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Redundant Role of the Syk Protein Tyrosine Kinase in Mouse NK Cell Differentiation¹

Francesco Colucci^*, Martin Turner, Edina Schweighoffer, Delphine Guy-Grand^*, Vincenzo Di Bartolo, Margarita Salcedo, Victor L. J. Tybulewicz and James P. Di Santo^2,*

^* Institut National de la Santé et de la Recherche Médicale, Unit 429, Hôpital Necker-Enfants Malades, Paris, France; National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom; and Institute Pasteur, Paris, France ² Current address: Babraham Institute, Babraham, Cambridge, U.K.

	Abstract

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Syk and ZAP-70 subserve nonredundant functions in B and T lymphopoiesis. In the absence of Syk, B cell development is blocked, while T cell development is arrested in the absence of ZAP-70. The receptors and the signaling molecules required for differentiation of NK cells are poorly characterized. Here we investigate the role of the Syk protein tyrosine kinase in NK cell differentiation. Hemopoietic chimeras were generated by reconstituting alymphoid (B^-, T^-, NK^-) recombinase-activating gene-2 x common cytokine receptor -chain double-mutant mice with Syk^-/- fetal liver cells. The phenotypically mature Syk^-/- NK cells that developed in this context were fully competent in natural cytotoxicity and in calibrating functional inhibitory receptors for MHC molecules. Syk-deficient NK cells demonstrated reduced levels of Ab-dependent cellular cytotoxicity. Nevertheless, Syk^-/- NK cells could signal through NK1.1 and 2B4 activating receptors and expressed ZAP-70 protein. We conclude that the Syk protein tyrosine kinase is not essential for murine NK cell development, and that compensatory signaling pathways (including those mediated through ZAP-70) may sustain most NK cell functions in the absence of Syk.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The Syk family of protein tyrosine kinases (PTKs)³ contains two members, p72syk (Syk) and ZAP-70 (1), which share structural features, including a consensus tyrosine kinase domain and two Src homology type 2 domains. The Src homology type 2 domains are required for the association of Syk and ZAP-70 to the immunoreceptor tyrosine-based activation motif (ITAM) contained within the cytoplasmic domain of activating cell surface receptors on hemopoietic cells (2). Syk is expressed in many hemopoietic cell types, although its levels are greatest in B cells (3). Expression of ZAP-70, in contrast, is restricted to T and NK cells (3). The coexpression of Syk and ZAP-70 in the same lymphoid cell subsets (T and NK cells) suggests that these two related PTKs could subserve redundant functions. Nevertheless, the intracellular targets of the Syk and ZAP-70 kinases are not entirely elucidated, and it remains possible that Syk and ZAP-70 phosphorylate a unique set of protein substrates.

Syk is primarily activated following engagement of ITAM-containing receptors present in the B cell receptor (BCR) complex and in the Fc receptors (FcR) for IgG (FcRI, FcRII, FcRIII) and IgE (FcRI) (1, 4). Engagement of platelet integrins has been shown to activate Syk (5), and Syk appears complexed to the IL-2R ß-chain (6), a functional component of the IL-2 and IL-15 receptors (reviewed in Ref. 7). Thus, Syk activation potentially intervenes in signaling from Ag receptors, FcRs, integrins and cytokine receptors.

Gene-targeting experiments have unraveled some of the essential roles of the Syk PTK in vivo. Mice deficient in Syk (Syk^-/-) die in utero or during the prenatal period from excessive hemorrhage (8, 9). This phenotype could be explained by the failure of collagen-activated Syk^-/- platelets to phosphorylate PLC2, which, in turn, results in compromised platelet secretion and aggregation (10). However, collagen signaling in platelets is also defective in Fc-deficient mice, which do not show a bleeding diathesis, and bleeding times in chimeric Syk^-/- mice are normal (10). Alternatively, a defect in the function of other hemopoietic cell types (perhaps macrophages) in the absence of Syk could lead to microvascular destruction (10).

