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[Cancer Research 65, 9099-9107, October 1, 2005]
© 2005 American Association for Cancer Research

Immunology

Src Homology 2–Containing Inositol 5'-Phosphatase 1 Negatively Regulates IFN-{gamma} Production by Natural Killer Cells Stimulated with Antibody-Coated Tumor Cells and Interleukin-12

Robin Parihar1, Rossana Trotta2, Julie M. Roda3, Amy K. Ferketich4, Susheela Tridandapani2, Michael A. Caligiuri1,2 and William E. Carson, III1,5

Departments of 1 Molecular Virology, Immunology, and Medical Genetics and 2 Internal Medicine; 3 Integrated Biomedical Graduate Program; Departments of 4 Biostatistics and 5 Surgery, Arthur G. James Comprehensive Cancer Center and Solove Research Institute, Ohio State University, Columbus, Ohio

Requests for reprints: William E. Carson III, Ohio State University College of Medicine, N924 Doan Hall, 410 West 10th Avenue, Columbus, OH 43210. Phone: 614-293-6306; Fax: 614-688-4366; E-mail: carson-1{at}medctr.osu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that natural killer (NK) cells secrete a distinct profile of immunomodulatory cytokines in response to dual stimulation with antibody-coated tumor cells and interleukin-12 (IL-12). This NK cell cytokine response is dependent on synergistic signals mediated by the activating receptor for the Fc portion of IgG (Fc{gamma}RIIIa) and the IL-12 receptor (IL-12R), both constitutively expressed on NK cells. The phosphatase Src homology 2–containing inositol 5'-phosphatase 1 (SHIP1) is known to exert inhibitory effects on Fc receptor (FcR) signaling via its enzymatic activity on phosphatidylinositol 3-kinase (PI3-K) products within many cells of the immune system, most notably mast cells, B cells, and monocytes. However, its activity in the context of FcR activation on NK cells has not been fully explored. The current study focused on the regulation of Fc{gamma}RIIIa-induced NK cell cytokine production by SHIP1. Inhibitor studies showed that NK cell IFN-{gamma} production following FcR stimulation in the presence of IL-12 depended, in part, on the downstream products of PI3-K. Overexpression of wild-type (WT) SHIP1, but not a catalytic-deficient mutant, via retroviral transfection of primary human NK cells, resulted in a >70% reduction of NK cell IFN-{gamma} production in response to costimulation. In addition, NK cells from SHIP1–/– mice produced 10-fold greater amounts of IFN-{gamma} following culture with antibody-coated tumor cells plus IL-12 compared with NK cells from WT mice. Further, activation of the mitogen-activated protein kinase (MAPK) family member extracellular signal-regulated kinase (Erk; a downstream target of PI3-K) was significantly enhanced within SHIP1–/– NK cells compared with WT NK cells following costimulation. Pharmacologic inhibition of Erk activity, but not Jnk MAPK activity, led to significantly decreased IFN-{gamma} production from both SHIP1–/– and WT NK cells under these conditions. These results are the first to show a physiologic role for SHIP1 in the regulation of NK cell cytokine production and implicate PI3-K in the induction of MAPK signal transduction following costimulation of NK cells via the FcR and the IL-12R.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer (NK) cells are large granular lymphocytes that participate in the innate immune response to virally infected and malignant cells (1). NK cells typically recognize target cells via NK cell cytotoxicity receptors that recognize MHClow or MHCnegative cells or nonclassic MHC molecules (e.g., MICA/B, ULBP, etc.). NK cells can also interact with antibody-coated target cells via their constitutive and abundant expression of an activating, low-affinity receptor for the Fc portion of IgG, known as Fc{gamma}RIIIa or CD16 (2). NK cells are uniquely equipped to mediate antibody-dependent effector functions because they contain abundant cytolytic granules, prominently express cellular adhesion molecules, and rapidly secrete large quantities of immunomodulatory cytokines following activation (1). These properties provide NK cells with the ability to directly lyse cellular targets as well as coordinate the developing adaptive immune response.

Cross-linking of the Fc{gamma}RIIIa results in tyrosine phosphorylation of the intracellular immunoreceptor tyrosine-based activation motif (ITAM) by members of the Src kinase family, with subsequent recruitment of Src homology 2–containing signaling molecules that bind the phosphorylated ITAM, most notably the Syk kinase family of molecules (3). Subsequent signaling events include the activation of phosphatidylinositol 3-kinase (PI3-K), the enzymatic activity of which leads to production of phosphatidylinositol-3,4,5-trisphosphate (PIP3) and recruitment of pleckstrin homology domain–containing molecules that further mediate positive signals downstream via phospholipases, protein kinase B (PKB), and PKC, and the Ras–mitogen-activated protein kinase (MAPK) pathway (4–9). Because most Fc receptor (FcR)-bearing immune cells coexpress both activating and inhibitory FcRs that bind IgG with comparable affinity and specificity, coengagement of activating and inhibitory FcRs by IgG or immune complexes usually occurs, thereby establishing thresholds for activation/repression and determining the magnitude of effector responses (10, 11). The majority of NK cells, however, do not routinely express inhibitory FcRs, and thus much less is known about inhibitory signals that regulate or attenuate NK cell effector responses following Fc{gamma}RIIIa engagement (12).

