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The Journal of Immunology, 2005, 175: 749-754.
Copyright © 2005 by The American Association of Immunologists

Phospholipase C-{gamma}2 Is a Critical Signaling Mediator for Murine NK Cell Activating Receptors1

Ilaria Tassi*, Rachel Presti*, Sungjin Kim{dagger}, Wayne M. Yokoyama{dagger}, Susan Gilfillan* and Marco Colonna2,*

* Department of Pathology and Immunology, and {dagger} Department of Medicine and Howard Hughes Medical Institute, Washington University School of Medicine, St Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Phospholipase C-{gamma} (PLC{gamma}) is a key regulator of intracellular Ca2+ mobilization. Two isoforms of PLC{gamma} have been identified, PLC{gamma}1 and PLC{gamma}2. Previously, in vitro studies indicated that activating NK cell receptors signal through both isoforms. However, PLC{gamma}2 deficiency alone was sufficient to induce a substantial impairment of NK cell-mediated cytotoxicity in vitro. Why PLC{gamma}2 is more important than PLC{gamma}1 for NK cell activation and whether PLC{gamma}2 is also critical for NK cell development, secretion of IFN-{gamma}, and clearance of viral infections in vivo is not known. In this study, we report that PLC{gamma}2 is the predominant isoform expressed in murine NK cells. PLC{gamma}2 deficiency did not affect NK cell numbers in bone marrow and spleen, but acquisition of Ly49 receptors by NK cells was partially impaired. PLC{gamma}2-deficient NK cells exhibited a dramatic impairment of cytolytic function and IFN-{gamma} production upon ligation of activating receptors, whereas they did secrete IFN-{gamma} in response to cytokines. Consequently, mice lacking PLC{gamma}2 controlled murine CMV infection substantially less effectively than did wild-type animals, and this defect was most evident in the spleen, where viral clearance mostly depends on NK cell lytic function. These results demonstrate that PLC{gamma}2 is crucial for development of the NK cell receptor repertoire and signaling of activating NK cell receptors, mediating optimal NK cell function in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer cells express multiple activating cell surface receptors with different specificities that deliver intracellular signals through distinct signaling adaptor proteins (1, 2, 3, 4). One major signaling pathway is initiated by the cytoplasmic ITAMs present in several adaptor proteins. In mouse, these include the {gamma}-chain of FcRs (FcR{gamma}), which mediates signaling for the low-affinity receptor for IgG (Fc{gamma}RIIIA), among others. Another ITAM-containing adapter protein, death-associated protein 12 (DAP12)3/killer cell-activating receptor-associated proteins, associates with a number of activating receptors, including Ly49D and Ly49H, which recognize the Chinese hamster MHC class I molecule Hm1-C4 (5) and the murine CMV (MCMV)3-encoded class I-like molecule m157 (6, 7), respectively. Engagement of these receptors results in tyrosine phosphorylation of the associated adaptors’ ITAMs, which recruit the protein tyrosine kinase Syk (1, 2, 3, 4, 8). This kinase activates multiple downstream signaling mediators, including linker for activation of T cells, SH2 domain-containing leukocyte protein of 76 kDa, c-Abl-SH3 domain-binding protein-2, phospholipase C-{gamma} (PLC{gamma}), PI3K, Vav family guanine nucleotide exchange factors, the low-m.w. GTP-binding protein Rho-Rac, and the Erk kinases. Collectively, these signaling mediators trigger gene transcription and the cellular programs for exocytosis of lytic granules that allow NK cells to lyse target cells and produce proinflammatory chemokines and cytokines, particularly IFN-{gamma}.