Due to the lethal nature of Syk deficiency, any role for the Syk PTK in lymphoid development has been addressed in somatic or hemopoietic chimeras using recombinase-activating gene-2 (RAG2) mutant mice as hosts (8, 9). In these studies B cell development failed to progress beyond the pro-B cell stage due to an inability of Syk-deficient early B cells to couple pre-BCR ligation to downstream intracellular processes (8, 9, 11). In contrast, ß T cell development appeared normal in Syk^-/- mice, although overall T cell numbers were reduced (9), and the dendritic epidermal T cell subpopulation was not detected (8, 12). Reciprocally, in ZAP-70-deficient mice (13, 14) and humans (15) the development of ß T cells, T cells, and NK1.1+ ß T cells is abnormal. In contrast, NK and B lymphopoiesis in ZAP-70 mutants are not affected. The T cell defect in ZAP-70 deficiency is caused by abnormal signaling following TCR triggering, thereby highlighting the analogous functions of Syk and ZAP-70 in BCR and TCR pathways, respectively.

Syk could intervene in the differentiation of NK cells via 1) signal transduction through the IL-2R ß-chain in response to IL-15, a vital growth and differentiation factor for NK cell progenitors and mature NK cells (16); 2) activating NK cell surface receptors, including those involved in natural cytotoxicity; or 3) FcRIII activation for Ab-dependent cellular cytotoxicity (ADCC) (reviewed in Ref. 17). Recently, Brumbaugh et al. (18) have suggested that Syk plays a major role in mediating the natural cytotoxicity of human NK cells. However, a role for Syk in NK cell differentiation could not easily be assessed in the previously reported Syk^-/- chimeras, because the host RAG2 mice can generate their own functional NK cells (19).

We have developed a novel alymphoid mouse strain harboring both the RAG2 and common cytokine receptor -chain (_c) mutations. RAG2/_c mutant mice have certain advantages for the study of lymphoid development in vivo, including the complete absence of all mature T, B, and NK cells and a reduced number of lymphoid precursors (20). The lymphoid system in RAG2/_c mice can be stably and efficiently reconstituted using normal or mutant hemopoietic stem cells (HSC) from adult bone marrow (BM) or fetal liver (FL), and thus RAG2/_c mutant mice provide a new tool to study the role of any gene in NK cell differentiation in vivo. In this report we have analyzed the role of the Syk PTK in murine NK cell development and function.

	Materials and Methods

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Mice and generation of hemopoietic chimeras

Mice with a null mutation in the _c (21) were from the fourth generation backcross to the C57BL/6 background. RAG2 mice (19) from the 10th generation backcross to C57BL/6 were provided by Dr. B. Rocha (Paris, France). Mice doubly deficient in RAG2 and _c (RAG2/_c; H-2^b) were obtained by intercrossing as previously described (20). C57BL/6 and C57BL/6.ß₂m^-/- mice were obtained from CDTA/CNRS (Orleans, France).

Mice heterozygous for the Syk^tm1Tyb mutation (8) that had been backcrossed five generations onto the B10.D2 background (H-2^d) were intercrossed to generate day 15.5–16.5 Syk^-/- and control (Syk^+/+ or Syk^+/-) embryos. The observation of a vaginal plug was designated day 0.5. FL cell suspensions were obtained by passage of the tissue through a 23-gauge needle. RAG2/_c mice (>6 wk of age) were irradiated with 0.3 Gy from a cobalt source and 4 h later were injected i.v. with 5 x 10⁶ FL cells. No obvious differences were seen between Syk^+/+ or Syk^+/- FL chimeras, which will be referred to as Syk⁺ chimeras. All mice received tetracycline and bactrim in the drinking water for the period following FL cell transfer. The Syk genotypes of the embryos were determined by Southern blotting as previously described (8).

Flow cytometry

Single-cell suspensions were prepared from spleen, BM, thymus, and liver. Erythrocytes were lysed in ammonium chloride, and cells were resuspended in PBS with 3% FCS and 0.01% sodium azide. mAbs directly conjugated to FITC, PE, Tricolor, or biotin were used for immunofluorescence analysis as previously described (20), including Abs specific for CD2, CD3, CD4, CD8, TCRß, TCR, CD11b (Mac-1), CD16 (FcRII/III), CD19, CD24, B220, IgM, CD90 (Thy-1), CD117 (c-Kit), CD122 (IL2Rß), NK1.1, DX5, 2B4, Ly49A, Ly49G2, CD132 (_c), H-2K^d, H-2K^b, and Gr-1 (all from PharMingen, San Diego, CA).