One of the key proteins that negatively regulates inhibitory FcR-dependent effects in monocytes, B cells, and mast cells is the Src homology 2–containing inositol 5'-phosphatase 1 (SHIP1; ref. 13). This is a hematopoietic cell-specific enzyme that cleaves the 5'-phosphate from the PI3-K product PIP3 to yield PI-3,4-P2. This reduces the ability of certain pleckstrin homology–containing proteins involved in mediating activation signals to target to the plasma membrane and become activated (14). Although SHIP1 has been studied extensively in the context of inhibitory FcRs (15, 16), its role in NK cell responses initiated by the activation of Fc{gamma}RIIIa is less well understood. Of note, Galandrini et al. (17, 18) have shown that Fc{gamma}RIIIa cross-linking on primary human NK cells induced transient translocation of SHIP1 into lipid raft microdomains, where it associated with the {zeta}-chain of the FcR complex and negatively regulated antibody-dependent cellular cytotoxicity (ADCC). Whereas interleukin 12 (IL-12) has long been known for its ability to stimulate NK cell ADCC, it was only recently that our group was able to show that IL-12 also has the ability to markedly enhance the NK cell cytokine response to immobilized IgG, both in preclinical studies and also in the context of a phase I clinical trial of IL-12 and an anti–breast cancer antibody (trastuzumab; refs. 19, 20). To date, however, the role of SHIP1 in regulating NK cell cytokine production, another critical NK cell effector function, has not been fully addressed.

In the current report, we have examined the role of SHIP1 in regulating cytokine production by primary NK cells undergoing Fc{gamma}RIIIa activation. We show that SHIP1 is rapidly phosphorylated following stimulation of primary human NK cells by Fc{gamma}RIIIa cross-linking in the presence of IL-12, suggesting recruitment of SHIP1 activity. Studies in which SHIP1 was overexpressed in primary NK cells and in vivo studies analyzing NK cells from SHIP1–/– mice confirmed the critical role for SHIP1 phosphatase activity in the inhibition of cytokine production by FcR-stimulated NK cells. Examination of NK cell intracellular signaling pathways by the combination of immunoblot analyses and inhibitor studies revealed that SHIP1 specifically regulated the activation of the MAPK extracellular signal-regulated kinase (Erk) 1/2, factors found to be essential for FcR-mediated NK cell cytokine production. These data provide a functional role for SHIP1 in the regulation of Fc{gamma}RIIIa-mediated cytokine production within human NK cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cytokines and antibodies. Recombinant human IL-12 (rhuIL-12) and recombinant murine IL-12 (rmuIL-12) were provided by Genetics Institute, Inc. (Cambridge, MA), and were resuspended in 1x PBS plus 0.1% bovine serum albumin. Anti-Fc{gamma}RIIIa antibody 3g8 was obtained from Medarex (Annandale, NJ). Rabbit polyclonal phospho-Erk, phospho-SHIP1, and total SHIP1 antibodies were purchased from Cell Signaling Technologies, Inc. (Beverly, MA). Total Erk antibody was purchased from Santa Cruz, Inc. (Santa Cruz, CA).

Isolation of human and murine natural killer cells. NK cells were isolated directly from fresh peripheral blood leukopacs (American Red Cross, Columbus, OH) by 30-minute incubation with RossetteSep cocktail (Stem Cell Technologies, Vancouver, BC) followed by Ficoll Hypaque density gradient centrifugation. Isolated NK cells were >96% CD56+ by fluorescence-activated cell sorting (FACS) analysis. Human NK cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated pooled human AB serum (HAB; C-six Diagnostics; Germantown, WI), 100 units/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (10% HAB medium). Murine NK cells were isolated from splenocyte populations of both wild-type (WT) and SHIP1–/– mice. Splenocytes were isolated via mechanical digestion of spleens followed by lysis of RBC. Splenocytes were then incubated with phycoerythrin-labeled anti-DX5 and anti-NK1.1 antibodies (BD PharMingen, San Diego, CA) for 30 minutes at 4°C. Cells were washed and incubated with an antiphycoerythrin antibody-coated magnetic microbeads (Miltenyi Biotech, Auburn, CA) for an additional 30 minutes at 4°C according to the instructions of the manufacturer. NK cells were isolated via positive selection through an LS+ magnetic column (Miltenyi Biotech). Isolated murine NK cells were >95% DX5+/NK1.1+ by FACS analysis.

In vitro costimulation assay. For in vitro costimulation experiments, wells of a 96-well flat-bottomed plate were coated with huIgG or muIgG (for stimulation of murine splenocytes) by incubation with 100 µg/mL of IgG in cold PBS overnight at 4°C. Plates were then washed and human NK cells, murine splenocytes, or purified murine NK cells were plated at 2 x 105 cells/well with IL-12 as previously described (19). At the indicated time points, cell-free culture supernatants were harvested and analyzed for levels of human IFN-{gamma} (Endogen, Inc, Rockford, IL) or murine IFN-{gamma} (R&D Systems, Minneapolis, MN) by ELISA. In the signaling inhibition experiments, NK cells were pretreated for 8 hours before addition into IgG-coated wells with 0 (DMSO vehicle control), 1, 10, or 25 µmol/L of specific inhibitors of signal transduction molecules: Erk (U0126 or PD098059) or Jnk [Jnk inhibitor, anthra[1,9cd]pyrazol-6(2H)-1,9-pyrazolo anthrone], both from Calbiochem (La Jolla, CA).

