Bcl10–/– mice were used. Values of p were calculated using two-tail two-sample Student’s t tests.

 
The substantial reduction of cytokine generation could be due to the inabilities of the Bcl10–/– NK cells to produce the cytokines (e.g., up-regulation of the cytokine mRNAs) or simply due to a defect in the secretion. To distinguish between these possibilities, IFN-{gamma} production in response to anti-NKG2D mAbs was measured by intracellular staining. IL-2-activated NK cells were stimulated, Fc-blocked, stained for NK1.1, permeabilized, and quantified for intracellular IFN-{gamma}. Percent cytokine-positive cells among the gated NK1.1+ population were shown in Fig. 5A. Bcl10–/– NK cells generated significantly lower levels of IFN-{gamma} compared with WT cells (Fig. 5A). These results demonstrate that Bcl10 specifically transmits a signal from NKG2D to cytokine generation and, therefore, the reduced levels are due to defects in the production and not likely in the secretion.


Figure 5
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  		FIGURE 5. Reduced intracellular cytokine generation in Bcl10–/– NK cell is not due to increased cell death and PMA/ionomycin could not fully rescue cytokine generation. A, Levels of intracellular IFN-{gamma} were quantified in NK cells derived from WT and Bcl10–/– mice after plate-bound anti-NKG2D mAb activation. Data presented are representative of two independent experiments. B, Activation-induced NK cell death was quantified after 16 h of incubation with plate-bound anti-NKG2D mAb followed by anti-annexin-V and 7-AAD staining. Data presented are representative of three independent experiments. C, IL-2-activated NK cells were incubated with PMA (P; 150 ng/ml), ionomycin (I; 400 ng/ml) or both (P+I) for 12 h and the culture supernatants were collected and enumerated for the quantity of IFN-{gamma}. Averages of IFN-{gamma} levels with SDs from three animals each category are shown. Values of p were calculated using two tail two-sample Student’s t tests.

 
Yet another possibility is that the reduced cytokine generation in Bcl10–/– NK cells could be due to activation-induced cell death. To find out whether cell death played a role, we stimulated the NK cells with anti-NKG2D mAb for 16 h and analyzed the viability with anti-annexin-V mAb and a fluorescent DNA stain, 7-aminoactinomycin-D (7-AAD) that binds to ssDNA in dying cells. Fig. 5B shows no significant differences in the staining of either annexin-V or 7-AAD between the WT and Bcl10–/– NK cells. Therefore, we conclude that the activation-induced cell death is not a probable cause for the lower level of cytokine generation in Bcl10–/– NK cells. Activation of Carma1 by PKC results in the recruitment of Bcl10 to the lipid rafts (40). Pharmacological agents such as PMA can activate PKC. To test whether cytokine generations are strictly dependent on the Carma1/Bcl10 pathway, we activated the NK cells with PMA, ionomycin, or both for 12 h and quantified the level of IFN-{gamma} in the supernatants (Fig. 5C). Our results show neither PMA nor ionomycin alone could activate NK cells to generate cytokines. PMA along with ionomycin did result in the generation of IFN-{gamma} from the WT NK cells. However, Bcl10–/– NK cells failed to generate similar levels of IFN-{gamma} in response to PMA and ionomycin. These results are consistent with earlier studies that Bcl10 is a major effector molecule downstream of the PKC/Carma1 pathway and the lack of Bcl10 severely disrupts this signaling axis (22).

Pharmacological inhibition of NF-{kappa}B and PKC significantly reduces NKG2D and Ly49D-mediated generation of cytokines and chemokines in NK cells

To further confirm the role of Bcl10 in the generation of cytokines and chemokines, we preincubated the NK cells with BAY 11-7085 (NF-{kappa}B inhibitor) and GF 109203X (PKC inhibitor) before activating with anti-NKG2D (Fig. 6A) or anti-Ly49D (Fig. 6B) Abs. These inhibitors were selected because 1) the Carma1/Bcl10 pathway is primarily responsible for NF-{kappa}B activation (41) and 2) PKC plays a vital role in initiating this activation pathway downstream of both the BCR and the TCR (42).


