Pepro Tech
Originally published online as doi:10.1189/jlb.0304179 on July 7, 2004

Published online before print July 7, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
jlb.0304179v1
76/4/896    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zompi, S.
Right arrow Articles by Colucci, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zompi, S.
Right arrow Articles by Colucci, F.
(Journal of Leukocyte Biology. 2004;76:896-903.)
© 2004 by Society for Leukocyte Biology

The absence of Grb2-associated binder 2 (Gab2) does not disrupt NK cell development and functions

Simona Zompi*, Hahiua Gu{dagger} and Francesco Colucci*,1

* Cytokines and Lymphoid Development Unit, Department of Immunology, The Pasteur Institute, Paris, France; and
{dagger} Cancer Biology Program, Beth Israel-Deaconess Medical Center, Boston, Massachusetts

1Correspondence: Department of Immunology, Institut Pasteur, 25-28 rue Dr Roux, 75015 Paris, France. E-mail: cecco{at}pasteur.fr


arrow
ABSTRACT
 
Scaffolding molecules bind simultaneously and link together various components of signal-transduction pathways. Grb2-associated binder 2 (Gab2) is a scaffolding protein required for Fc{gamma}R-initiated allergic responses in mast cells and Fc{gamma}R-mediated phagocytosis in macrophages, where it links IgE and IgG receptors to the phosphatidylinositol-3 kinase (PI-3K) pathway. The Fc{gamma}R expressed by natural killer (NK) cells triggers antibody-dependent cellular cytotoxicity (ADCC). We show here that mouse NK cells express Gab2 and that although PI-3K was required for ADCC, this Fc{gamma}R-mediated function was normal in Gab2–/– NK cells. Moreover, NK cell development, spontaneous cytotoxicity, and responses to and production of cytokines were not perturbed in Gab2–/– mice. Considering the striking differences between the signaling requirements of Fc{gamma}R in macrophages and NK cells, our findings suggest that the organization of signal transduction downstream of the same FcR can be cell type-specific. Conversely, Gab family members Gab1, Gab2, and Gab3 may play specific roles in different leukocytes. As pharmacological targeting of Gab2 in mast cells is a potential strategy to treat allergy, our results suggest prudence, as NK cells may participate in IgE-mediated anaphylaxis in a Gab2-independent manner.

Key Words: cell-mediated cytotoxicity • intracellular signaling • FcR functions


arrow
INTRODUCTION
 
Natural killer (NK) cells are a subset of lymphocytes able to kill tumor cells and infected cells without prior stimulation. NK cells also produce proinflammatory and antimicrobial cytokines and chemokines that modulate immune responses [1 ]. Although we appreciate their effector functions in innate immunity and their regulatory functions in shaping adaptive immunity, the intracellular signals that regulate NK cells functions are not fully understood [2 ]. NK cells express, at their surface, multiple molecules capable of triggering cytotoxicity, among which is a low-affinity Fc receptor for immunoglobulin G (IgG; Fc{gamma}R)IIIA, also known as CD16, which is responsible for antibody (Ab)-dependent cellular cytotoxicity (ADCC) [3 4 ]. In addition to ADCC, ligation of CD16 on NK cells induces cytokine release, notably interferon-{gamma} (IFN-{gamma}) and tumor necrosis factor {alpha}.

Like all multichain immune-recognition receptors, CD16 lacks intrinsic enzymatic activity. The association of these receptors with adaptor signaling subunits that contain immune receptor-based tyrosine-activating motifs (ITAMs) allows signal transduction. Human CD16 can couple with homodimers or heterodimers composed of the signaling chains CD3{zeta} or Fc receptor for IgE (Fc{epsilon}R)I{gamma}, whereas mouse CD16 can only bind to Fc{epsilon}RI{gamma}. Ligation of CD16 by Ab-antigen (Ag)-immune complexes or by specific monoclonal Ab (mAb) activates membrane-associated Src tyrosine kinases, which phosphorylate tyrosine residues on the ITAMs. Syk family tyrosine kinases Syk and {zeta}-associated protein-70 (ZAP-70) are then recruited to the phosphorylated ITAMs and become activated. Syk tyrosine kinases are essential for signal transduction through CD16 and other ITAM-bearing receptors in NK cells [2 ]. Phosphatidylinositol-3 kinase (PI-3K) enzymes are also activated by CD16 and have been shown to participate in granule exocytosis and ADCC activity in human NK cells [5 6 ]. It is clear that the Syk and PI-3K pathways may be crucial in different forms of NK cell-mediated cytotoxicity [7 8 9 10 ]; however, it is not known if and how they are linked together.

A potential molecular link between Syk and PI-3K in NK cells is Grb2-associated binder 2 (Gab2), a member of the daughter of sevenless(Dos)/Gab family of scaffolding proteins, which includes mammalians Gab1, Gab2, and Gab3, Drosophila homologue Dos, and Caenorhabditis elegans homologue suppressor of clear 1. These adaptor molecules are devoid of any catalytic function but contain several binding sites for signaling molecules such as Grb2, Shc, Src homology-2-containing tyrosine phosphatase, and the p85 subunit of the PI-3K as well as a highly conserved pleckstrin homology domain, which mediates recruitment to the plasma membrane through interaction with phospholipids. In vitro studies have shown the implication of Gab2 in growth factor, cytokine, and multichain immune-recognition receptor signaling [11 12 13 ].