Another NK cell activating receptor, NKG2D, mediates "natural cytotoxicity" against tumor cells that, like YAC-1, express the NKG2D ligands Rae1 and murine UL16-binding protein-like transcript 1 (9, 10). NKG2D associates with a unique transmembrane adapter protein, DAP10 (11), which lacks cytoplasmic ITAMs but contains a YINM motif that binds the p85 regulatory subunit of PI3K (11, 12) and Grb2 (12). DAP10 couples engagement of NKG2D with a Syk-independent pathway that involves a more restricted array of downstream mediators than do the ITAM-mediated pathways (13). However, these mediators—PI3K, SH2 domain-containing leukocyte protein of 76 kDa, Vav1, Rho family GTPases, and PLC{gamma}—are sufficient for triggering release of lytic granules and inducing some IFN-{gamma} production. In mouse, NKG2D exists as two isoforms with distinct cytoplasmic domains that mediate differential association with adaptors: the long isoform (NKG2D-L), associates only with DAP10, whereas the short isoform (NKG2D-S) associates with both DAP10 and DAP12 (14, 15). The relative predominance of NKG2D-L/DAP10 and NKG2D-S/DAP10-DAP12 signaling varies with NK cell activation (14, 15). NKG2D-L/DAP10 is particularly evident in NK cells isolated ex vivo and after prolonged culture with IL-2, whereas NKG2D-S/DAP10-DAP12 prevails early after IL-2 activation.

A common signaling mechanism shared by the ITAM and DAP10-mediated pathways is modulation of intracellular calcium ion concentration (Ca2+), which is necessary for reorientation of microtubules and the actin cytoskeleton, exocytosis of lytic granules, and transcriptional activation of cytokine genes. Intracellular Ca2+ is primarily regulated by PLC{gamma}, which hydrolyzes membrane phosphatidylinositols into diacylglycerol and inositol 1,4,5-trisphosphate (16, 17, 18). Inositol 1,4,5-trisphosphate triggers the mobilization of Ca2+ from internal stores, resulting in a transient intracellular Ca2+ flux. This is followed by sustained Ca2+ influx from the extracellular medium through Ca2+-permeable channels in the plasma membrane. Two isoforms of PLC{gamma}, PLC{gamma}1 and PLC{gamma}2, have been identified. PLC{gamma}1 is ubiquitously expressed, whereas PLC{gamma}2 is predominantly expressed in hemopoietic cells (19). Activation of PLC{gamma}1/2 is mediated by PI3K, which phosphorylates the inositol ring of phosphatidylinositides in D3, generating, among others, phosphatidylinositol 3,4,5-trisphosphate (20, 21, 22). Phosphatidylinositol 3,4,5-trisphosphate binds the pleckstrin homology domain of PLC{gamma} and other proteins, including the Tec family kinases, promoting their recruitment to the cell membrane and consequent activation. Tec kinases, as well as Src and Syk kinases, further contribute to PLC{gamma} activation and Ca2+ mobilization by phosphorylating PLC{gamma} (20, 21, 22).

Early biochemical studies in human NK cells demonstrated that engagement of Fc{gamma}RIIIa results in tyrosine phosphorylation and activation of both PLC{gamma}1 and PLC{gamma}2 (23, 24, 25, 26). Later studies in the rat NK cell line RNK-16 showed that Ly49D activates PLC{gamma}1 and induces Ca2+ mobilization (8). Moreover, cross-linking of NKG2D/DAP10 in human NK cells triggers phosphorylation of PLC{gamma}2, whereas pharmacological blockade of PLC{gamma} inhibits NKG2D-mediated killing (13). Cumulatively, these in vitro studies suggest that NK cell activating receptors use both PLC{gamma}1 and PLC{gamma}2. However, analysis of PLC{gamma}2-deficient mice revealed substantial impairment of Fc{gamma}RIIIa-mediated cytotoxicity as well as a significant reduction in "natural cytotoxicity" against the classical NK cell target YAC-1, despite the presence of intact PLC{gamma}1 (27). This suggests that, at least in mice, PLC{gamma}2 is more important than PLC{gamma}1 for NK cell activation. Alternatively, PLC{gamma}2 may have a major role in some signaling pathways, whereas others are regulated by PLC{gamma}1. A precedent for the differential requirement of related isoforms in NK cell signaling was recently reported for Vav (28). Moreover, although previous reports indicate that PLC{gamma} is required for optimal NK cell-mediated cytotoxicity in vitro, it is unclear whether PLC{gamma} is necessary for NK cell development or other NK cell functions, particularly secretion of IFN-{gamma}. Finally, whether PLC{gamma} is required for NK cell function in vivo is not known. To address these questions, we investigated NK cell development in PLC{gamma}2-deficient mice as well as signaling via the ITAM- and DAP10-mediated pathways in PLC{gamma}2-deficient NK cells both in vitro and in vivo. Our results demonstrate that PLC{gamma}2 is the predominant PLC{gamma} isoform in murine NK cells. PLC{gamma}2 is not required for the development of normal NK cell numbers in bone marrow and spleen, but does play a role in the acquisition of NK cell receptor repertoire, particularly the Ly49 receptors. PLC{gamma}2 is essential for both ITAM- and DAP10-mediated pathways that trigger cytotoxicity and IFN-{gamma} secretion. In contrast, PLC{gamma}2 is not necessary for NK cell secretion of IFN-{gamma} in response to cytokines. Hence, mice lacking PLC{gamma}2 control MCMV infection less effectively than do wild-type (WT) animals, particularly in the spleen, where viral clearance predominantly depends on NK cell-mediated cytotoxicity.