FcR clustering and Western blotting. Primary human NK cells and transfected human NK cells were activated by clustering Fc{gamma}RIIIa with F(ab')2 fragments of monoclonal antibody (mAb) 3g8 and goat F(ab')2 antimouse Ig secondary antibody, in the presence of 10 ng/mL huIL-12. Murine NK cells were similarly activated by clustering Fc{gamma}RIIIa with mAb 24G.2 and goat F(ab')2 anti-rat Ig secondary antibody in the presence of 10 ng/mL muIL-12. Resting and activated human NK cells were lysed in TN1 buffer [50 mmol/L Tris (pH 8.0), 10 mmol/L EDTA, 10 mmol/L Na4P2O7, 10 mmol/L NaF, 1% Triton X-100, 125 mmol/L NaCl, 10 mmol/L Na3VO4, 10 µg/mL each of aprotinin and leupeptin]. These whole cell lysates were incubated on ice for 30 minutes to clear the nuclear fraction, centrifuged and postnuclear lysates were then boiled in SDS sample buffer [60 mmol/L Tris (pH 6.8), 2.3% SDS, 10% glycerol, 0.01% bromphenol blue, and 1% 2-mercaptoethanol] for 5 minutes before SDS-PAGE. For determination of Erk activation, 5 x 104 WT or SHIP1–/– NK cells were directly lysed by boiling in 20 µL of SDS sample buffer. Proteins were separated by 8% SDS-PAGE, transferred to nitrocellulose filters, probed with the primary antibody of interest, and developed by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).

Mice. Female SHIP1–/– (C57BL/6 background) mice and WT littermates were a kind gift from Dr. G. Krystal (Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada; ref. 21). All mice were utilized in experiments at 4 to 6 weeks of age. All SHIP1–/– and WT mice used within individual experiments were age- and sex-matched and derived from the same litter. However, mice from different experiments may have come from different mouse litters and may have differed in age as well as their response to IL-12 plus immobilized IgG. Mice were maintained in the animal facility at Ohio State University Heart and Lung Research Institute with free access to food and water. All protocols were approved by Ohio State University Comprehensive Cancer Center Animal Care and Use Committee and mice were treated in accordance with the institutional guidelines for animal care.

Retroviral transfer of SHIP1 constructs. The PINCO retroviral vector contains full-length Maloney long terminal repeats and carries the green fluorescent protein (GFP) cDNA under the control of the cytomegalovirus promoter (22). PINCO WT SHIP1 retroviral vector and pTRE-D675A-SHIP1 vector were kindly provided by Dr. Martin Sattler (Dana-Farber Cancer Institute, Boston, MA) and Dr. Larry R. Rohrschneider (Fred Hutchinson Cancer Research Center, Seattle, WA; refs. 23, 24). To generate a PINCO-D675A SHIP1 vector, the pTRE-D675A-SHIP1 plasmid was EcoRI-digested and the fragment containing the D675A SHIP1 mutant was subcloned into the EcoRI sites of the PINCO vector. This PINCO vector was transiently transfected into the viral packaging cell line, Pheonix, by the calcium-phosphate chloroquine method (25). Culture supernatants containing the viral particles were collected after 24 hours. Target cells (primary human NK cells isolated as described above) were infected with viral particles by culture with the viral supernatants supplemented with 4 µg/mL polybrene (Sigma, St. Louis, MO) and 900 IU/mL huIL-2 (Roche Pharmaceuticals, Nutley, NJ), as described previously (26). The efficiency of infection was then evaluated by flow cytometry for GFP fluorescence. Although the efficiency was routinely between 5% and 15%, a >90% pure population of similarly infected cells (i.e., copy number) was acquired via cell sorting on the basis of the level of GFP expression. This infected population was viable and divided actively upon restimulation with low-dose IL-2. Recent work by our group has verified that GFP fluorescence within infected NK cells serves as an adequate surrogate for SHIP1 protein expression under the conditions of the PINCO viral transfection system (27). The phosphorylation status of transfected SHIP1 following stimulation of NK cells via the IL-12R or FcR was not determined. Because SHIP1 is phosphorylated following its translocation to the membrane, we cannot exclude the possibility that SHIP1 phosphorylation is important for IFN-{gamma} production induced by costimulation of the IL-12R and FcR.

Intracellular flow cytometry. The in vivo production of IFN-{gamma} by murine NK cells in response to antibody-coated tumor plus IL-12 costimulation was analyzed using a FITC-conjugated mAb to murine IFN-{gamma} (BD PharMingen) and phycoerythrin-conjugated mAbs to surface markers specific for murine NK cells (DX5 and NK1.1), as previously described (28). P815 murine tumor cells were incubated at 4°C in PBS plus 10% fetal bovine serum at 1 x 107 cells/mL with either rabbit anti-murine IgG (1 mg/mL) or normal murine control IgG (1 mg/mL) for 45 minutes. Cells were then washed twice in sterile PBS and 4 x 106 cells in PBS were given i.p. to each mouse concurrently with a separate i.p. injection of 1 µg muIL-12 in PBS. Each group consisted of 10 WT or SHIP1–/– C57BL/6 mice and control groups received injections of PBS, control IgG-treated tumor alone, control IgG-treated tumor plus IL-12, or rabbit anti-murine IgG-coated tumor alone. Serum was harvested from each mouse at 24 hours and analyzed for cytokine levels by ELISA. Mice were immediately euthanized, splenocytes were harvested as described above, and cultured for 6 hours in brefeldin-A in 96-well plates, permeabilized, and stained with phycoerythrin-labeled DX5 and NK1.1 antibodies (pan-NK) and FITC-labeled anti–muIFN-{gamma} mAb. Cells were analyzed by flow cytometry for levels of intracellular IFN-{gamma} within the NK cell and non-NK cell populations. The percentage of positively staining cells and mean specific fluorescence intensity (Fsp) were calculated for IFN-{gamma} within the specified cell populations.