Generation of lymphokine-activated NK (LAK) cells

Splenocytes were passed through nylon wool columns to remove most B cells and macrophages. Nylon wool-nonadherent cells were cultured in flat-bottom 24-well plates at 5 x 10⁶ cells/ml in complete medium (RPMI 1640 with 10% FCS, 10^-5 M ß-ME, 100 µg/ml streptomycin, and 100 U/ml penicillin) supplemented with 20 ng/ml of human IL-15 (R&D Systems, Minneapolis, MN) or 1000 U/ml of human IL-2 (PeproTech, Rocky Hill, NJ). After 3–4 days the nonadherent cells were removed, and the adherent lymphokine-activated killer (A-LAK) cells were refed and cultured until days 8–10. A-LAK cultures produced in this manner routinely contained >95% NK1.1⁺/CD3^- cells.

Cell-mediated cytotoxicity

A ⁵¹Cr release assay was used to measure NK activity in vitro as previously described (22). Target cells (YAC-1, EL-4, P815, mouse fibroblasts (L cells) or their Qa-I^b-transfected derivatives (F12) (23), or Con A-activated B6 and ß₂m^-/- blasts) were labeled with 100 µCi of ⁵¹Cr (ICN Pharmaceutical, Costa Mesa, CA), and 2.5–5 x 10³ targets were incubated with graded numbers of effector cells in 200 µl of medium for 4 h. For natural cytotoxicity, effector cells were NK-enriched, B cell-depleted splenocytes from poly(I:C)-primed mice (40 h after injection with 0.2 mg of poly(I:C)/mouse). A-LAK cells were used as effectors for 1) Ab-dependent cell cytotoxicity (ADCC) using EL-4 cells with or without Thy 1.1 mAb (20); 2) reverse ADCC (rADCC) using FcR⁺ P815 cells in the presence or the absence of NK1.1, 2B4, or CD16 mAbs; or 3) MHC inhibition and recognition of "missing self" using Con A-activated B6 and ß₂m^-/- blasts or L and F12 cells. Radioactivity released into the cell-free supernatant was measured, and the percent specific lysis was calculated as follows: 100 x [(experimental release - spontaneous release)/(maximum release - spontaneous release)].

In vitro cytokine production and ZAP-70 immunodetection

Splenic NK cells were FACS sorted using the pan-NK cell mAb DX5, which is coexpressed on 95% of splenic NK cells and shows no NK cell-activating or -blocking activities (PharMingen). Purified DX5⁺ NK cells (10⁵ cells/200 µl) were cultured in flat-bottom microtiter plates in human IL-15 (20 ng/ml) or human IL-2 (1000 U/ml). Stimuli included mIL-12 (2 ng/ml; PeproTech), or mAbs NK1.1 (clone PK136; 20 µg/ml), 2B4 (5 µg/ml), CD16 (75 µg/ml), or control Gr-1 (10 µg/ml). Wells were precoated with mAbs (50 µl/well) for 4 h before adding NK cells. After 24 h, the cell-free supernatants were collected, and the amount of IFN- was quantitated by ELISA (Genzyme, Cambridge, MA).

Analysis of ZAP-70 protein expression in FACS-purified NK1.1⁺/CD3^- NK cells was performed by immunoblotting as previously described (24).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Lymphoid development in Syk^-/- FLRAG2/_c hemopoietic chimeras