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

PLC{gamma}2–/– mice (27) on a C57BL/6 background were kindly provided by J. Ihle (St. Jude Children’s Research Hospital, Memphis, TN). C57BL/6 mice were purchased from Taconic Farms.

Antibodies

Fluorochrome-conjugated mAbs included the following: allophycocyanin-anti-NK1.1 (PK136); PerCP-Cy5.5-anti-CD3 (145-2C11); PerCP-Cy5.5-anti-CD19 (1D3); PE-Cy5-anti-TCR{beta} (H57-597); PE-anti-IFN-{gamma} (XMG1.2); PE-anti-Ly49E/C (4D12); PE-anti-Ly49F (HBF-719); FITC-anti-Ly49I (YLI-90); and FITC-anti-NKG2A/C/E (20d5). All these Abs were from BD Pharmingen PE-anti-NK62D (Cx5) was purchased from ebioscience. PE-anti-Ly49H (3D10) and FITC-anti-Ly49A (JR9) and FITC-anti-Ly49D (4E4) have been previously described (7, 29). We also used PE-anti-CD43 (S7); FITC-anti-Mac-1 (m1/70); PE-anti-IL-2/IL-15R{beta} (CD122, TM-{beta}1); PE-anti-2B4 (CD244); and PE-anti-c-Kit (2B8) from BD Pharmingen (data not shown).

Cell preparation and flow cytometry

Single-cell suspensions were prepared from bone marrow and spleen. For flow cytometry, cells were first incubated with mAb 2.4G2 (American Type Culture Collection) to block nonspecific Ab binding and then stained with combinations of the indicated fluorochrome-conjugated mAbs. Stained cells were analyzed with a FACSCalibur (BD Biosciences). In some experiments, dead cells were excluded by staining with propidium iodide.

Biochemical analysis

WT NK cells were cultured 7 days in IL-2 and used for Western blot analysis. The cells were lysed in lysis buffer (1% Triton X-100, 50 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM EGTA, and 1.5 mM MgCl2) and protease inhibitors (5 mM NaF, 1 mM sodium orthovanadate, 10 µg/ml aprotinin, 4 µg/ml leupeptin, and 1 mM PMSF). After 15 min on ice, lysates were centrifuged for 15 min at 14,000 rpm, and supernatants were analyzed by SDS-PAGE, followed by immunoblotting with Abs specific for PLC{gamma}1 (sc-81) and PLC{gamma}2 (sc-407) (Santa Cruz Biotechnology).