Figure 6
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  		FIGURE 6. Pharmacological inhibitors specific for NF-{kappa}B and PKC inhibit anti-NKG2D and anti-Ly49D-mediated cytokine generation in NK cells. NK cells were incubated for 1 h with varying concentrations of inhibitors for NF{kappa}B (BAY 11-7085) or PKC (GF 109203X), washed, and activated with plate-bound (A) anti-NKG2D or (B) anti-Ly49D Abs. Culture supernatants were used to quantify the indicated cytokines (IFN-{gamma}, GM-CSF) or chemokines MIP-1{alpha}, MIP-1beta, or RANTES.

 
Blockade of NF-{kappa}B significantly reduced the ability of NK cells to generate cytokines IFN-{gamma} and GM-CSF along with chemokines MIP-1{alpha}, MIP-1beta, and RANTES. Even concentrations of BAY 11-7085 that are less than the previously defined IC50 of 5 µM were highly effective in significantly reducing the generation of all cytokines and chemokines tested (Fig. 6). These results strongly demonstrate that the generation of these cytokines and chemokines are dependent on the activation, translocation, and the transcriptional control of NF-{kappa}B.

Next, we analyzed the role of PKC in NKG2D- and Ly49D-mediated cytokine generation. We used the bisindolylmaleimide PKC inhibitor, GF 109203X, which is known to inhibit the conventional PKC isoforms in B and T cells (43). Results presented in Fig. 6 demonstrates that GF 109203X significantly blocked the generation of cytokines and chemokines within the defined IC50 range (1–10 µM). GF 109203X has been known to have a specificity for conventional PKC isoforms PKC-{alpha}, -beta, -{gamma}, and -{epsilon}. Therefore, it is possible that a unique PKC isoform that is exclusively blocked by GF 109203X is responsible for mediating the activation of the Carma1/Bcl10 pathway. These observations are of importance because the dominant PKC isoform that regulates NK cell activation has yet to be determined. Collectively, our results demonstrate that Bcl10 plays a critical and nonredundant role in transducing signals from NKG2D, Ly49D, and NK1.1 receptors for the generation of cytokines and chemokines.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
NK cells are a unique subset of lymphocytes that are critical for the innate immune responses (44). Through NKG2D or Ly49D receptors, NK cells mediate cytotoxicity against tumors and virally infected cells. Activation of NKG2D or Ly49D also generates a large quantity of inflammatory cytokines (45). These effector functions of NK cells are coordinated by signals originating from both inhibitory and stimulatory receptors (24, 46, 47, 48). Although key signaling molecules associated with these receptors have been defined, the exclusive signaling molecules that are responsible for effector functions such as cytotoxicity or cytokine generation are yet to be revealed (10, 11, 12, 13, 14). Our results provide strong evidence that Bcl10 is a focal point at which the signaling divergence for cytotoxicity and cytokine generation can occur in NK cells (Fig. 7).


Figure 7
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  		FIGURE 7. Carma-1/Bcl10 pathway represents a "molecular switch" for cytokine generation. NKG2D receptor recruits two distinct adaptor proteins to transduce activation signals. DAP10 with a YINM motif recruits PI3K. DAP12 with an ITAM triggers Syk and Zap70. Both DAP10-PI3K and DAP12-Syk/Zap70 pathways redundantly regulate cytotoxicity via Vav1–3 that activates ERK, resulting in degranulation. However, the DAP12-Syk/Zap70 pathway is exclusively responsible for NKG2D-mediated cytokine production via PLC{gamma}2->DAG->PKC->Carma-1->Bcl10->Malt-1->NF-{kappa}B. Whether PI3K recruitment to DAP10 results in PLC{gamma}2 activation in NK cells is currently not known (dotted arrow). Current data implies that Carma-1/Bcl10 pathway is a major signaling pathway downstream of NKG2D/DAP12-Syk/Zap70 for cytokine/chemokine generation.