Statistics. Statistical analyses of ELISA cytokine levels and percentage of specific lysis values were done using an exact Wilcoxon rank sum test to compare differences between the two experimental groups (WT NK cells versus SHIP1–/– NK) with P < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of phosphatidylinositol 3-kinase abrogates natural killer cell IFN-{gamma} production. We first wished to determine whether cytokine production by NK cells in response to Fc{gamma}RIIIa clustering in the presence of IL-12 was dependent on the activity of PI3-K. Pretreatment of primary human NK cells with two separate chemical inhibitors of PI3-K activity significantly reduced IFN-{gamma} production in a dose-dependent manner (Fig. 1). In contrast, an inhibitor of Jnk signaling had no effect on NK cell cytokine production in response to these dual stimuli (data not shown). These data suggested that the ability of NK cells to produce IFN-{gamma} in response to FcR stimulation in the presence of cytokines such as IL-12 depended, in part, on the downstream products of PI3-K.



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  		Figure 1. Inhibition of PI3-K abrogates NK cell IFN-{gamma} production. NK cells were pretreated with increasing concentrations of two different biochemical inhibitors of PI3-K—wortmannin (A) and LY294002 (B)—and then cultured in the presence of immobilized IgG and IL-12 (10 ng/mL). Culture supernatants were harvested after 24 hours and analyzed for IFN-{gamma} content by ELISA. *P < 0.01 versus DMSO-treated condition.

 
SHIP1 is phosphorylated in primary natural killer cells. Having established a role for PI3-K signaling in NK cell IFN-{gamma} production, the phosphorylation of SHIP1 protein in primary NK cells was assessed following costimulation with a Fc{gamma}RIIIa cross-linking antibody and IL-12. Although phosphorylation of SHIP1 has not been shown to increase the enzymatic activity of this protein, it has been shown to be involved following the localization of SHIP1 to the plasma membrane where it mediates its enzymatic activity (29). Cross-linking of the NK cell FcR resulted in rapid and potent phosphorylation of SHIP1, whereas IL-12 stimulation alone resulted in lower levels of phosphorylation (Fig. 2). Although SHIP1 was rapidly activated in response to FcR cross-linking and IL-12 in every donor tested (n = 8), levels of phosphorylated SHIP1 in costimulated NK cells were either lower than those obtained in response to FcR cross-linking alone, or began returning to baseline more rapidly than the single stimulus condition (Fig. 2B). Thus, although phosphorylation of SHIP1 was actively induced in NK cells following Fc{gamma}RIIIa activation, costimulation with IL-12 did not seem to significantly enhance SHIP1 phosphorylation.



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  		Figure 2. SHIP1 is phosphorylated in primary NK cells after FcR stimulation. Purified human NK cells were activated by Fc{gamma}RIIIa cross-linking in the presence of IL-12 and for the indicated time points and (A) analyzed by immunoblot for phosphorylated (P)-SHIP1 and total SHIP1 protein. Control conditions consisted of control antibody-treated NK cells (–), NK cells activated via FcR cross-linking alone (FcR), or NK cells activated with IL-12 alone (IL-12). B, levels of P-SHIP1 were quantified by densitometry, normalized to levels of total SHIP1, and presented as fold increase in P-SHIP1 versus baseline for each stimulation condition. Results from one representative donor of eight examined are shown.

 
Overexpression of wild-type SHIP1 in primary human natural killer cells inhibits IFN-{gamma} production in response to Fc{gamma}RIIIa and interleukin-12 costimulation. The effect of SHIP1 overexpression on IFN-{gamma} production by primary human NK cells following costimulation with immobilized antibody and IL-12 was next examined. Overexpression of WT SHIP1 via retroviral transfection of primary human NK cells (Fig. 3A) resulted in a 73% reduction of NK cell IFN-{gamma} secretion in response to dual stimulation (Fig. 3B). In contrast, overexpression of a catalytic mutant form of SHIP1 did not result in a similar reduction, suggesting the importance of the catalytic activity of SHIP1 in its inhibitory effect on NK cytokine production. Of note, there may be alternate pathways by which SHIP1 can regulate cytokine production independently of its catalytic activity on PI3-K products. SHIP1 has been shown to recruit adaptor molecules (e.g., dok, csk, shc) that are able to exert inhibitory effects on monocytes and B cells following FcR engagement (13, 30, 31). However, the observation that NK cells transfected with the catalytic-inactive SHIP1 did not exhibit reduced IFN-{gamma} secretion compared with that of mock-transfected NK cells suggested that SHIP1 primarily regulates cytokine production within NK cells through its catalytic activity. Of note, overexpression of WT SHIP1 in human NK cells inhibited in vitro IFN-{gamma} production in response to FcR stimulation, but had no effect on IFN-{gamma} production by IL-12–activated NK cells. These results suggest that the phosphatase activity of endogenous SHIP1 in human NK cells is not saturated by the substrates that are generated in response to IL-12. Thus, the additional SHIP1 phosphatase activity that is achieved via transfection would not affect NK cell production of IFN-{gamma}. This does not seem to be the case for FcR-generated substrates; hence, the ability of overexpressed WT SHIP1 to significantly further inhibit FcR-mediated IFN-{gamma} secretion.