Cytotoxicity assays

Murine NK cells were purified from spleens with DX5 microbeads (Miltenyi Biotec), yielding >90% NK1.1+CD3– cells. Purified NK cells were cultured for different intervals in IL-2 and tested against target cells (YAC-1, RMA-S, RMA-S-Rae1{gamma}, CHO, Baf/3, Baf/3-Hm1-C4, Baf/3-m157, EL4) by standard 4-h chromium release assay as previously described (28). Cytotoxicity was blocked with anti-Ly49D (4E5; BD Pharmingen) or anti-NKG2D Abs. To measure Ab-dependent cellular cytotoxicity, EL-4 cells were incubated with anti-Thy1.2 mAb (30H12) before cytotoxicity.

Induction and detection of IFN-{gamma}

To induce IFN-{gamma} in vitro, spleen cells (107) from untreated mice were incubated in six-well plates coated with mAb against NK1.1, Ly49D, NKG2D (2–5 µg/ml) (A10) (29) for 1 h, and then further incubated in the presence of brefeldin A (Sigma-Aldrich) for an additional 5–8 h. Alternatively, spleen cells were incubated with YAC-1 cells (10:1 ratio) or IL-12 (1 ng/ml) plus IL-18 (10 ng/ml). Cells were then stained with allophycocyanin-anti-NK1.1 and FITC-anti-Ly49A to gate NK cells. In addition, cells were stained with PE-Cy5-anti-CD3, PE-Cy5-anti-CD19, and PE-Cy5-anti-TCR{beta} to exclude T and B cells. Stained cells were fixed with 2% paraformaldehyde and permeabilized in saponin buffer (5% FCS, 0.5% saponin, and 10 mM HEPES). Intracellular IFN-{gamma} was detected with PE-conjugated anti-IFN-{gamma} mAb.

Mouse infections and plaque assays

C57BL/6 and PLC{gamma}2–/– mice were housed in a biosafety level 2 facility at Washington University in accordance with all federal and university policies. MCMV (ATCC VR-1399) was grown and titered on NIH 3T12 fibroblasts (ATCC CCL 164). Salivary gland-passaged stocks (sgMCMV) were prepared by infecting BALB/c mice with 105 PFU of tissue culture-passaged MCMV, and salivary glands were harvested and homogenized in DMEM (Invitrogen Life Technologies) with 10% BCS (HyClone). Mice were infected with 5 x 105 PFU of sgMCMV i.p., and organs were harvested 3 days postinfection and frozen in 1 ml of DMEM at –80°C. Titers were determined by plaque assay on 3T12 fibroblasts. After mechanical disruption by bead-beating (BioSpec), serial 10-fold dilutions were made and plated in triplicate on 3T12 monolayers in six-well plates (Costar). After infection in minimal medium at 37°C for 1 h, monolayers were overlaid with 3 ml of 5% DMEM-0.5% Noble agar and incubated for 6 days (37°C, 5% CO2) with one overlay of 2 ml at 3 days. At 6 days, plates were overlaid with 2.5% neutral red in DMEM-0.5% Noble agar. Plaques were counted 12 h later. Statistical significance of differences in viral titers between groups of mice was determined by unpaired t test using the Prism software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Murine NK cells preferentially express PLC{gamma}2

Human NK cells express both the PLC{gamma}1 and PLC{gamma}2 isoforms (24, 25, 26). However, expression of the two PLC{gamma} isoforms in murine NK cells has not been determined. Therefore, we purified NK1.1+CD3– NK cells from splenocytes, cultured them in vitro for 7 days with IL-2, and assessed PLC{gamma}1 and PLC{gamma}2 expression by immunoblot analysis of cell lysates. Murine NK cells expressed high levels of PLC{gamma}2, whereas PLC{gamma}1 was barely detectable (Fig. 1). PLC{gamma}1 was present in other murine cells, including the pro-B cell Baf/3 and the mastocytoma cell P815, as well as in human NK cells and T cells (Fig. 1). We conclude that murine NK cells predominantly express PLC{gamma}2.