Although lymphoid and hematopoietic development was normal in Gab2–/– mice, mast cell responses to in vitro and in vivo stimulation of the high-affinity Fc{epsilon}RI were defective [14 ]. Thus, allergic reactions, such as anaphylaxis or cutaneous reactions, were markedly impaired. Gab2 was shown to play a key role in PI-3K activation in downstream Fc{epsilon}RI, suggesting that at least in mast cells, the ITAM-bearing and Syk-dependent Fc{epsilon}RI is linked to the PI-3K pathway by Gab2 [14 ].

A similar organization has been described in macrophages where Gab2 plays a key role in downstream PI-3K-dependent FcR signaling [15 ]. FcR triggering in macrophages initiates phagocytosis. Mouse macrophages express three receptors for IgGs: the high-affinity receptor Fc{gamma}RI (CD64), capable of binding monomeric IgG, and the low-affinity Fc{gamma}RIII (CD16), which only binds multimeric IgGs. The third FcR expressed by mouse macrophages is the inhibitory Fc{gamma}RIIB (CD32), also a low-affinity receptor (reviewed in ref. [16 ]). Fc{gamma}R-mediated phagocytosis was defective in Gab2–/– bone marrow-derived macrophages (BMM): This correlated with a decreased activation of the PI-3K pathway in Gab2–/– macrophages, as attested by impaired phosphorylation of Akt, a downstream target of PI-3K [15 ].

Thus, Gab2 is a key component of FcR signal transduction in mast cells and in macrophages. In these two cell types, Gab2 links together multichain immune-recognition receptors and the PI-3K pathway. We hypothesized that Gab2 could be an important player in NK cell-mediated ADCC in downstream CD16 signaling and could also participate in several aspects of NK cell biology. First, macrophages and NK cells share some of the crucial molecules involved in FcR signal transduction. For example, Src, Syk, and PI-3K are key components of the activation cascades that trigger Fc{gamma}R-mediated phagocytosis in macrophages and Fc{gamma}RIII-mediated ADCC in NK cells [5 6 17 18 ]. In addition to its potential role in signal transduction downstream of Fc{gamma}R in NK cells, the various Gab family members have been associated with other receptors that mediate functions relevant to NK cell biology. In particular, Gab2 participates in signal transduction downstream of c-kit and fetal liver tyrosine kinase 3 ligand (Flt3; reviewed in ref. [19 ]), two receptor tyrosine kinases involved in NK cell development [20 ]. Moreover, Gab2 participates in the signal transduction of the receptors for interleukin (IL)-2 and IL-15, two crucial cytokines for NK cell expansion in vitro and NK cell development in vivo, respectively [20 ].

With these premises, we analyzed the expression pattern of Gab family members in NK cells and studied NK cell development and effector functions in Gab2–/– mice. We found that NK cells expressed Gab2 protein, albeit at lower levels compared with mast cells and macrophages. Like mast cells and in contrast to macrophages, NK cells did not express transcripts for Gab1. Gab2 and Gab3 transcripts were present in NK cells. Gab2–/– NK cells developed with no apparent phenotype, responded to CD16 triggering by mediating normal ADCC and by producing and secreting IFN-{gamma}, were activated by and proliferated in response to IL-2 in vitro, and could kill tumor cells by spontaneous cytotoxicity. These results are compatible with a redundant role of Gab2 in NK cells and with a cell-specific organization of FcR signal-transduction pathways.


arrow
MATERIALS AND METHODS
 
Mice
Gab2–/– mice were described previously [14 ]. We crossed the original Gab2–/– mice (129/jxC57BL/6 background) for six generations onto the C57BL/6 background. C57BL/6, C57BL/6.Gab2–/–, and C57BL/6.Rag2–/– mice were kept at the Central Animal Facilities of The Pasteur Institut (Paris, France) and were used for experiments at 6–12 weeks of age. All protocols for animal experiments were reviewed by the Central Animal Facilities of The Pasteur Institut and were done in accordance with guidelines approved by the French Ministry of Agriculture.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA of various cells or spleen was extracted using the RNAble kit (Eurobio). RT-PCR was performed using specific oligonucleotides. Primers for Gab1 and Gab3 have been published previously [21 ]. We designed primers for Gab2 (Gab2 forward, 5'-CAACCTGAACTTGTGTGAGC-3'; Gab2 reverse, 5'-TGGTGGACAAGGTAGGCTGC-3'), which do not recognize Gab1 and Gab3 cDNA sequences found in the database. Standard PCR conditions were used to amplify Gab2 for 38 cycles and Gab 1 and Gab 3 for 35 cycles. Positive control for PCR amplification was done using hypoxanthine guanine phosphoribosyl transferase (HPRT) primers.