Reconstitution of RAG2/_c mice with Syk^-/- FL cells generated the same developmental arrest at the BM pre-B cell stage that was originally described using RAG2^-/- blastocyst complementation (8, 9). Few IgM⁺B220⁺ B cells could be detected in the BM of Syk^-/ chimeras, but these cells were almost completely absent in the spleen (Fig. 1A, Table I, and data not shown). In contrast, normal numbers of BM and splenic IgM⁺B220⁺ B cells were present in Syk⁺ chimeras. Consistent with previous reports (8, 9), Syk^-/- chimeras showed no obvious defect in thymocyte development as defined by CD4 and CD8 expression (Fig. 1B), although thymocyte numbers were reduced 2-fold in Syk^-/- thymi (data not shown). Mature CD4 and CD8 T cells were found in the peripheral lymphoid organs of these mice, which expressed normal levels of TCR-ß (data not shown). Moreover, T cells and NK T cells could be readily detected in the thymus, spleen, and liver and lining the gut intestinal tract of Syk^-/- FLRAG2/_c chimeras (F.C., D.G.-G., A. Wilson, M.T., E.S., V.L.J.T., and J.P.D., manuscript in preparation).

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Lymphoid development in the absence of Syk. BM cells (A) were stained with FITC-anti-IgM and PE-anti-B220, while thymocytes (B) were stained with FITC-anti-CD4 and PE-anti-CD8. T cell subsets are normally represented in Syk-/- chimeras, while B cells (B220+ IgM+) are barely detectable in BM and are absent in spleen, as previously described (9 ). C, Splenocytes were stained with FITC-anti-IL-2Rß, PE-anti-NK1.1, biotinylated anti-CD19, and biotinylated anti-TCRß. B cells and ß T cells were electronically excluded from the analysis, and expression of IL-2Rß vs NK1.1 is shown. IL-2Rß+ NK1.1+ NK cells represented between 30–60% of non-B, non-T cells in the spleens of Syk-/- chimeras. D, The donor origin (H-2d) of NK cells was verified in TCRß- CD19- NK1.1+ splenocytes. The NK1.1- TCRß- CD19- H-2d- cells are of host origin and stain positively for H-2b (data not shown).

NK cell development in the absence of Syk

Syk-deficient FL cells reconstituted NK cell development in RAG2/_c chimeras. Flow cytometric analysis revealed that NK1.1⁺ IL-2Rß⁺ NK cells were readily detectable in the CD19^- TCRß^- fraction of splenic, hepatic, and BM cells from Syk⁺ and Syk^-/- chimeras (Fig. 1C and data not shown). Absolute numbers of NK cells were similar between the two groups of mice (<2-fold reduction was found in Syk^-/- chimeras; Table I). The phenotype of Syk^-/- NK cells was analyzed by flow cytometry using a panel of NK cell-associated markers. Both Syk⁺ and Syk^-/- NK1.1⁺ cells demonstrated a normal percentage and expression level of CD2, CD11b, CD16, B220, DX5, 2B4, CD90, and CD117 (data not shown). Finally, the donor origin of NK cells was confirmed by PCR amplification of the RAG2 gene in sorted NK cells (data not shown) and by the cell surface expression of H-2K^d molecules by NK1.1⁺ splenocytes in the H-2^b RAG2/_c hosts (Fig. 1D). Reciprocal staining with an anti-H-2K^b mAb confirmed that all lymphoid cells were donor derived (data not shown). These results demonstrate that the Syk PTK is not essential for murine NK cell development in vivo.

The signal transduction pathways required for the generation of NK cells are poorly characterized. Cytokine receptor signaling via the IL-15/IL-15R complex is required for NK cell development, presumably at an early stage during the commitment of HSC to the NK lineage (16). A potential role for Syk in IL-15 responses has been implied as well, because an association of Syk with the IL-2R ß-chain has been documented (6), and this chain is required for signaling via IL-2 or IL-15. Previous studies, however, have demonstrated that Syk-deficient T cells can respond to IL-2 in vitro (8), and in this study we did not observe any manifestations of IL-2 deficiency (such as deregulated T cell homeostasis, lymphoproliferation, or colitis) in Syk^-/- chimeras (data not shown). Moreover, IL-15 responses of Syk-deficient NK cells appear normal; IL-15-stimulated A-LAK cultures from Syk⁺ and Syk^-/- chimeras were generated similarly, and IL-15 induced a comparable level of proliferation from Syk⁺ and Syk^-/- cells (data not shown). These results suggest that Syk association with the IL-2R ß-chain is not essential for many of the functions of IL-2 or IL-15. Finally, our data extend the list of nonessential PTKs for murine NK cell development, which includes Lck, ZAP-70, Fyn, and now Syk (13, 25, 26). It remains possible that the receptors controlling NK cell development do not require any PTK signals.