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  		FIGURE 1. Murine WT NK cells predominantly express the PLC{gamma}2 isoform. Analysis of PLC{gamma}1 (left panel) and PLC{gamma}2 (right panel) expression by immunoblot shows that PLC{gamma}2 is the major isoform expressed in murine WT NK cells. Each lane of the immunoblot was loaded with the same amount of protein. Human CD56+CD3– NK cells were purified by cell sorting from PBMC of a healthy donor and cultured in IL-2. Murine NK cells were purified from spleens with DX5 microbeads and cultured in IL-2.

 
Defective Ly49 repertoire in PLC{gamma}2–/– NK cells

PLC{gamma}2 is critical for normal development of B cells but is not required for T cell development (27). Whether PLC{gamma}2 deficiency affects NK cell development is not known. NK cells develop in the bone marrow through sequential stages that are distinguished by cell surface expression of various markers, including the IL-2 and IL-15 receptor common {beta} subunit (CD122), the cytokine receptor c-Kit, the integrin CD11b (Mac-1), the leukosialin CD43, and the receptors 2B4 (CD244), NKG2A-C-E, and Ly49s (30, 31, 32). Although NK cells were present in equivalent numbers in spleen and bone marrow of PLC{gamma}2–/– and WT mice (data not shown), NK cells expressing inhibitory and activating Ly49 receptors were significantly reduced in PLC{gamma}2–/– mice (Fig. 2). All of the other markers, including NKG2D, NKG2A/C/E, CD122, c-Kit, CD43, and CD11b, were expressed on similar proportions of NK cells and at comparable levels in PLC{gamma}2–/– and WT spleen and bone marrow NK cells (Fig. 2 and data not shown). Moreover, PLC{gamma}2–/– and WT NK cells exhibited normal levels of perforin and granzymes transcripts (data not shown). Thus, PLC{gamma}2 appears to be required only for NK cell acquisition of a normal Ly49 receptor repertoire. However, a partial impairment of Ly49 repertoire does not block NK cell development, functional maturation, and acquisition of other NK cell activating and inhibitory receptors.



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  		FIGURE 2. Ly49 receptors in PLC{gamma}2–/– NK cells. The percentages of NK cells expressing Ly49A, Ly49I, and Ly49D are partially reduced in PLC{gamma}2–/– mice. Total splenocytes of PLC{gamma}2–/– and WT mice were stained with Abs against NK1.1 to gate NK cells, anti-CD3, and -CD19 to gate out T and B cells, and Abs against various Ly49 receptors, NKG2A/C/E, and NKG2D. Plots represent expression of Ly49s, NKG2A/C/E, and NKG2D receptors in NK1.1+CD3–CD19– cells. PLC{gamma}2–/– and WT NK cells expressed comparable levels of CD122, CD11b, CD43, c-Kit, and 2B4 in spleen and bone marrow (data not shown).

 
Lack of PLC{gamma}2 impairs Fc{gamma}RIIIA-, DAP12-, and DAP10-mediated cytotoxicity

NK cells lacking PLC{gamma}2 do not effectively lyse Ab-coated target cells, which indicates that PLC{gamma}2 is required for Fc{gamma}RIIIA/FcR{gamma}-mediated signaling (27). Consistent with this report, we found that PLC{gamma}2–/– NK cells inefficiently lysed EL-4 lymphoma cells coated with an anti-Thy1.2 Ab (Fig. 3). To assess the role of PLC{gamma}2 in DAP12-mediated killing, we focused on Ly49D and Ly49H, both of which exclusively signal through DAP12. Ly49D mediates lysis of cells expressing the Chinese hamster class I molecules Hm1-C4, whereas Ly49H recognizes cells expressing the MCMV class I-like molecule m157. The ability of PLC{gamma}2–/– NK cells to lyse CHO cells expressing Hm1-C4, Baf/3 cells transfected with either Hm1-C4 or m157 was dramatically reduced in comparison to WT NK cells (Fig. 3). Thus, PLC{gamma}2 is required for DAP12-mediated signaling. Moreover, because both FcR{gamma} and DAP12 contain a cytoplasmic ITAM, we conclude that PLC{gamma}2 is required for ITAM-mediated signaling.