Western blot analysis
IL-2-activated wild-type (WT) or Gab2–/– NK cells were used for Western blot analysis. The cells were solubilized in buffer containing 1% Nonidet P-40 (Boehringer Mannheim, Mannheim, Germany), 25 mM Tris HCl, pH 7.5, 150 mM NaCl, 10 mM NaF, 1 mM sodium vanadate, and protease inhibitors from Boehringer Manheim. After 15 min on ice, the extracts were centrifuged for 30 min at 27,000 g, and supernatants were boiled in Laemmli sample buffer and used for Western blot.

Flow cytometry
Cell suspensions (0.5–1x106 cells/sample) from spleen, peritoneal cavity, liver, and BM were stained with different mAb conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), biotin, or allophycocyanin (APC; from PharMingen, San Diego, CA). Biotin-conjugated mAb were revealed by using streptavidin–peridinin chlorophyll protein (PharMingen). Analysis was performed on a FACSCalibur flow cytometer using Cell Quest software (Becton Dickinson, San Jose, CA).

NK cell cytotoxicity assays
A standard 4-h 51Cr release assay was used to measure NK activity in vitro. Target cells [yeast artificial chromosome-1 (YAC-1), resistance-modulating agent (RMA), Chinese hamster ovary (CHO)] were labeled with 100 µCi 51Cr (ICN Pharmaceutical, Costa Mesa, CA). Red cell-depleted splenocytes were used fresh (after passage on nylon wool column to enrich for NK cells) or cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 5 x 10–5 M ß-mercaptoethanol (ß-ME), 100 µg/ml streptomycin, 100 U/ml penicillin, and 1000 U/mL human IL-2 (R&D Systems, Minneapolis, MN) for 5–8 days. Before the assay, cells were stained with anti-NK1.1 mAb conjugated to PE or APC and anti-CD3 mAb conjugated to FITC. The number of effector cells was adjusted so as to have equivalent numbers of NK1.1+CD3– NK cells in the assay. For ADCC, RMA lymphoma cells were incubated with 20 µg/mL anti-Thy-1 mAb (anti-CD90.2) for 20 min at 4°C prior to the 4-h 51Cr release assay done using IL-2-activated NK cells as effectors. RMA cells belong to the T cell lineage and originate from C57BL/6 mice and therefore express the CD90.2 Ag. As they express major histocompatibility complex (MHC) class I, they are relatively resistant to NK cell cytotoxicity. Coating RMA cells with anti-CD90.2 makes them susceptible to NK cell-mediated lysis, overcoming the resistance imposed by MHC class I. For PI-3K-inhibition experiments, IL-2-activated NK cells were incubated for 30 min at 37°C with vehicle alone [dimethyl sulfoxide (DMSO)] or with various concentrations of the specific PI-3K inhibitor LY294002 and thereafter, used for the ADCC assay against RMA targets that had been previously coated or not with anti-CD90.2 at 20:1 or for spontaneous cytotoxicity assay against YAC-1 targets at 5:1.

Cytokine production
NK1.1+CD3– NK cells were electronically sorted with a FACStar Plus from fresh spleen and were cultured in RPMI 1640 supplemented with 10% FCS and 5 x 10–5 M ß-ME in the presence of 1000 U/ml human recombinant IL-2 for 5–6 days. Tissue-culture plates were coated with 20 µg/mL mAb diluted in phosphate-buffered saline for 16 h at 4°C. NK cells (105) were incubated in RPMI 1640 containing 10% FCS and IL-2 (1000–2000 U/ml) and were cultured for 24 or 48 h in plates containing the immobilized antibodies or IL-12 (5 ng/ml). The amount of IFN-{gamma} released in the culture supernatants was determined by using the mouse IFN-{gamma}-specific enzyme-linked immunosorbent assay kit OptEIA (PharMingen) and was used according to the manufacturer’s instructions.

Statistical analysis
Student’s t-test was done using Excel software. P > 0.05 was considered statistically nonsignificant.


arrow
RESULTS
 
Gab2 and Gab3 but not Gab1 are expressed in mouse NK cells
The three members of the Gab family are differentially expressed in various tissues and cell types [19 ]; however, the expression pattern in NK cells is not known. RT-PCR analysis of NK1.1+CD3– cells purified from fresh splenocytes revealed that NK cells express Gab2 and Gab3 but not Gab1 mRNA (Fig. 1a ). Splenic NK1.1+CD3– NK cells activated in vitro for 6–8 days with IL-2 showed a similar pattern of expression (data not shown). Moreover, the absence of Gab2 did not cause qualitative changes in the expression patterns of the various Gab family members. Thus, Gab2–/– NK cells, like WT NK cells, expressed Gab3 but not Gab1 transcripts (Fig. 1a) .



