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RIII1
* Department of Molecular Genetics, Graduate School of Medicine, Chiba University, Chiba, Japan; and
Laboratory of Cell Signaling, RIKEN, Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIII (CD16), which is responsible for IgG-dependent cell cytotoxicity and for production of several cytokines and chemokines. Whereas FcRIII on NK cells is composed of both FcRIII
and FcR chains, that on mast cells is distinct from NK cells and made of FcRIII, FcR
, and FcR. Mast cells show degranulation and release several mediators, which cause anaphylactic responses upon cross-linking of FcRIII as well as Fc
RI with aggregated IgE. In this paper, we examined whether IgE activates NK cells through FcRIII on their cell surface. We found that NK cells produce several cytokines and chemokines related to an allergic reaction upon IgE stimulation. Furthermore, NK cells exhibited cytotoxicity against IgE-coated target cells in an FcRIII-dependent manner. These effects of IgE through FcRIII were not observed in NK cells from FcR-deficient mice lacking FcRIII expression. Collectively, these results demonstrate that NK cells can be activated with IgE through FcRIII and exhibit both cytokine/chemokine production and Ab-dependent cell cytotoxicity. These data imply that not only mast cells but also NK cells may contribute to IgE-mediated allergic responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI induces mast cells to release various mediators and results in an anaphylactic reaction. Because FcRI-deficient (-/-) mice were found to be resistant to cutaneous and systemic IgE-mediated anaphylaxis, a prominent role for FcRI exclusively expressed on mast cells in classical type I hypersensitivity was established (1). By contrast, IgG was shown in the 1960s to induce passive anaphylaxis in vivo (2). The studies using FcRI-/-, FcR-/-, or FcRIIB-/- mice confirmed the importance of IgG in systemic and in passive cutaneous anaphylaxis. In OVA-induced systemic anaphylaxis, both IgG1 and FcRIII are more important than IgE and FcRI (3). Furthermore, FcRIIB-/- mice showed enhanced IgG1-mediated passive cutaneous anaphylaxis (4). These observations revealed that not only the FcRI but also the FcR system (3, 4) could regulate type I hypersensitivity depending on the Ag.
It was demonstrated that IgE binds to FcRIII on a mast cell line in vitro and causes degranulation (5). Furthermore, when IgE-mediated anaphylaxis was compared between FcRIII-/- and wild-type (wt)3 mice, the anaphylaxis was more severe in wt mice than in FcRIII-/- mice (6). These studies suggested the importance of FcRIII for not only IgG- but also IgE-mediated anaphylaxis. In contrast, it has been shown that systemic anaphylaxis occurs in mast cell-deficient W/Wv and Sl/Sld mice, indicating that some cells other than mast cells could induce anaphylactic responses in their absence (7, 8, 9). Moreover, Choi et al. (10) showed that systemic anaphylaxis induced by penicillin occurred in mast cell-deficient mice and was correlated with the serum IgE level but not with the IgG level. Although NK cells, as well as macrophages (M
) and neutrophils, are known to highly express FcRIII, its function in IgE-mediated anaphylactic responses has not been analyzed.
NK cells mediate natural cytotoxicity against a variety of tumor cells and virus-infected cells. They also produce cytokines and chemokines upon recognition of target cells, without prior sensitization (11). NK cells have also been implicated in eosinophilic airway inflammation in mice (12). In addition, a specific correlation between NK cell function and total serum IgE levels had also been observed (13). Although these analyses suggested the possible involvement of NK cells in allergic reactions, direct evidence of the functional participation of NK cells in allergic responses has not yet been reported.
Stimulation of mast cells through FcRIII with oligomeric IgE induced serotonin secretion in vitro (5). This study provided the first evidence that IgE could bind FcRIII and stimulates mast cells via this receptor. The composition of FcRIII on mast cells (FcRIII, FcR, and FcR chains) is different from that on other cell types, which is composed of only FcRIII and FcR (14, 15). Therefore, it still remains unclear whether FcRIII-expressing cells other than mast cells can be activated by IgE.
In this study, we investigated the function of FcRIII as an IgE receptor on normal NK cells and demonstrated that NK cells can be activated with IgE through their FcRIII. NK cells exhibited secretion of several cytokines and chemokines and mediated Ab-dependent cell cytotoxicity (ADCC) against IgE-coated targets. From these data, the possible involvement of NK cells in type I hypersensitivity is discussed.