 
Earlier studies have shown that Bcl10 plays a critical role in the development and functions of T and B cells (22, 23). Bcl10 deficiency specifically affected the development of double-positive thymocytes and severely impaired TCR-mediated activation, proliferation, IL-2 production, and up-regulation of activation markers (22). These effects were in part due to the lack of TCR-induced NF-{kappa}B activation. Bcl10 deficiency also resulted in a significant reduction in FO, MZ, and peritoneal B1 B cells (23). Bcl10–/– B cells exhibited a severe defect in BCR-mediated proliferation, consistent with the finding that BCR-induced NF-{kappa}B activation is disrupted in these cells. MZ B, but not FO B, cells were impaired in their ability to respond to LPS in the absence of Bcl10. Most importantly, Bcl10–/– mice were unable to generate a successful humoral response to various Ags (22, 23).

Irrespective of these defects in the T and B cells, our study demonstrates that the lack of Bcl10 did not have any remarkable effect on the total number and the development of NK cells. We found that the expression of activating NKG2D, Ly49D, and inhibitory Ly49 receptors was normal in Bcl10–/– mice. Although NK and T cells have been proposed to share common intrathymic progenitors (7, 8), developmental requirements for the two lineages differ. Molecular requirements for NK cell development were found only in growth factor-signaling pathways, whereas T cell maturation requires not only intact cytokine signaling, but also pre-TCR- and TCR-mediated signal transductions. Activation through IL-2R-{gamma} by IL-15 plays a critical step in early NK cell development (49). Targeted gene disruption of IL-15 or its transcription factor, IFN regulatory factor-1 resulted in the severe reduction in NK cell population (50). Treatment of IRF-1–/– mice with IL-15 restored NK cell development by activating through its receptor IL-2R-{gamma} (20, 21). Along these lines, IL-2R-{gamma}–/– mice also exhibited severe NK cell development (51). Our present data indicating the normal NK cell development in Bcl10–/– mice suggests that Bcl10 does not participate in the IL-15-mediated signaling pathway that has been shown to be critical for NK cell development.

One of the major effector functions of the NKG2D or Ly49D receptor is to mediate cytotoxicity against tumor and virally infected cells. NKG2D recognizes H60, Rae-1, and Mult-1 in mice (4, 5, 6, 24), while Ly49D interacts with classical MHC class I, H2-Dd (2, 52). Our data demonstrates that the lack of Bcl10 affected the ability of the fresh NK cells to mediate cytotoxicity by activations via NKG2D. The reduced cytotoxic potential of fresh NK cells to recognize induced-self could be due to a developmental defect of NK cells. It is also possible that the perforin/granzyme pathway is in part dependent on the Bcl10-mediated pathway in fresh NK cells. Nevertheless, lack of Bcl10 did not affect the ability of the IL-2-activated NK cells to mediate cytotoxicity. These observations are of importance because fresh NK cells are known to express only NKG2D/DAP10 complexes (36). In contrast, IL-2-activated NK cells express both NKG2D/DAP10 and NKG2D/DAP12 complexes (12). There are two possible explanations for our observations. First, DAP12-Syk/Zap70 recruits adaptor proteins such as Vav1, 2, 3 that leads to the activation of ERK, a key regulator of cytotoxic granule release (53). Second, the DAP10-PI3K-mediated pathway can independently enforce the NKG2D-mediated target cell lysis via Grb2/Vav-1 recruitment that results in ERK activation (Fig. 7). Our observation implies that the Bcl10-associated activation pathway is not responsible for regulating the ERK-mediated cytotoxic granule release in IL-2-cultured NK cells.