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  		Figure 3. Overexpression of WT SHIP1 in primary human NK cells inhibits IFN-{gamma} production in response to Fc{gamma}RIIIa and IL-12 costimulation. Primary human NK cells were transfected with WT human SHIP1 or a catalytic-inactive mutant of SHIP1 (D675A) using the PINCO retroviral transfection system. A, following infection, NK cells (>96% CD56+) were sorted based on expression of the reporter gene coexpressed with SHIP1, GFP. Shown are representative flow results (inset, percentage of CD56+ cells that are also GFP+) from an NK cell population before infection (top left) or following infection with the indicated constructs and sorting for GFP-expressing NK cells. B, equal numbers of sorted NK cells were cultured separately in the presence of immobilized IgG and IL-12. Culture supernatants were harvested after 24 hours and analyzed for IFN-{gamma} content by ELISA. *P < 0.001 versus vector-only control condition. Results shown are representative of three separate experiments.

 
Natural killer cells from SHIP1–/–mice exhibit enhanced IFN-{gamma} production and antibody-dependent cellular cytotoxicity activity. To further explore the role of SHIP1 in the negative regulation of NK cell function, mice deficient in SHIP1 protein were used (Fig. 4A; ref. 21). Spleen cells derived from both WT and SHIP1–/– mice were cultured separately with IL-12 on plates coated with immobilized IgG. Significantly enhanced levels of IFN-{gamma} secretion were observed from SHIP1–/– splenocytes compared with WT cells (Fig. 4B). Examination of the cellular source of IFN-{gamma} in the whole splenocyte cultures by intracellular flow cytometry revealed that over 51% of the SHIP1–/– NK cells were producing IFN-{gamma} compared with 18% of the WT NK cells at the same time point (Fig. 4C). When ex vivo purified NK cells from the whole splenocyte populations of these mice were utilized in the immobilized IgG assay, IFN-{gamma} secretion by SHIP1–/– NK cells was over 10-fold greater than that of their WT counterparts (Fig. 5A). In fact, there was a statistically significant increase in the IFN-{gamma} response in both the immobilized IgG alone and IL-12 alone stimulation conditions, suggesting a role for SHIP1 in both FcR and IL-12R signaling pathways in NK cells. Examination of ADCC activity mediated by WT and SHIP1–/– NK cells further confirmed the notion that SHIP1 may regulate components of both signaling pathways. In comparison to WT NK cells, SHIP1–/– NK cells exhibited enhanced ADCC activity against antibody-coated tumor cell targets, both in the presence and absence of IL-12 (Fig. 5B). Importantly, at the lower effector-to-target ratios, SHIP1–/– NK cells exhibited significantly greater ADCC activity following stimulation with IL-12. This finding is an extension of the work conducted by Galandrini et al. (17) because this group did not examine the role of SHIP1 in ADCC conducted by cytokine-activated NK cells.



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  		Figure 4. Splenocytes from SHIP1–/– mice exhibit enhanced IFN-{gamma} production. A, the absence of SHIP1 protein in splenocytes derived from SHIP1–/– mice was confirmed by immunoblot analysis. B, splenocytes were cultured in the presence of immobilized IgG and IL-12. Supernatants were harvested at 24 hours and analyzed for IFN-{gamma}. *P < 0.05 versus medium, IgG, and IL-12 stimulation conditions. The P value for the comparison of IFN-{gamma} levels from WT and SHIP1–/– splenocytes for the IgG + IL-12 stimulation condition is shown in the inset. C, splenocyte populations from WT and SHIP1–/– mice following stimulation with immobilized IgG + IL-12 were analyzed via flow cytometry for IFN-{gamma} production by DX5+ NK cells. Shown is a representative histogram showing IFN-{gamma} fluorescence within NK cells from the indicated mouse (P = 0.005).

 


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  		Figure 5. Purified NK cells from SHIP1–/– mice exhibit significantly enhanced IFN-{gamma} production and ADCC activity in vitro. A, equal numbers of enriched NK cells derived from the spleens of WT and SHIP1–/– (KO) mice were cultured in the presence of immobilized IgG and IL-12. Supernatants were harvested at 24 hours and analyzed for IFN-{gamma} content. *P < 0.05 versus medium, IgG, and IL-12 alone stimulation conditions. The P value for the comparison of IFN-{gamma} levels from WT and SHIP1–/– NK cells for each stimulus condition is shown inset. B, NK cells from both WT and SHIP1–/– spleens were cultured for 18 hours in medium alone or medium supplemented with 10 ng/mL muIL-12. Cells were then analyzed for ADCC activity against P815 target cells in the absence or presence of rabbit anti-mouse lymphocyte polysera (antibody source). 51Cr release was measured after a 4-hour incubation period. The experiment shown is representative of three separate determinations. * P = 0.002 versus WT at the same effector-to-target ratio.

 
Suppression of IFN-{gamma} production in natural killer cells by SHIP1 occurs via inhibition of Erk activation. Fc{gamma}RIIIa cross-linking on NK cells has previously been shown to induce the activation of the MAPK signaling family, proteins found to be critical for NK cell lytic function and cytokine production (9, 32). Activation of the MAPK proteins Erk1/2 following target cell ligation by NK cells has been shown to require the activity of PI3-K (33). Inhibition of PI3-K led to a down-regulation of Erk1/2 activity and decreased NK lytic activity against tumor targets. Therefore, we examined the role of SHIP1 in regulating downstream MAPK activation within primary NK cells following Fc{gamma}RIIIa clustering and investigated its effect on IFN-{gamma} production. Cross-linking of the activating FcR on purified NK cells derived from WT animals in the presence of IL-12 led to synergistic phosphorylation of Erk1/2 compared with either stimulus alone (Fig. 6A). Interestingly, this synergistic activation of Erk1/2 in response to FcR/IL-12R costimulation was significantly greater for SHIP1–/– mice than for their WT counterparts. Normalization of phosphorylated Erk levels to total Erk levels within each of the cellular preparations (i.e., within each lane) by densitometry revealed that the addition of IL-12 to FcR stimulation led to greater synergy for Erk phosphorylation in SHIP1–/– NK cells than in WT cells (54.6-fold phospho-Erk induction versus no stimulation condition in SHIP1–/– NK cells compared with 21.8-fold induction versus no stimulation condition in WT NK cells, P = 0.031). Furthermore, inhibition of Erk1/2 signaling by chemical inhibitor pretreatment resulted in almost complete abrogation of the IFN-{gamma} response from both WT and SHIP1–/– NK cells (Fig. 6B). Inhibitors of another MAPK protein Jnk (Fig. 6C) had no significant effect on IFN-{gamma} production in either WT or SHIP1–/– NK cells undergoing costimulation. These results suggested that SHIP1, through its enzymatic activity on PI3-K products, negatively regulates FcR-dependent IFN-{gamma} production by inhibiting the downstream activation of Erk1/2.