Natural cytotoxicity mediated by Syk^-/- NK cells

To analyze the effector functions of Syk^-/- NK cells, the lytic capacity of freshly isolated splenic NK cells was tested in vitro. Syk-deficient NK-enriched splenocytes demonstrated normal levels of natural cytotoxicity against YAC-1 targets (Fig. 2A). This result contrasts with a recent report suggesting that Syk is essential for the natural cytotoxicity of human NK cells (18). Although species-specific differences in NK cell function may account for these differences, one must consider the possibility that the dominant negative (DN) Syk construct used in the studies of Brumbaugh et al. (18) might have inhibited not only Syk, but other signaling pathways (including, perhaps, ZAP-70) as well. Along these lines, studies using a DN LAT construct revealed an essential role for LAT in human NK cell functions (27), while LAT-deficient mice appear to have normal NK activities (28).

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Natural cytotoxicity and ZAP-70 expression in Syk-/- NK cells. A, A classical 4-h 51Cr release assay was performed to evaluate NK cell lytic capacity. Specific lysis was determined at the indicated E:T cell ratios using NK-enriched (B cell-depleted) spleen cells from poly(I:C)-treated mice as effectors and 51Cr-labeled YAC-1 cells as targets. One representative experiment of three is shown. B, Sorted NK cells from Syk+ or Syk-/- RAG2/c chimeras or control Jurkat T cells were lysed, and total cellular proteins were analyzed by anti-ZAP-70 immunoblotting. The arrow indicates the migration of ZAP-70, and m.w. standards are indicated on the right.

The ability of Syk and ZAP-70 to partially compensate for each other in T cell development has been demonstrated in ZAP-70 mutant mice, in which intrathymic differentiation is arrested at the double-positive stage (13, 14). Enforced expression of Syk in ZAP-70-deficient mice can rescue T cell development (29). In B cells, the absence of Syk arrests development with no compensation by ZAP-70, due to its lack of expression in early B cell progenitors (3). However, ZAP-70 can restore BCR signaling in a Syk-deficient B cell line (30), demonstrating that compensation is possible. Redundancy between Syk- and ZAP-70-mediated signaling may also operate during NK cell development and for NK cell effector functions. In this respect, NK cells are the only mouse lymphocyte subset in which Syk family PTKs are nonessential for differentiation. In contrast, ß T cells, T cells, and B cells are strictly dependent either on ZAP-70 (ß T cells and some T cells (13, 14) or on Syk (some T cells and B cells (8, 9, 12).

Function of inhibitory receptors in Syk^-/- NK cells

Previous studies have suggested a role for PTKs in the function of NK cell receptors, including members of the Ly49 family. In particular, the role of signaling molecules in the shaping of the NK cell repertoire (31) or in calibrating Ly 49 receptor expression (32) has not been defined. Ly49A binds H-2D^d and H-2D^k, while Ly49G2 binds to H-2D^d and H-2L^d (reviewed in Ref. 31). Because the donor FL cells were H-2^d, the resultant RAG2/_c chimeras harbored cells bearing either H-2^b (host) or H-2^d (donor). This allowed us to determine whether Syk played a role in the regulation of expression of Ly49 receptors by H-2D^d (33).

We examined the expression levels of Ly49A and Ly49G2 receptors and the frequencies of these Ly49⁺ NK subsets in Syk and Syk^-/- RAG2/_c chimeras. As shown in Fig. 3A, the percentages of Ly49A⁺ or Ly49G2⁺ NK cells were not altered in the absence of Syk. Moreover, both Ly49A and Ly49G2 levels were similarly decreased in Syk⁺ and Syk^-/- NK cells compared with that observed in NK cells derived from C57BL/6 mice (Fig. 3A). These results demonstrate that the Syk PTK signaling is not required for Ly49 receptor calibration and suggest that expression of H-2^d molecules by hemopoietic cells is sufficient to shape the NK cell repertoire.