Apart from mediating cytotoxicity, NK cells also generate a large array of inflammatory cytokines such as IFN-{gamma}, GM-CSF, TNF-{alpha}, lymphotoxin, and chemokines such as CCL1, CCL4, MIP-1{alpha}, MIP-1beta, and RANTES (39). Gene transcriptions of many of these soluble mediators are regulated by NF-{kappa}B. If this is the case, does Bcl10 regulate the generation of these cytokines and chemokines? In T and B cells, AgR-mediated activation of PLC{gamma} generates 1,2-diacylglycerol (DAG), which activates PKC leading to the recruitment of Carma1 and Bcl10 to the immunological synapse (54). Bcl10 subsequently promotes the oligomerization of Malt1 (55, 56) that binds to TRAF6, an ubiquitin E3 ligase. TRAF6 activation catalyzes the poly ubiquitination of NF-{kappa}B essential modulator (NEMO)/IKK{gamma}, the regulatory subunit of IKK. NEMO facilitates the recruitment of the IKK to the kinase complex, which allows the phosphorylation of IKKbeta leading to NF-{kappa}B activation. Earlier studies using knockout mice for specific members of NF-{kappa}B family demonstrated that only the lack of c-Rel reduced the ability of NK cells to generate IFN-{gamma} and lack of p50 (NF-{kappa}B1) did not (57). Our earlier studies have demonstrated that NKG2D uses PLC{gamma}2 as a critical regulator of downstream signaling events for mediating NK cell effector functions (32). NK cells lacking PLC{gamma}2 were also severely impaired both in their ability to generate cytokines and mediating cytotoxicity. Therefore, we propose that NKG2D-mediated signaling also uses the Carma1-Bcl10 pathway for the nuclear translocation of NF-{kappa}B and thereby successful generation of cytokines and chemokines.

NKG2D recruits two distinct adaptor proteins to transduce activation signals (10, 11, 12). One of these adaptor proteins DAP10 with a YINM motif associates with a longer isoform of NKG2D and recruits PI3K (13). The second adaptor molecule DAP12 with an ITAM associates with the shorter isoform of NKG2D and triggers Syk family PTK, Syk, and Zap70 (15). In contrast, Ly49D uses only DAP12 adaptor protein to transduce activation signals (17). Recent genetic studies suggest that DAP10-PI3K and DAP12-Syk/Zap70 pathways redundantly regulate cytotoxicity (13, 16) whereas DAP12-Syk/Zap70 pathway exclusively responsible for NKG2D-mediated cytokine production (14). Absence of DAP10 (15, 24), DAP12, or Syk/Zap70 (16), did not impair the ability of NK cells to mediate cytotoxicity. However, lack of DAP12 or Syk/Zap70 affected cytokine generation in addition to cytotoxicity (16). Our current results obtained using pharmacological inhibitors that block the function of PKC and NF-{kappa}B further confirm the role of the Carma1/Bcl10 pathway in cytokine generation in NK cells. Although it is well-established that PKC-beta in BCR and PKC-{theta} in TCR-mediated activations are responsible for phosphorylating Carma1, the particular PKC isoform that mediate this downstream of NKG2D is yet to be defined (58, 59). Based on these results, we conclude that the Carma-1/Bcl10 pathway is a major signaling pathway downstream of NKG2D/DAP12 or Ly49D/DAP12 complexes for cytokine/chemokine generation.

These previous and present observations need further in-depth analyses. Function of Bcl10 depends on the upstream activation of Carma1 by PKC (22, 60). In T cells, activation of PKC can occur either through Syk/Zap70->PLC{gamma}->DAG->PKC or by PI3K-> PDK->PKC pathways (40, 58, 61). However, earlier studies have shown only the NKG2D/DAP12 complexes were exclusively responsible for the cytokine generation implying that the Syk/Zap70->PLC{gamma}->DAG->PKC pathway is responsible for the initiation of the Carma1/Bcl10-signaling cascade (16). Reasons for the failure of NKG2D/DAP10-mediated the PI3K->PDK-> PKC pathway to activate Carma1/Bcl10 cascade is currently not clear. Yet, reduction in the ability of Bcl10–/– fresh NK cells to mediate cytotoxicity indicates a partial role for this signaling protein in granule release. It is also important to point out that the human NKG2DR uses only DAP10 and not DAP12 adaptor proteins (11). In this context, it is important to note that studies by Sutherland et al. (62), illustrated that pharmacological agents specific for PI3K blocked the ability of human NK cells to generated IFN-{gamma}. How the NKG2D/DAP10/PI3K pathway connects to the Carma1/Bcl10 pathway in NK cells is yet to be determined. Thus, future work should be focused on the downstream events of NKG2D/DAP10 complexes and distinct the molecular mechanisms in human and murine NK cells responsible for cytotoxicity and cytokine generation.