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  		Figure 6. Purified NK cells from SHIP1–/– mice exhibit enhanced levels of activated Erk and Erk-dependent IFN-{gamma} production. A, NK cells from both WT and SHIP1–/– (KO) splenocyte populations were activated by FcR cross-linking in the presence of muIL-12. Cells were analyzed for phosphorylated (P)-Erk and total Erk by immunoblot. A representative pair of mice from five analyzed are shown. B, equal numbers of purified NK cells (2.5 x 105/well) derived from a population of pooled NK cells (n = 3 mice) were pretreated with a specific Erk inhibitor or inhibitors of another MAPK family member Jnk (C) and then cultured in the immobilized IgG assay. Culture supernatants were harvested after 24 hours and analyzed for murine IFN-{gamma}. Cells were counted via trypan blue exclusion following inhibitor pretreatment and following the IgG + IL-12 culture period to ensure no difference in cell viability between control-treated and inhibitor-treated NK cells from the WT and KO populations. *P < 0.01 versus medium, IgG, and IL-12 conditions for DMSO-treated cells. The P value for the comparison of IFN-{gamma} levels from WT and KO NK cells for the IgG + IL-12 stimulation condition is shown inset.

 
Natural killer cells from SHIP1–/– mice exhibit greater IFN-{gamma} production in vivo following administration of antibody-coated tumor cells plus interleukin-12. To confirm the biological role of SHIP1 in the negative regulation of NK cell cytokine production, an in vivo costimulation assay was used in which age-matched SHIP1–/– [knockout (KO)] or WT littermates received i.p. injections of PBS, control IgG-treated P815 cells, rabbit anti-mouse lymphocyte antibody-coated P815 cells, muIL-12 and control IgG-treated P815 cells, or muIL-12 plus rabbit anti-mouse lymphocyte antibody-coated P815 tumor cells. After 24 hours, serum was harvested and analyzed for IFN-{gamma} content by ELISA. SHIP1–/– mice injected with IL-12 plus antibody-coated tumor cells exhibited significantly enhanced levels of serum IFN-{gamma} compared with SHIP1–/– mice injected with either stimulus alone and a 4.2-fold greater amount of IFN-{gamma} than WT animals given the same stimulus (Fig. 7A). To define the cellular source of cytokine production, splenocytes were concurrently harvested, cultured for an additional 6 hours in brefeldin-A, and analyzed by flow cytometry for levels of intracellular IFN-{gamma} within the NK cell population. As shown in Fig. 7B, splenocytes from WT and SHIP1–/– mice exhibited very little IFN-{gamma} production following PBS administration, suggesting that basal production of IFN-{gamma} is not enhanced in SHIP1–/– mice. Of note, SHIP1–/– animals had on average 2-fold greater number of NK cells producing IFN-{gamma} following administration of antibody-coated tumor plus IL-12 than their WT counterparts (23.9% versus 66.1%; Fig. 7B). Furthermore, SHIP1–/– NK cells also produced greater amounts of IFN-{gamma} than WT NK cells [specific fluorescence (Fsp) of IFN-{gamma} in SHIP1–/– NK cells = 32.5 versus IFN-{gamma} Fsp in WT NK = 3.82]. Of note, the absolute number of total NK cells (Fig. 7B, top two quadrants) within SHIP1–/– mice routinely decreased following administration of antibody-coated tumor cells plus IL-12, but not following administration of PBS or control IgG-P815 tumor alone. This decrease may reflect activation-induced NK cell death in an environment (the SHIP1–/– mouse) where attenuation signals for potent cellular activation are absent. Indeed, analysis of the peripheral blood and spleen 24 hours following administration of antibody-coated tumor plus IL-12 revealed a greater decrease in DX5+ NK cells within SHIP1–/– mice (61.8% and 33.7% decrease within the spleen and blood versus control IgG-P815 tumor administration, respectively) compared with WT mice (37.5% and 9.4% decrease versus control IgG-P815 tumor alone, respectively). Of note, the percentage of NK cells in the liver increased following administration of antibody-coated tumor plus IL-12, but was not significantly different between SHIP1–/– mice (30.6% increase versus control IgG-P815 tumor alone) compared with WT mice (38.6% increase versus control IgG-P815 tumor alone). Despite this overall decrease in circulating NK cell number, a higher IFN-{gamma}–specific fluorescence and a greater overall quantity of circulating IFN-{gamma} protein was observed in SHIP1–/– mice. Of note, significant production of IFN-{gamma} was observed in a non-NK cell compartment. Cell types other than NK cells may respond to the combination of IL-12 and immobilized IgG. Granulocytes and monocytes both express FcR for IgG and these cell populations have also been shown to express the IL-12R (34, 35). Thus, the response to IL-12R and FcR triggering in vivo could be influenced by the activity of these other cell compartments. In a similar system, we have previously observed IFN-{gamma} production in a subset of T cells in response to FcR plus IL-12R costimulation (19). The source of this extra-NK cell cytokine production is currently under investigation. Nevertheless, these results suggested that individual NK cells from SHIP1–/– mice produced greater amounts of IFN-{gamma} in response to antibody-coated tumor cells and IL-12 costimulation.