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Materials and Methods |
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C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). FcR-/- mice with C57BL/6 background were generated as previously described (16).
Preparation of NK cells
Murine NK cells were isolated as previously described (17). Briefly, splenocytes were mixed with anti-CD4 mAb (GK1.5) and anti-CD8 mAb (53.6.7) followed by incubation with magnetic beads coupled with goat anti-mouse and rat IgG Abs (Perceptive Biosystems, Framingham, MA). The residual cells were then stained with PE-anti-NK1.1 mAb and FITC-anti-CD3 mAb (BD PharMingen, San Diego, CA), and NK1.1+ CD3- cells were sorted by FACStarPlus (BD Biosciences). The purity of the sorted cells was always >99%. Purified NK cells were cultured in RPMI 1640 containing 10% FCS, 2 mM glutamine, kanamycin (100 µg/ml), and 5 x 10-5 M 2-ME and 1000 U/ml human IL-2 (provided by Dr. J. Hamuro (Ajinomoto, Kawasaki, Japan)) for 7 days. Bone marrow-derived mast cells (BMMC) were obtained by culture of bone marrow cells for 5 wk in complete medium with 15% WIHI-3 culture supernatant as a source of IL-3. M were separated by cell sorting of Mac-1+ cells from the peritoneal cavity from thioglycolate-injected mice.
Stimulation of NK cells
A 96-well plate was incubated overnight at 4°C with graded concentrations of either mouse IgG1 (anti-biotin; Zymed, San Francisco, CA) or IgE (anti-DNP; Sigma-Aldrich, St. Louis, MO), and then washed with PBS. Cultured NK cells (2 x 105) were stimulated in the plate immobilized with IgG1 or IgE for 2 days for ELISA. Anti-FcRII/III mAb (20 µg/ml; 2.4G2; BD PharMingen) was added for blocking of the FcRIII. For real-time PCR analysis, NK cells were stimulated on the 96-well plate immobilized with 50 µg/ml IgE or IgG mAb. After 6- or 12-h stimulation, the cells were harvested and lysed.
RT-PCR
Total cellular RNA was extracted with an RNeasy mini kit (Qiagen, Tokyo, Japan) and was reverse transcribed using Superscript II (Invitrogen, Tokyo, Japan) and random hexamers (Invitrogen) as primer. Real-time PCR was conducted by iCycler thermocycler (Bio-Rad, Hercules, CA) using SYBR green PCR master mix (Qiagen) and 300 pmol/ml each primer pair. Amplification condition was as follows: 95°C for 5 min, 50 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 30 s. For the analysis of the FcR expression in NK cells, RNA was extracted by guanidinium-isothiocyanate method, and cDNA was prepared by RT-PCR. cDNA was amplified by PCR under the following conditions: 94°C for 15 s, 57°C for 30 s, and 72°C for 1 min with 22–24 cycles for -actin or 30–34 cycles for other primers. The following primers were used: FcRI, 5'-CTGCTACTTTGGGTTCCAG and 3'-CCGAATCTGAAGAAAACTGA; FcRIIB, 5'-CCCAAGTCCAGCAGGTCTTTA and 3'-CTGTTTCTTCATCCAGGGCTT; FcRIII, 5'-GTTTAAGGCCACAGTCAATG and 3'-GGTTGGCTTTTGGGATAG; FcRI, 5'-TGAGTGCCACCGTTCAAGA and 3'-CAAACAGAATCGCCACCAAC; FcR, 5'-CAGACATGGCGGACATT and 3'-GTTGTTCATATAAGCGAAGTT; FcR, 5'-AAGAATTCCAGCGCCGCAGCCCCCAGCG and 3'-GGAATTCGCTGCCTTTCGGACCTGGAT; and -actin, 5'-ACCCACACTGTGCCCATCTA and 3'-TCATGGATGCCACAGGATTC. To detect several cytokines and chemokines, the following primers were used: IFN-, 5'-CCTCAGACTCTTTGAAGTCT and 3'-CAGCGACTCCTTTTCCGCTT; TNF-, 5'-ATGAGCACAGAAAGCATGATCCGCGAC and 3'-TCACAGAGCAATGACTCCAAAGTAGACCTG; GM-CSF, 5'-CCGCTCACCCATCACTGTCAC and 3'-AGGCTGTCTATGAAATCCGCA; and macrophage-inflammatory protein (MIP)-1, 5'-CAAGTCTTCTCAGCGCCATA and 3'-TCTTTGGAGTCAGCGCAGAT.