View larger version (31K):
[in this window]
[in a new window]
  		Figure 1. NK cells express Gab2 and Gab3 but not Gab1. (a) Total RNA was extracted from freshly purified or IL-2-activated (6–8 days cultures) WT and Gab2–/– NK cells. Total splenocytes and the pre-B cell lymphoma BaF3 cell line, which express all three Gab family members, were used as controls. (b) Whole cell lysates of purified NK cells and BMM from WT (+/+) and Gab2–/– (–/–) mice were immunoblotted with anti-Gab2 Ab (upper panel) and anti-Shc antibodies (lower panel) for loading control. (c) Whole cell lysates of 5 x 107-purified NK cells from WT (+/+) and Gab2–/– (–/–) mice were immunoprecipitated (IP) with anti-Gab2 Ab and subsequently immunoblotted with anti-Gab2 Ab.

Gab2 protein was expressed in NK cells as shown by Western blot, although the level of expression was lower than that found in BMM (Fig. 1b) and in mast cells (not shown). Mast cells of Gab2–/– mice have been reported to express a truncated version of the Gab2 protein that can, in principle, compensate for at least some functional activities of the full-length Gab2 protein [14 ]. The sharp band showing in NK cell extract from Gab2–/– mice is most probably a cross-reactive one, as no truncated form of Gab2 was detected in NK cells purified from Gab2–/– mice (Fig. 1c) . Gab2 is phosphorylated upon engagement of the receptors for IL-2 and IL-15 [22 ]. IL-2-activated NK cells, unlike resting T cells, presented a migration shift compatible with phosphorylation of Gab2 (data not shown).

NK cell development in Gab2–/– mice
Gab2 participates in the signal-transduction machinery of the receptors for several cytokines including some that are crucial for the biology of NK cells such as stem cell factor, Flt3L, IL-2, and IL-15 (reviewed in refs. [19 , 20 ]). Nevertheless, the percentages and absolute numbers of NK1.1+CD3– NK cells in WT and Gab2–/– mice were not significantly different in any of the organs analyzed. Thus, NK cell development appears to proceed normally in the absence of Gab2 (Fig. 2a ). Moreover, Gab2–/– NK cells responded with a vigorous proliferation to IL-2 stimulation in vitro.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. Gab2 is dispensable for NK cell development. (a) Cells from spleen, BM, liver, and peritoneal cavity were stained for NK1.1 and CD3. Absolute numbers of NK1.1+CD3– cells were counted. Each data point represents a single mouse. Results are pooled from four independent experiments. (b) Spleen cells were stained with NK1.1–PE and CD3–FITC. Figures show percentages of NK cells (NK1.1+CD3–) positive for different NK receptors. No significant differences were detected between WT and Gab2–/– NK cells. Results are from one representative of four independent experiments. (c) Spleen cells were stained as described in b. Figure shows mean fluorescence intensity (MFI) of CD16 expression in NK1.1+CD3– NK cells. No significant difference was detected between WT and Gab2–/– NK cells. Results are from one representative of four independent experiments.

NK cell differentiation follows a series of steps that can be identified by studying the combinations of surface markers expressed sequentially on the developing NK cell [20 ]. WT and Gab2–/– NK cells showed comparable expressions of various NK cell-surface markers such as members of the Ly49 and NKG2 families, DX5 (CD49b), Mac-1 (CD11b), 2B4 (CD124), and CD69 (Fig. 2b and data not shown). Thus, NK cells of Gab2–/– mice show a mature phenotype. In line with the results obtained with the expression of all the other markers tested, no difference in Fc{gamma}RIII (CD16) expression was found between WT and Gab2–/– NK cells (Fig. 2c : MFI=26.3±3.5 and 23.4±2.5 in WT and Gab2–/–, respectively; P=0.11; n=4).