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  		Figure 7. NK cells from SHIP1–/– mice exhibit greater IFN-{gamma} production in vivo following administration of antibody-coated tumor cells plus IL-12. Age-matched WT or SHIP1–/– (KO) mice received i.p. injections of PBS (not shown), control IgG-treated P815 cells, rabbit anti-mouse lymphocyte antibody-coated P815 cells, IL-12, and control IgG-treated P815 cells, or muIL-12 plus rabbit anti-mouse lymphocyte antibody-coated P815 tumor cells. A, after 24 hours, serum was harvested and analyzed for IFN-{gamma} content by ELISA. *P < 0.001 versus PBS injection of same genotype of mouse. The P value for the comparison of serum IFN-{gamma} levels from WT and SHIP1–/– mice is shown inset. B, splenocytes were harvested, cultured for 6 hours in brefeldin-A, permeabilized, and stained with phycoerythrin-labeled DX5 and NK1.1 antibodies (pan-NK) and FITC-labeled anti–muIFN-{gamma} mAb. Cells were analyzed by flow cytometry for levels of intracellular IFN-{gamma} within the NK cell populations. Data from a representative set of mice is shown. P = 0.0079 versus percentage of IFN-{gamma}+ NK cells within WT group of animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that SHIP1 negatively regulates IFN-{gamma} secretion in response to costimulation with immobilized antibody and IL-12 in primary NK cells. The significant reduction in IFN-{gamma} secretion observed from primary human NK cells transfected with WT SHIP1, coupled with the enhanced IFN-{gamma} secretion observed in NK cells derived from SHIP1–/– mice, support this notion. We have also shown that the reduction in IFN-{gamma} secretion is at least partly dependent on the phosphatase activity of SHIP1, and thus on restraining the levels of the PI3-K product PIP3. This is consistent with our finding that the PI3-K inhibitors, LY294002 and wortmannin, blocked IFN-{gamma} production in primary human NK cells. Our results suggest that SHIP1, through its enzymatic activity on PI3-K products, negatively regulates FcR-dependent IFN-{gamma} production by inhibiting the downstream activation of Erk1/2. These data provide the first demonstration that SHIP1 can inhibit IFN-{gamma} production induced in NK cells by Fc{gamma}RIIIA stimulation, or IL-12 and Fc{gamma}RIIIA costimulation, thereby expanding the physiologic role of this phosphatase in attenuating NK effector functions. Additionally, these data strongly suggest that SHIP1 integrates signals generated by IL-12 and Fc{gamma}RIIIA costimulation that, in turn, synergistically activate Erk1/2 and induce IFN-{gamma} production in NK cells.

We have previously shown that NK cells produce a broad array of cytokines following activation by antibody-coated targets and that costimulation with immune stimulatory cytokines such as IL-2 and, especially IL-12, can synergistically enhance this cytokine response (19, 36). Further, NK cells costimulated with human HER2-overexpressing breast cancer cells coated with a therapeutic anti-HER2 mAb (trastuzumab) and IL-12 produced abundant quantities of several immunomodulatory cytokines, including IFN-{gamma}, granulocyte macrophage colony-stimulating factor, macrophage inflammatory protein-1{alpha}, and tumor necrosis factor-{alpha}, both in an in vitro and in an in vivo tumor model (19). In a phase I clinical trial in which trastuzumab and IL-12 were administered to patients with HER2-overexpressing malignancies, detectable levels of IFN-{gamma} and other NK cell-derived cytokines were observed only in patients who exhibited clinical benefit during therapy (20). In contrast, no correlation was observed between clinical benefit and in vitro ADCC activity mediated by patient NK cells, further emphasizing the role of NK cell–derived cytokines in possibly mediating an antitumor response. Unfortunately, it remained unclear why only a subset of patients exhibited this potent cytokine production. To address this important question, we examined the mechanisms by which NK cell effector functions are attenuated following engagement of antibody-coated targets. The current observation that SHIP1 can negatively regulate the ability of IL-12 to enhance Fc{gamma}RIIIa-mediated cytokine production in primary NK cells indicates an important biological role for SHIP1 in attenuating activation signals within NK cells. In addition, these data imply that SHIP1 could serve as a potential target for pharmacologic intervention during immune therapies in which NK cells might participate. Nevertheless, care must be taken when assessing the applicability of this approach to the clinical situation because sustained activation of the potent activating molecules that SHIP1 normally suppresses in B cells have led to autoimmune susceptibility (37, 38).