Similar to our current observations with Bcl10, earlier studies have documented a critical function for CD45 in exclusively regulating cytokine generation from NK cells (40, 63). CD45 is a transmembrane protein tyrosine phosphatase. CD45 dephosphorylates COOH terminal of Src family kinases, leading to a conformational modification in these kinases that is a requisite for their autophosphorylation of a catalytic tyrosine (64, 65). NK cells lacking CD45-mediated normal levels of cytotoxicity against a variety of tumor targets including YAC-1 (H60+), CHO (H2-Dd+), RMA/S (MHC class I deficient), and RMA cells stably transfected with Rae-1beta (63, 66, 67). However, CD45–/– NK cells were severely impaired in their ability to generate IFN-{gamma}, GM-CSF, MIP-1{alpha}, MIP-1beta, and RANTES (63, 68). Recently, Mason et al. (67) observed a high basal level DAP12 phosphorylation demonstrating that DAP12 itself could be a substrate of the phosphatase activity of CD45. These studies equivocally support the hypothesis that the signaling events for cytotoxicity and cytokine generation can be separated. Based on these observations and our current results, we conclude that CD45 regulates upstream signaling events by activating Src family kinases (or DAP12 itself), while Bcl10 represents a downstream component of the same activation pathway. Therefore, we conclude that CD45 helps in the successful activation of NKG2D/DAP12 complexes either direct or indirect mechanisms. This leads to the recruitment and activation of PLC{gamma}2 resulting in the generation of secondary messengers IP3 and DAG. DAG in turn activates PKC, which is a critical player in the initiation of the Carma1/Bcl10 signaling cascade and eventual activation and nuclear translocation of NF-{kappa}B is responsible for the transcription and generation of cytokines and chemokines. However, as indicated in this current study, cytotoxicity does not depend on the Carma1/Bcl10 pathway and as demonstrated by other studies may depend on the Vav-1-mediated activation of MAPK such as JNK, ERK, and p38 (16, 69). A possible role for ERK and p38 in the generation of IFN-{gamma} upon activation through 2B4 receptors have also been recently demonstrated (70). However, their role in NKG2D-mediated cytokine generation has yet to be established.

In conclusion, Bcl10 is a critical connecting link between the NKG2D- or Ly49D-associated DAP12-Syk/Zap70 complexes to the downstream cytokine gene transcriptions. The signaling pathway for cytokine and chemokine generation in NK cell is via PLC{gamma}2->DAG->PKC->Carma1->Bcl10->Malt1 leading to the activation and nuclear translocation of NF-{kappa}B. Our study provides strong evidence that the divergence of receptor-mediated cytokine generation and cytotoxicity in NK cells occurs upstream of Bcl10. Thus, Bcl10 has exclusive and nonredundant functions during the innate immune responses.


   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 H.C. is the recipient of Breast Cancer Show House Postdoctoral Fellowship, from the Cancer Center of the Medical College of Wisconsin. This work was supported in part by American Cancer Society Scholar Grants RSG-02-172-LIB (to S.M.) and RSG CCG-106204 (to D.W.), Roche Organ Transplantation Research Foundation Grant 111662730 (to S.M.), and National Institutes of Health Grants R01 A1064826-01, U19 AI062627-01, NO1-HHSN26600500032C (to S.M.), R01 AI52327 (to R.W.), and R01 HL073284 (to D.W.). Back

2 Address correspondence and reprint requests to Dr. Subramaniam Malarkannan, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226; E-mail address: subra.malar{at}bcw.edu or Dr. Demin Wang, Blood Research Institute, Milwaukee, WI 53226; E-mail address: demin.wang{at}bcw.edu Back

3 Abbreviations used in this paper: MHC I, MHC class I; PTK, protein tyrosine kinase; Malt, mucous-associated lymphoid tissue lymphoma; IKK, I{kappa}B kinase; FO, follicular; MZ, marginal zone; WT, wild type; PKC, protein kinase C; BM, bone marrow; PLC{gamma}, phospholipase C{gamma}; 7-AAD, 7-aminoactinomycin D; DAG, 1,2-diacylglycerol. Back

Received for publication December 21, 2006. Accepted for publication July 9, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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