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  		FIGURE 3. ITAM-mediated cytotoxicity in PLC{gamma}2–/– NK cells. Cytotoxicity of WT and PLC{gamma}2–/– NK cells after 7 days of culture in IL-2 against EL-4 lymphoma cells, CHO cells, and Baf/3 cells expressing Hm1-C4 (C4) or m157. NK cells were incubated with EL-4 in the presence or in the absence of an anti-Thy1.2 Ab to test Ab-mediated cytotoxicity. Deficiency of PLC{gamma}2 dramatically reduces NK cell mediated-cytotoxicity against all target cells.

 
To determine the role of PLC{gamma}2 in DAP10-mediated killing, we studied the function of NKG2D, which is the only NK cell receptor known to signal through DAP10. In mouse, NKG2D signals through DAP10 and DAP12. However, the relative involvement of DAP10 and DAP12 varies during NK cell culture with IL-2 (14, 15). NKG2D preferentially signals through DAP10 in NK cells isolated ex vivo. Early after IL-2 activation, NKG2D mainly signals through DAP12. After prolonged culture with IL-2, DAP10 signaling again becomes predominant. Given this, we analyzed NKG2D-mediated cytotoxicity in PLC{gamma}2–/– and WT NK cells ex vivo and at late time points during culture with IL-2. Target cells included YAC-1 cells as well as the class I-deficient RMA-S cells transfected with the NKG2D ligand Rae1{gamma} (RMA-S-Rae1{gamma}). Lack of PLC{gamma}2 severely reduced the ability of NK cells to lyse cells expressing NKG2D ligands, whether NK cells were isolated ex vivo or cultured for 14–20 days in IL-2 (Fig. 4). Thus, PLC{gamma}2 is absolutely required for NKG2D/DAP10-mediated signaling. NKG2D-mediated lysis was also significantly reduced at day 7 of NK cell culture, confirming that DAP12 signaling requires PLC{gamma}2 (data not shown).



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  		FIGURE 4. NKG2D-DAP10-mediated cytotoxicity in PLC{gamma}2–/– NK cells. Cytotoxicity of WT and PLC{gamma}2–/– NK cells ex vivo or after 14 and 20 days of culture in IL-2 against YAC-1 (upper panels) or RMA-S and RMA-S-Rae1{gamma} (lower panels). Deficiency of PLC{gamma}2 reduces NK cell mediated-cytotoxicity ex vivo and at any time point of culture.

 
The significant defect in NK cell-mediated lysis observed in PLC{gamma}2–/– mice could be due to impairment of integrin signaling, resulting in a defect of NK cell-target cell adhesion. To test this possibility, we mixed WT and PLC{gamma}2–/– NK cells with YAC-1. After 20-min incubation at 37°C we measured formation of conjugates by two-color flow cytometry. Under these conditions, the conjugation frequency of PLC{gamma}2–/– NK cells with target cells was similar to that obtained with WT NK cells (data not shown). Thus, PLC{gamma}2–/– NK cells do not have an obvious defect in adhering to target cells.

NK cell secretion of IFN-{gamma} is partially dependent on PLC{gamma}2

NK cells produce IFN-{gamma} in response to a variety of stimuli, including engagement of activating receptors and exposure to proinflammatory cytokines secreted by macrophages and dendritic cells during viral infections (33). To examine the role of PLC{gamma}2 in NK cell secretion of IFN-{gamma}, we isolated splenocytes from PLC{gamma}2–/– and WT mice and stimulated them with plate-bound Abs specific for the activating receptors NK1.1, Ly49D, and NKG2D, which signal through distinct associated adaptors. NK1.1 signals through FcR{gamma}, as does Fc{gamma}RIIIA (34); Ly49D associates exclusively with DAP12 (1, 2), whereas NKG2D uses both DAP10 and DAP12 (14, 15). NK cells were also stimulated by conjugation with YAC-1 cells, which trigger NKG2D as well as-yet-undefined activating receptors. Assessment of IFN-{gamma} in NK cells by intracellular staining revealed that PLC{gamma}2–/– NK cells produced very little or no IFN-{gamma} in comparison to WT NK cells after each stimulation tested (Fig. 5). In contrast, incubation of splenocytes with both IL-12 and IL-18 effectively induced comparable amounts of IFN-{gamma} in PLC{gamma}2–/– and WT NK cells (data not shown). We conclude that PLC{gamma}2 is required for NK cell production of IFN-{gamma} induced by activating receptors, whereas cytokine-induced IFN-{gamma} production is independent of PLC{gamma}2.