View larger version (37K): [in this window] [in a new window]   FIGURE 3. Inhibitory receptors are normally regulated and function efficiently in the absence of Syk. A, Spleen cells were stained with anti-Ly49A or Ly49G2, anti-NK1.1, and a combination of anti-CD19 and anti-TCRß Abs. CD19+ B cells and TCRß+ T cells were electronically eliminated from the analysis, and the percentage and mean fluorescence intensity of the Ly49 molecules were determined on the gated NK1.1+ cells. Mean fluorescence intensities and SDs for three B6 mice and four to six Syk+ or Syk-/- chimeras are shown. The percentages of NK1.1+ cells expressing Ly49A were 16 ± 4.1 in B6 mice (n = 3), 3.1 ± 1.3 in Syk+ chimeras (n = 6), and 3.6 ± 0.6 in Syk-/- chimeras (n = 6). The percentage of NK1.1+ cells expressing Ly49G2 were 46.3 ± 1.5 in B6 mice (n = 3), 44.1 ± 7.7 in Syk+ chimeras (n = 4), and 31.3 ± 4.5 in Syk-/- chimeras (n = 6). B, Syk+ and Syk-/- A-LAK cells were tested for their ability to distinguish between targets expressing classical and nonclassical MHC molecules. Con A-activated (5 µg/ml for 48 h) B6 (filled circles) and ß2m-/- (filled triangles) splenocytes were used as targets. Qa-1b-transfected (F12 cells, empty circles) or control (L cells, empty triangles) fibroblasts were used to test inhibitory receptors of the CD94/NKG2 family.

A specific NK cell effector function denoted ADCC is elicited when Ig-coated target cells are recognized by NK cells via their sole FcR, FcRIII, consisting of CD16 associated with the Fc chain. The essential role of Fc in ADCC is demonstrated by Fc-deficient mice, which fail to express FcRIII and as such have no demonstrable ADCC activity (34). Syk interacts with the ITAM of Fc and is activated during ADCC (35). To address the role of Syk in ADCC, lymphokine-activated NK cells from Syk⁺ or Syk^-/- chimeras were tested for their ability to lyse Ab-coated target cells in vitro. Syk⁺ NK cells lysed Ab-coated EL-4 thymoma cells with high efficiency (Fig. 4A and Table II). In contrast, Syk^-/- NK cells showed abnormal ADCC responses, corresponding to about 50% of normal (Fig. 4A and Table II). The defective ADCC was not attributable to an overall decrease in Syk^-/- NK cell effector function, as these same lymphokine-activated NK cells efficiently lysed several different targets, including YAC-1, P815, class I-deficient blasts, and L cells (Figs. 3B and 4, A and B, and Table II). Based on these results, it appears that Syk plays an important role in FcRIII triggering in NK cells, but that ADCC can proceed in vitro in the absence of Syk.

View larger version (28K): [in this window] [in a new window]   FIGURE 4. Signaling through FcRIII, NKR-P1, and 2B4 in Syk-/- NK cells. A, A-LAK cells where tested for the ability to lyse Ab (anti-Thy-1)-coated EL-4 thymoma targets () or control EL-4 cells (•). The specific lysis was determined at the indicated E:T cell ratios after a standard 4-h 51Cr release assay. , YAC-1 killing. The mean and SD of four independent experiments are shown. B, FcR+ P815 cells were used as targets for rADCC. A-LAK cells were preincubated for 15 min at room temperature with 5 µg/ml of 2B4, 20 µg/ml of NK1.1, 75 µg/ml of CD16 mAbs, or medium alone. A representative of three independent experiments is shown.