PI-3K but not Gab2 is crucial for spontaneous cytotoxicity and ADCC
The signals that activate ADCC are better characterized than those that activate spontaneous cytototoxicity [2 23 ]. For example, the PI-3K pathway has been shown to be essential for ADCC in human NK cells [5 6 ], but its role in spontaneous cytotoxicity is controversial [2 5 9 ]. We show here that IL-2-activated mouse NK cells depend on functional PI-3K to mediate effective spontaneous cytotoxicity and ADCC (Fig. 3a and 3b). In addition, NK cells of mice lacking an isoform of the catalytic subunit of PI-3K had defective FcR responses upon Ab cross-linking, direcly implicating PI-3K downstream of FcR in mouse NK cells (S. Zompi and F. Colucci, unpublished observation), recapitulating findings in human NK cells [6 ]. As Gab2 plays crucial roles in PI-3K activation downstream of Fc{epsilon}R in mast cells and of Fc{gamma}R in macrophages, it could play similar roles downstream of NK cell-activating receptors. However, Gab2–/– NK cells could spontaneously kill YAC-1 lymphoma cells as efficiently as WT NK cells (Fig. 4a ); thus, Gab2 is not essential for this PI-3K-dependent NK cell function. Moreover, ADCC activity of Gab2–/– NK cells was comparable with that of WT NK cells (Fig. 4b) . Thus, in contrast to mast cells [14 ] and macrophages [15 ], the multichain immune-recognition FcR expressed by NK cells is functional in the absence of Gab2.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 3. Spontaneous cytotoxicity and ADCC depend on PI-3K. (a) Spontanous cytotocixity: IL-2-activated NK cells from Rag2–/– mice were first incubated with the indicated doses of the PI-3K inhibitor LY294002 and thereafter, coincubated with YAC-1 tumor cells for a 4-h chromium release assay at an effector-to-target ratio of 5:1. Data are means ± SD from a representative of three independent experiments that gave similar results. (b) ADCC: IL-2-activated NK cells from Rag2–/– mice were first incubated with the indicated doses of the PI-3K inhibitor LY294002 and thereafter, coincubated with RMA tumor cells that had been previously coated ({blacksquare}) or not ({diamond}) with anti-CD90.2, for a 4-h chromium release ADCC assay at an effector-to-target ratio of 20:1. Data are means ± SD from a representative of three independent experiments that gave similar results.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Spontaneous cytotoxicity and ADCC do not depend on Gab2. (a) Spontaneous cytotoxicity: NK cell-enriched, fresh splenocytes (left panel) or IL-2-activated NK cells (right panel) from WT (control, {blacksquare}) and Gab2–/– mice ({circ}) were used in a 4-h chromium release assay at the indicated NK:target cell ratios against YAC-1 targets. Data are means ± SD from a representative of three independent experiments that gave similar results. (b) ADCC: IL-2-activated WT (control, {blacksquare}) and Gab2–/– ({circ}) NK cells (day 8 cultures) were coincubated with RMA tumor cells that had been previously coated or not with anti-CD90.2 for a 4-h chromium release ADCC assay at the indicated NK:target cell ratios. Data are means ± SD from a representative of three independent experiments that gave similar results.

Finally, we tested and excluded the possibility that the Ly49D-activating receptor could require Gab2, as cytotoxicity against CHO target cells was similar in WT and Gab2–/– NK cells (data not shown).

IFN-{gamma} production in the absence of Gab2
NK cells produce IFN-{gamma} upon stimulation by proinflammatory cytokines IL-12 and IL-18 or upon stimulation of different receptors, including CD16. These physiological situations can be reproduced in vitro by stimulating NK cells with natural ligands such as IL-12 or plate-bound antibodies. Gab2–/– NK cells could produce as much IFN-{gamma} as WT NK cells in response to all the different stimuli tested, including IL-12, anti-CD16, anti-2B4, and anti-NK1.1 Ab (Fig. 5 ). Similar results were obtained by stimulating NK cells with nonsaturating doses of anti-CD16, (data not shown). Therefore, cytokine production by NK cells is not perturbed in the absence of Gab2.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 5. Gab2 is dispensable for IFN-{gamma} production. Sorted IL-2-activated NK cells of Gab2–/– and control WT mice were stimulated with 5 ng/ml IL-12, 20 µg/ml anti-CD16, anti-NK1.1, and anti-2B4 or were left with no stimulus. Data are means ± SD of triplicates from one representative of two independent experiments giving similar results.


arrow
DISCUSSION
 
We found that NK cells express the scaffolding proteins Gab2 and Gab3, albeit Gab2 was expressed at lower levels compared with macrophages and mast cells. NK cells did not express Gab1 transcripts. Gab2 plays a pivotal role in Fc{epsilon}R-initiated, allergic responses in mast cells [14 ] and Fc{gamma}R-mediated phagocytosis in macrophages [15 ]. Moreover, Gab2 has been implicated in lymphocyte signal transduction, where it mediates a negative control over T cell receptor signaling [24 ] and transduces signals downstream of the receptors for IL-2 and IL-15 (IL-2R and IL-15R, respectively) in T cells [22 ]. In striking contrast, our results show that Gab2 is not an essential player in the transduction of crucial signals that regulate NK cell development and differentiation, nor NK cell functions, including expansion and activation in IL-2, spontaneous cytotoxicity, Fc{gamma}R-mediated ADCC, and cytokine production. Gab2–/– NK cells do not express the truncated form of Gab2 found in Gab2–/– mast cells, ruling out any possible contribution to functional activity.

The lack of Gab1 transcripts in NK cells and the absence of an obvious phenotype in Gab2–/– NK cells make Gab3 the most likely family member involved in the biology of NK cells. Normal numbers of DX5+Mac-1+ NK cells were found in spleen and BM of Gab3–/– mice [25 ], indicating that Gab3 may also be dispensable for NK cell development. However, NK cell functions were not assessed in Gab3–/– mice. The analysis of NK cell functions in Gab3–/– mice combined with the analysis of NK cell development and functions in compound Gab2–/–3–/– mice will address the questions of whether Gab scaffolding proteins are required for NK cell biology; if so, which aspects of NK cell development and functions they regulate; and whether Gab2 and Gab3 are functionally redundant.