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  		FIGURE 5. IFN-{gamma} production in PLC{gamma}2–/– NK cells. A, Splenocytes were activated in vitro with mAbs against NK1.1, Ly49D, and NKG2D coated on plastic. Alternatively, splenocytes were stimulated with YAC-1 cells as described in Materials and Methods. Plots represents intracellular staining of IFN-{gamma} gated on NK1.1+Ly49+/–CD3–CD19–TCR{beta}– cells within PLC{gamma}2–/– and WT splenocytes.

 
PLC{gamma}2 is required for the early control of MCMV infection

To investigate the role of PLC{gamma}2 in NK cell antiviral activity in vivo, we infected WT C57BL/6, PLC{gamma}2–/– mice, and PLC{gamma}2+/– littermates with MCMV and harvested spleens and livers 3 days postinfection. Viral titers in the spleens from PLC{gamma}2–/– mice were ~100-fold higher than in those from WT and PLC{gamma}2+/– mice, and this difference was highly significant (Fig. 6); viral titers in the liver were slightly higher in PLC{gamma}2–/– than in WT and PLC{gamma}2+/– mice, but this difference was not statistically significant (Fig. 6). Because spleen viral titers at this early time point of infection reflect the efficiency of NK cell lysis of MCMV-infected cells through the m157/Ly49H pathway (6, 7, 35, 36), PLC{gamma}2 most likely plays a significant role in NK cell control of viral replication by transducing Ly49H-mediated signals. Differences in liver viral titers in PLC{gamma}2–/– mice were not as impressive as those observed in the spleen, most likely because IFN-{gamma} primarily controls MCMV infection in the liver (37, 38) and PLC{gamma}2–/– NK cells can still secrete IFN-{gamma} in response to IL-12 and IL-18.



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  		FIGURE 6. PLC{gamma}2 deficiency increases susceptibility to early MCMV infection. WT C57BL/6, age-matched PLC{gamma}2–/– or PLC{gamma}2+/– mice were infected with 5 x 105 PFU of MCMV i.p. Three days postinfection, mice were sacrificed, and spleen and liver were harvested and titered by plaque assay. Viral titers in the spleens from PLC{gamma}2–/– mice were significantly higher than in those from WT and PLC{gamma}2+/– mice (p < 0.0001 and p < 0.001, respectively). Viral titers in the liver were not significantly higher in PLC{gamma}2–/– than in WT and PLC{gamma}2+/– mice. Titers of individual mice are shown. The figure represents one of three experiments with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our study shows that PLC{gamma}2 is the predominant PLC{gamma} isoform in murine NK cells, as it is in B cells. Although pro-B cell differentiation is blocked and mature B cells are reduced in numbers (27) in PLC{gamma}2–/– mice, PLC{gamma}2 is not required for NK cell expansion from bone marrow progenitors, because PLC{gamma}2-deficient mice harbored normal numbers of NK cells in spleen and bone marrow. Moreover, PLC{gamma}2-deficient cells expressed many markers indicative of maturation, including CD122, CD11b, CD43, c-Kit, NKG2D, NKG2A/C/E, perforin, and granzymes. However, we observed reduced percentages of NK cells expressing Ly49 receptors. Because acquisition of Ly49 receptors represents a relatively late event in the sequential process that leads to development of NK cells (39, 40, 41), a PLC{gamma}2-mediated signal may be important for promoting the transition to the final developmental stage, which involves complete acquisition of Ly49 receptors. NK cell receptors acquired at an earlier stage of development, such as NKG2D and NK1.1, could trigger this PLC{gamma}2-mediated pathway upon engaging cognate ligands on stromal cells.