NK1.1, 2B4, and CD16 signaling in Syk-deficient NK cells

Cross-linking of the NKR-P1C molecule (defined by the mAb PK136; anti-NK1.1) on murine NK cells activates NK cell lytic functions and induces IFN- production (38). Previous studies have demonstrated that the Fc -chain is required for NK1.1-dependent signaling (39). Cross-linking of the 2B4 receptor enhances cytotoxicity, granule exocytosis, and IFN- production (40), while CD16 ligation on NK cells can induce IFN- production (41). To assess whether Syk is essential in any of these signaling pathways, Syk^-/- NK cells were tested for their capacity to mediate rADCC or to produce IFN- upon cross-linking of NK1.1, 2B4, or CD16 receptors. As shown in Fig. 4B, Syk^-/- LAK cells mediated rADCC after preincubation with either NK1.1 or 2B4 mAbs. In contrast, while cross-linking of CD16 in Syk^-/- NK cells resulted in some rADCC activity, the levels were clearly reduced relative to those in Syk⁺ LAK cells. Similarly, cross-linking of NK1.1 or 2B4 molecules on purified Syk^-/- NK cells resulted in IFN- production comparable with that by Syk⁺ NK cells, while IFN- production from Syk^-/- NK cells following CD16 ligation was defective, reaching only half the control levels (Fig. 5).

View larger version (23K): [in this window] [in a new window]   FIGURE 5. IFN- production by purified NK cells. Splenic DX5+ NK cells were FACS sorted and expanded in IL-2 or IL-15. After 6–8 days, 2 x 104 cells were plated in 200 µl of complete medium supplemented with human IL-2 (1000 U/ml), or on plates precoated with 2B4, NK1.1, CD16, or Gr-1 (control) mAbs. Stimulation with IL-12 served as a positive control. After 24 h, the supernatants were collected, and the amount of IFN- was quantitated by ELISA. Results are expressed as the mean and SD of three independent experiments.

Concluding remarks

The analysis of Syk-deficient NK cells in our novel alymphoid mouse strain demonstrates that this PTK plays a nonessential role in murine NK cell development and is dispensable for crucial NK cell effector functions, including natural cytotoxicity, generation of LAK activity, and cytokine production. Stimulation of a variety of NK surface receptors (including the IL-2/15R, NK1.1, 2B4, Ly49, and CD94/NKG2 complex) resulted in normal responses by Syk^-/- NK cells. Although the signaling pathways that permit NK cells to function in the absence of Syk remain unknown, a number of observations suggest that this may be achieved by the Syk-related PTK ZAP-70. We demonstrate that Syk^-/- NK cells express ZAP-70 protein. Moreover, previous studies have demonstrated that activation of NK cells can result in engagement of both Syk and ZAP-70 signaling pathways (reviewed in Ref. 17). These observations are consistent with interchangeable roles of Syk and ZAP-70 in NK cell differentiation. The analysis of NK cell development and NK cell effector functions in mice deficient in both Syk and ZAP-70 will allow these hypotheses to be addressed directly.

	Acknowledgments

 
We thank Dr. P. J. Dyson (London, U.K.) for kindly providing the F12 cells, Drs. O. Acuto (Pasteur Institute, Paris, France) and P. Hoglund (Strasbourg, France) for helpful discussions, and M. Malassis for technical assistance.

	Footnotes

 
¹ This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, the Association pour le Recherche sur le Cancer, the Ligue Nationale Contre le Cancer, the Fondation pour la Recherche Médicale, and the Medicinska Forskningsrådet (to F.C. and J.P.D.) and by the Medical Research Council, U.K. (to V.L.J.T.).

² Address correspondence and reprint requests to Dr. James P. Di Santo, Institut National de la Santé et de la Recherche Médicale, Unit 429, Pavillon Kirmisson, Hôpital Necker Enfants Malades, 149 rue de Sèvres, 75015 Paris, France. E-mail address:

³ Abbreviations used in this paper: PTK, protein tyrosine kinase; ITAM, immunoreceptor tyrosine-based activation motif; BCR, B cell Ag receptor; ADCC, Ab-dependent cell cytotoxicity; rADCC, reverse ADCC; HSC, hemopoietic stem cells; BM, bone marrow; FL, fetal liver; LAK, lymphokine-activated killer cells; A-LAK, adherent LAK; FcR, fragment c receptor; _c, common -chain; RAG, recombinase-activating gene; DN, dominant negative.

Received for publication February 22, 1999. Accepted for publication May 28, 1999.

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Top Abstract Introduction Materials and Methods Results and Discussion References

 

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