From our results stem a number of considerations about the differential requirements for Gab2 in NK cells as compared with other leukocytes, including T lymphocytes, macrophages, and mast cells. First, stimulation of IL-2R and IL-15R in human T and NK cells induces Gab2 phosphorylation, which is Src-independent and Janus tyrosine kinase-3-dependent [22 ]. IL-2R and IL-15R, in human and mouse, share the ß{gamma} signaling complex but express unique high-affinity {alpha}-chains. Mouse NK cells, unlike human NK cells, do not express the high-affinity {alpha}-chain of the IL-2R; therefore, NK cell responses to IL-2 are actually mediated by the IL-15 ß{gamma} receptor complex. As Gab2–/– NK cells expanded normally and became fully activated following IL-2 stimulation in vitro, our results suggest that Gab2 is not essential for transducing signals downstream of IL-15R, which is a crucial cytokine for NK cell development [20 ]. The result showing that Gab2–/– NK cells develop to normal numbers and display normal expression of differentiation markers is in line with the notion that Gab2 is not essential for IL-15R signaling in NK cells. In this context, it is worth noting that our results suggest that Gab2 is not required to transduce signals downstream of other cytokine receptors involved in NK cell development such as c-kit [26 ] and FLT3 [27 ], in spite of the association of Gab2 with these receptors (ref. [19 ] and references therein). In fact, Gab2 is required for c-kit signaling during mast cell development [28 ] but not NK cell development (this report).

Second, we found that signal transduction downstream of the NK cell FcR was apparently intact in Gab2–/– NK cells, whereas macrophages Fc{gamma}R and mast cells Fc{epsilon}R require Gab2 to function properly. However surprising this finding was, other instances of such cell-specific requirement downstream of Fc{gamma}R have been described previously. For example, although Src, Syk, and PI-3K kinases participate in the activation cascades that trigger Fc{gamma}R-mediated phagocytosis in macrophages and Fc{gamma}RIII-mediated ADCC in NK cells (refs. [5 , 17 18 ] and this report), different members of these kinase families may be involved in these signaling pathways. Thus, Fc{gamma}R preferentially associates with Lyn and Syk in macrophages, whereas it couples with Lck and ZAP-70 in NK cells (reviewed in ref. [16 ]). One possibility is that NK cell Fc{gamma}R preferentially uses Gab3, whereas macrophages use Gab2. Future experiments with Gab3–/– NK cells will clarify this issue.

Phagocytosis and ADCC engage different cellular events, i.e., formation of phagocytic cup versus degranulation of cytotoxic granules. Although the same Fc{gamma}R triggers these two effector functions, separate pathways and distinct signaling molecules may well regulate different downstream events. Conversely, Fc{epsilon}R and Fc{gamma}R induce similar downstream events in mast cells and NK cells, respectively, i.e., degranulation and cytokine gene transcription. Given that, it was striking to find intact ADCC and cytokine release in Gab2–/– NK cells, as opposed to defective degranulation and cytokine release in Gab2–/– mast cells [14 ]. Gab2 and Gab3 may be functionally redundant in NK cells, or NK cells preferentially use Gab3. Alternatively, the pathways that regulate cytokine induction downstream of FcRs are distinct in mast cells and NK cells.

The activating receptor NKG2D has been shown to be a major contributor to trigger NK cell-spontaneous cyototoxicity against YAC-1 targets. In fresh, nonstimulated NK cells, this receptor is constitutively associated with the adaptor DAP10, which lacks ITAM but possess a consensus motif for recruitment and activation of PI-3K. Our results confirm that PI-3K is a crucial component of spontaneous cytotoxicity against YAC-1 tumor cells and suggest that Gab2 is not essential for the NKG2D–DAP10–PI-3K pathway of spontaneous cytotoxicity.

Finally, our results may have some clinical implications. Mast cell-deficient mice display Fc{epsilon}R-independent allergic responses [29 ], and Fc{gamma}RIII–/– mice display reduced IgE-mediated anaphylaxis [30 ]. Therefore, cells other than mast cells and receptors other than Fc{epsilon}R can participate in IgE-mediated anaphylaxis. IgE can activate Fc{gamma}RIII in NK cells and initiate production of cytokines and chemokines [31 ]. This suggests a possible involvement of NK cells in type I hypersensitivity. In line with this, NK cells regulate allergen-induced eosinophilic airways inflammation [32 ]. In addition, IgE-mediated ADCC could also play a role in type II hypersensitivity. Pharmacological targeting of Gab2 in mast cells is a potential strategy to treat allergy; however, our results suggest prudence, as NK cells may participate in IgE-mediated anaphylaxis in a Gab2-independent manner.


arrow
ACKNOWLEDGEMENTS
 
This work was supported by Ligue National Contre le Cancer, Association pour la Recherche contre le Cancer, Institut Pasteur, INSERM (to F. C. and S. Z.), and National Institutes of Health (to H. G). We thank Cécile Pham for excellent secreterial assistance, Anne-Marie Balazuc and Anne Louise for FACS sorting, James P. Di Santo for support, Erwan Corcuff for skilled technical help, and Marc Däeron and Paul Leibson for critically reading the manuscript.