Although PLC{gamma}2 has a limited role in NK cell development, NK cell effector functions largely require PLC{gamma}2. Both ITAM- and DAP10-coupled activating receptors substantially depend on PLC{gamma}2 activation as a common signaling mechanism to trigger NK cell-mediated cytotoxicity. Supporting this conclusion, we found that PLC{gamma}2 deficiency causes a remarkable reduction in both Ab-dependent cytotoxicity and "natural cytotoxicity" in vitro. The impaired cytotoxicity is not due to a defect in NK cell adhesion to target cells. Importantly, the defect in NK cell lysis is functionally significant in vivo; PLC{gamma}2-deficient mice control MCMV infection in the spleen 100-fold less efficiently than do WT mice. In the context of previous studies demonstrating involvement of both PLC{gamma}1 and PLC{gamma}2 in human and rat NK cell activation, the magnitude of the defect caused by lack of PLC{gamma}2 in murine NK cells was unexpected and is most likely explained by our observation that murine NK cells, unlike human NK cells, express PLC{gamma}2 almost exclusively, along with barely detectable amounts of PLC{gamma}1. These results document another significant difference between the human and mouse NK cell signaling pathways, supplementing the previously reported difference in NKG2D signaling (14, 15). Although PLC{gamma}2 is absolutely essential for NK cell-mediated cytotoxicity, it is possible that the minimal amounts of PLC{gamma}1 partially compensate for the lack of PLC{gamma}2, mediating a residual cytotoxicity observed in some experiments. Consistent with this, it has been reported that PLC{gamma}1 is capable of mediating a significant signaling function in T cells even after a 90% reduction (42). Murine NK cell functions may also partially rely on PLC{gamma}2-independent pathways for exocytosis of lytic granules, such as the MAPK-mediated pathways (43, 44).

Previous studies in human NK cells showed that Fc{gamma}RIIIA-mediated secretion of IFN-{gamma} is significantly dependent on intracellular Ca2+ mobilization, whereas cytokine-induced secretion of IFN-{gamma} is Ca2+ independent (45). Because PLC{gamma}2 is required for intracellular Ca2+ signaling, we expected significantly reduced IFN-{gamma} secretion by PLC{gamma}2–/– NK cells in response to engagement of activating receptors. Indeed, we found that PLC{gamma}2–/– NK cells did not produce IFN-{gamma} in comparison with WT NK cells following ligation of activating cell surface receptors. In contrast, in the presence of IL-12 and IL-18, NK cells produced IFN-{gamma} in a PLC{gamma}2-independent fashion. The partial maintenance of IFN-{gamma} secretion in PLC{gamma}2–/– mice together with the predominant role of IFN-{gamma} in controlling MCMV infection in the liver (37, 38) explain why liver viral titers are not significantly increased by PLC{gamma}2 deficiency.

In summary, we have shown that PLC{gamma}2 is a downstream mediator crucial for signaling of all activating NK cell receptors. This signaling pathway promotes acquisition of the Ly49 cell receptors repertoire, receptor-mediated cytotoxicity, and receptor-mediated IFN-{gamma} secretion and, ultimately, is required for optimal NK cell surveillance of MCMV infection in vivo.


   Acknowledgments
 
We thank James N. Ihle (St. Jude Children’s Research Hospital, Memphis, TN), Roberta Faccio (Washington University School of Medicine, St. Louis, MO) for PLC{gamma}2–/– mice.


   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 National Institutes of Health Grant 5R01AI056139-03. Animal work was performed in a mouse facility supported by NCRP Grant CO6RRO12466. Back

2 Address correspondence and reprint requests to Dr. Marco Colonna, Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: mcolonna{at}pathbox.wustl.edu Back

3 Abbreviations used in this paper: DAP, DNAX-activating protein; MCMV, murine CMV; PLC, phospholipase C; WT, wild type. Back

Received for publication January 12, 2005. Accepted for publication May 5, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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