Previous reports investigating the role of SHIP1 in NK cells have shown that upon cross-linking of Fc{gamma}RIIIa, SHIP1 was phosphorylated and recruited to the {zeta}-chain of the FcR signaling complex via its association with the shc adaptor protein, where it exerted inhibitory effects on both NK natural cytotoxicity and ADCC activity (17, 18). Specifically, the shc/SHIP1 complex tightly regulated the mobilization of the cytolytic machinery within NK cells (18, 39). In fact, both the extent and duration of Ca2+ mobilization were enhanced in cells expressing a dominant-negative form of shc, suggesting that recruitment of SHIP1 is necessary for attenuating the activation signals associated with cytotoxicity (18). Importantly, subsequent work has shown that SHIP1 catalytic activity, and not only recruitment to FcR signaling chains, is important in regulating NK cell–mediated cytotoxicity in response to NK-sensitive and antibody-coated targets (17). NK cells overexpressing WT SHIP1 exhibited significantly reduced Fc{gamma}RIIIa-mediated killing of anti-CD16 mAb-coated tumor targets compared with control cells. Interestingly, overexpression of a form of SHIP1 deficient in catalytic activity did not result in a similar reduction, suggesting the importance of the enzymatic activity of SHIP1 in mediating the inhibitory effect on cellular cytotoxicity. The present data extend these initial observations with regard to regulation of NK lytic activity and, for the first time, suggest a role for SHIP1 in regulating NK cell cytokine production. Of note, recent work by Trotta et al. (27) reveals that SHIP1 overexpression also inhibits the production of IFN-{gamma} by CD56dim NK cells in response to stimulation with the monokines IL-12 plus IL-18. Similarly, NK cells from SHIP1–/– mice exhibited increased secretion of IFN-{gamma} following this treatment. To date, however, the signaling intermediates that mediate this response have yet to be established.

To gain some insight into the SHIP1-regulated pathways that modulate IFN-{gamma} secretion in primary NK cells and to delineate the contribution of each of these pathways to NK cell activation, we used specific inhibitors of signaling pathways that were known to be induced following Fc{gamma}RIIIa cross-linking. We found that the phosphorylation of Erk (but not Jnk) was significantly up-regulated in SHIP1–/– NK cells and apparently contributed to the increased IFN-{gamma} production observed in these cells. Inhibitor studies further suggested that the synergistic activation of Erk following FcR clustering in the presence of IL-12 (and the attendant synergistic IFN-{gamma} production) was highly dependent on PI3-K activity (Fig. 6B; data not shown). Thus, it seems that SHIP1 is one means by which the effects of PI3-K on downstream signal transduction pathways such as Erk are negatively regulated.

In the present study, inhibitors of both Erk1/2 and PI3-K attenuated IFN-{gamma} production by NK cells stimulated with IL-12 alone. Although signal transduction from the IL-12R complex seems to proceed via the activation of signal transducers and activators of transcription 4 in NK cells (40), there is evidence to suggest that the regulation of a novel gene (DC21) in human peripheral blood mononuclear cells by IL-12 is dependent upon PI3-K activity (41). This ability of the PI3-K signaling pathway to regulate gene expression in immune effectors may in part explain the negative effects of the PI3-K inhibitors wortmannin and LY294002 on IFN-{gamma} production by IL-12–stimulated NK cells. Also, recent reports have implicated Erk in the control of gene transcription via epigenetic mechanisms. Erk can mediate the phosphorylation of histone H3 in vitro and can modify the activity of histone deactylase 4 (42), suggesting a role for MAPK proteins in regulating chromatin structure. In addition, Erk and PI3-K both mediate prosurvival signals in lymphoid cells, and it is possible that NK cell viability in vitro was negatively influenced by inhibition of these pathways. Accordingly, inhibition of Erk and PI3-K in these NK cell assays may have led to reduced levels of IFN-{gamma} secretion by IL-12–stimulated NK cells via all or some of these pathways.

The role of specific MAPK proteins in regulating different activation pathways has been previously reported in T cells following activation by either CD2 or CD28 and apparently also applies to activated NK cells (43). Activator protein 1 and p38 are well-established regulators of IFN-{gamma} transcription in NK cells (9, 44). The role of Erk in this regulation is indirect, as MAPK proteins do not typically bind DNA. Thus, the mechanisms by which Erk leads to enhanced IFN-{gamma} transcription in these studies remain unclear. Of note, Kalesnikoff et al. (45) have shown the involvement of Erk in IgE-induced Fc{varepsilon}RI activation and subsequent IL-6 production by mast cells. Their data revealed that Erk stimulated trans-activation of NF-{kappa}B, a critical step in up-regulation of IL-6 gene transcription. The importance of Erk in mediating FcR effector functions was initially characterized by Trotta et al. (9, 46) in NK cells. Similar findings implicating Erk in FcR-induced cytokine production were observed for monocytic cells following cross-linking of Fc{gamma}RI or Fc{gamma}RIIa (47). Thus, our studies lend support to the involvement of Erk in the regulation of cytokine production following FcR activation and provide a potential mechanism by which cytokines, such as IL-12, can enhance the Fc{gamma}RIIIa-mediated effects within NK cells.

Taken together, these results suggest a model in which SHIP1 represses IFN-{gamma} production in primary NK cells, at least in part, by reducing PI3-K–generated products induced by FcR plus IL-12 costimulation. In addition, SHIP1 specifically inhibits the downstream activation of the MAPK proteins Erk1/2. These findings most likely reflect a normal physiologic mechanism for regulating the magnitude and duration of NK cell activation following Fc{gamma}RIIIa cross-linking. Pharmacologic manipulation of SHIP1 activity may, therefore, represent a means of enhancing NK cell responses in the context of infection and malignant disease.


   Acknowledgments
 
Grant support: Susan G. Komen Breast Cancer Foundation Dissertation Research Award (R. Parihar) and NIH grant P01 CA95426.

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.

We thank Huiqing Fang for expertise in animal care and genotyping.

Received 12/10/04. Revised 7/22/05. Accepted 7/26/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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