Received March 22, 2004; revised May 24, 2004; accepted May 28, 2004.


arrow
REFERENCES
 
    1
  1. Trinchieri, G. (1989) Biology of natural killer cells Adv. Immunol. 47,187-376[Medline]
    2. 2
  2. Colucci, F., Di Santo, J. P., Leibson, P. J. (2002) Natural killer cell activation in mice and men: different triggers for similar weapons? Nat. Immunol. 3,807-813[CrossRef][Medline]
    3. 3
  3. Lanier, L. L., Le, A. M., Phillips, J. H., Warner, N. L., Babcock, G. F. (1983) Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens J. Immunol. 131,1789-1796[Abstract]
    4. 4
  4. Perussia, B., Starr, S., Abraham, S., Fanning, V., Trinchieri, G. (1983) Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. I. Characterization of the lymphocyte subset reactive with B73.1 J. Immunol. 130,2133-2141[Abstract]
    5. 5
  5. Bonnema, J. D., Karnitz, L. M., Schoon, R. A., Abraham, R. T., Leibson, P. J. (1994) Fc receptor stimulation of phosphatidylinositol 3-kinase in natural killer cells is associated with protein kinase C-independent granule release and cell-mediated cytotoxicity J. Exp. Med. 180,1427-1435[Abstract/Free Full Text]
    6. 6
  6. Kanakaraj, P., Duckworth, B., Azzoni, L., Kamoun, M., Cantley, L. C., Perussia, B. (1994) Phosphatidylinositol-3 kinase activation induced upon Fc {gamma} RIIIA-ligand interaction J. Exp. Med. 179,551-558[Abstract/Free Full Text]
    7. 7
  7. Zompi, S., Hamerman, J. A., Ogasawara, K., Schweighoffer, E., Tybulewicz, V. L., Di Santo, J. P., Lanier, L. L., Colucci, F. (2003) NKG2D triggers cytotoxicity in mouse NK cells lacking DAP12 or Syk family kinases Nat. Immunol. 4,565-572[CrossRef][Medline]
    8. 8
  8. Billadeau, D. D., Upshaw, J. L., Schoon, R. A., Dick, C. J., Leibson, P. J. (2003) NKG2D-DAP10 triggers human NK cell-mediated killing via a Syk-independent regulatory pathway Nat. Immunol. 4,557-564[CrossRef][Medline]
    9. 9
  9. Jiang, K., Zhong, B., Gilvary, D. L., Corliss, B. C., Hong-Geller, E., Wei, S., Djeu, J. Y. (2000) Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells Nat. Immunol. 1,419-425[CrossRef][Medline]
    10. 10
  10. Jiang, K., Zhong, B., Gilvary, D. L., Corliss, B. C., Vivier, E., Hong-Geller, E., Wei, S., Djeu, J. Y. (2002) Syk regulation of phosphoinositide 3-kinase-dependent NK cell function J. Immunol. 168,3155-3164[Abstract/Free Full Text]
    11. 11
  11. Gu, H., Pratt, J. C., Burakoff, S. J., Neel, B. G. (1998) Cloning of p97/Gab2, the major SHP2-binding protein in hematopoietic cells, reveals a novel pathway for cytokine-induced gene activation Mol. Cell 2,729-740[CrossRef][Medline]
    12. 12
  12. Nishida, K., Yoshida, Y., Itoh, M., Fukada, T., Ohtani, T., Shirogane, T., Atsumi, T., Takahashi-Tezuka, M., Ishihara, K., Hibi, M., Hirano, T. (1999) Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors Blood 93,1809-1816[Abstract/Free Full Text]
    13. 13
  13. Gu, H., Maeda, H., Moon, J. J., Lord, J. D., Yoakim, M., Nelson, B. H., Neel, B. G. (2000) New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway Mol. Cell. Biol. 20,7109-7120[Abstract/Free Full Text]
    14. 14
  14. Gu, H., Saito, K., Klaman, L. D., Shen, J., Fleming, T., Wang, Y., Pratt, J. C., Lin, G., Lim, B., Kinet, J. P., Neel, B. G. (2001) Essential role for Gab2 in the allergic response Nature 412,186-190[CrossRef][Medline]
    15. 15
  15. Gu, H., Botelho, R. J., Yu, M., Grinstein, S., Neel, B. G. (2003) Critical role for scaffolding adapter Gab2 in Fc {gamma} R-mediated phagocytosis J. Cell Biol. 161,1151-1161[Abstract/Free Full Text]
    16. 16
  16. Ravetch, J. V., Bolland, S. (2001) IgG Fc receptors Annu. Rev. Immunol. 19,275-290[CrossRef][Medline]
    17. 17
  17. Fitzer-Attas, C. J., Lowry, M., Crowley, M. T., Finn, A. J., Meng, F., DeFranco, A. L., Lowell, C. A. (2000) Fc{gamma} receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn J. Exp. Med. 191,669-682[Abstract/Free Full Text]
    18. 18
  18. Colucci, F., Schweighoffer, E., Tomasello, E., Turner, M., Ortaldo, J. R., Vivier, E., Tybulewicz, V. L., Di Santo, J. P. (2002) Natural cytotoxicity uncoupled from the Syk and ZAP-70 intracellular kinases Nat. Immunol. 3,288-294[CrossRef][Medline]
    19. 19
  19. Liu, Y., Rohrschneider, L. R. (2002) The gift of Gab FEBS Lett 515,1-7[CrossRef][Medline]
    20. 20
  20. Colucci, F., Caligiuri, M. A., Di Santo, J. P. (2003) What does it take to make a natural killer? Nat. Rev. Immunol. 3,413-425[CrossRef][Medline]
    21. 21
  21. Wolf, I., Jenkins, B. J., Liu, Y., Seiffert, M., Custodio, J. M., Young, P., Rohrschneider, L. R. (2002) Gab3, a new DOS/Gab family member, facilitates macrophage differentiation Mol. Cell. Biol. 22,231-244[Abstract/Free Full Text]
    22. 22
  22. Gadina, M., Sudarshan, C., Visconti, R., Zhou, Y. J., Gu, H., Neel, B. G., O’Shea, J. J. (2000) The docking molecule Gab2 is induced by lymphocyte activation and is involved in signaling by interleukin-2 and interleukin-15 but not other common {gamma} chain-using cytokines J. Biol. Chem. 275,26959-26966[Abstract/Free Full Text]
    23. 23
  23. Lanier, L. L. (2003) Natural killer cell receptor signaling Curr. Opin. Immunol. 15,308-314[CrossRef][Medline]
    24. 24
  24. Pratt, J. C., Igras, V. E., Maeda, H., Baksh, S., Gelfand, E. W., Burakoff, S. J., Neel, B. G., Gu, H. (2000) Cutting edge: Gab2 mediates an inhibitory phosphatidylinositol 3'-kinase pathway in T cell antigen receptor signaling J. Immunol. 165,4158-4163[Abstract/Free Full Text]
    25. 25
  25. Seiffert, M., Custodio, J. M., Wolf, I., Harkey, M., Liu, Y., Blattman, J. N., Greenberg, P. D., Rohrschneider, L. R. (2003) Gab3-deficient mice exhibit normal development and hematopoiesis and are immunocompetent Mol. Cell. Biol. 23,2415-2424[Abstract/Free Full Text]
    26. 26
  26. Colucci, F., Di Santo, J. P. (2000) The receptor tyrosine kinase c-kit provides a critical signal for survival, expansion, and maturation of mouse natural killer cells Blood 95,984-991[Abstract/Free Full Text]
    27. 27
  27. McKenna, H. J., Stocking, K. L., Miller, R. E., Brasel, K., De Smedt, T., Maraskovsky, E., Maliszewski, C. R., Lynch, D. H., Smith, J., Pulendran, B., Roux, E. R., Teepe, M., Lyman, S. D., Peschon, J. J. (2000) Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells Blood 95,3489-3497[Abstract/Free Full Text]
    28. 28
  28. Nishida, K., Wang, L., Morii, E., Park, S. J., Narimatsu, M., Itoh, S., Yamasaki, S., Fujishima, M., Ishihara, K., Hibi, M., Kitamura, Y., Hirano, T. (2002) Requirement of Gab2 for mast cell development and KitL/c-Kit signaling Blood 99,1866-1869[Abstract/Free Full Text]
    29. 29
  29. Martin, T. R., Takeishi, T., Katz, H. R., Austen, K. F., Drazen, J. M., Galli, S. J. (1993) Mast cell activation enhances airway responsiveness to methacholine in the mouse J. Clin. Invest. 91,1176-1182
    30. 30
  30. Ujike, A., Ishikawa, Y., Ono, M., Yuasa, T., Yoshino, T., Fukumoto, M., Ravetch, J. V., Takai, T. (1999) Modulation of immunoglobulin (Ig)E-mediated systemic anaphylaxis by low-affinity Fc receptors for IgG J. Exp. Med. 189,1573-1579[Abstract/Free Full Text]
    31. 31
  31. Arase, N., Arase, H., Hirano, S., Yokosuka, T., Sakurai, D., Saito, T. (2003) IgE-mediated activation of NK cells through Fc {gamma} RIII J. Immunol. 170,3054-3058[Abstract/Free Full Text]
    32. 32
  32. Korsgren, M., Persson, C. G., Sundler, F., Bjerke, T., Hansson, T., Chambers, B. J., Hong, S., Van Kaer, L., Ljunggren, H. G., Korsgren, O. (1999) Natural killer cells determine development of allergen-induced eosinophilic airway inflammation in mice J. Exp. Med. 189,553-562[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
N. Kim, A. Saudemont, L. Webb, M. Camps, T. Ruckle, E. Hirsch, M. Turner, and F. Colucci
The p110delta catalytic isoform of PI3K is a key player in NK-cell development and cytokine secretion
Blood, November 1, 2007; 110(9): 3202 - 3208.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
jlb.0304179v1
76/4/896    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zompi, S.
Right arrow Articles by Colucci, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zompi, S.
Right arrow Articles by Colucci, F.