Measurement of IFN- production
The amount of IFN- produced was measured by ELISA (16) using anti-IFN- mAbs (R4-6A2 and XMG1.2; BD PharMingen).
Analysis of ADCC activity
ADCC activity was analyzed as previously described except for the target cell preparation (17). For preparing hapten-coupled streptavidin, 0.5 mg of streptavidin (1 mg/ml; Sigma-Aldrich) dialyzed with 0.1 M NaHCO3 overnight was reacted with 25 µg of DNP-X succinimidyl ester or 25 µg of dansyl-X succinimidyl ester (Molecular Probes, Eugene, OR) for 2 h at room temperature, and dialyzed with PBS. P815 cells were surface biotinylated (EZ-Link Sulfo-NHS-Biotin; Pierce, Rockford, IL), followed by incubation with 51Cr-labeled sodium citrate (Amersham Bioscience, Tokyo, Japan). The cells were labeled with DNP-streptavidin or dansyl-streptavidin, then incubated with 5 µg/ml anti-DNP IgE mAb (Sigma-Aldrich) and anti-DNP IgG1 mAb (F1E2; kindly provided by Dr. S. Taki (Shinshu University, Matsumoto, Japan)), or with 5 µg/ml anti-dansyl IgE and anti-dansyl IgG2b mAbs (BD PharMingen). A standard short-term 51Cr release assay was performed by mixing 1 x 104 IL-2-activated NK cells with graded numbers of the 51Cr-labeled target cells in the presence of 5 µg/ml each Ab in a U-bottom 96-well plate (BD Biosciences) for 4 h and then measuring the released 51Cr in the supernatant. Specific cytolysis was calculated as follows: ((E - C)/(M - C)) x 100 (E, 51Cr release by effector cells plus target cells; C, target cells alone; and M, the maximum release).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIII on NK cells
The expression of various FcR chains in our NK cell population was examined by RT-PCR analysis to exclude the possibility of a low expression of other FcR in our NK cell preparation (Fig. 1). cDNAs prepared from IL-2-cultured murine NK cells, peritoneal M, and BMMC were amplified with specific primers for each FcR chain. Consequently, the NK cell preparation expressed only FcRIII and FcR, but not FcRI, FcR, FcRI, or FcRIIB. These results demonstrate that NK cells expressed FcRIII, but not FcRI. FcRIII on NK cells associates only with FcR, but not with FcR (18). Thus, FcRIII on NK cells was composed of FcRIII and FcR, while FcRIII on mast cells was composed of FcRIII, FcR, and FcR as described (14, 15).
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production upon stimulation of NK cells with IgE
To analyze the possible stimulation of NK cells with IgE, murine NK cells were stimulated with immobilized IgG or IgE mAb for 2 days, because we determined that the maximum response for IFN- production by NK cells upon stimulation with immobilized IgG was 2 days (data not shown). IFN- secretion in the culture supernatants was analyzed by ELISA (Fig. 2A). Immobilized IgG induced a high level of IFN- from NK cells in a dose-dependent manner. To our surprise, a considerable amount of IFN- was detected in the supernatant of NK cells stimulated with immobilized IgE, also in a dose-dependent fashion. The efficiency of NK cell stimulation by IgE was less than that by IgG and the difference in the dose-response curve was 3- to 10-fold. This result shows that NK cells can be activated to produce IFN- upon stimulation with immobilized IgE.
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RIII
Next, we investigated the possibility that NK cell activation was mediated through FcRIII, because FcRIII has been reported to be a low affinity receptor for IgE on mast cells (5). When we added an anti-FcRII/III mAb 2.4G2 (20 µg/ml) to the culture of IgG- and IgE-stimulated NK cells, IFN- production upon stimulation with either IgG or IgE was totally abrogated by 2.4G2 mAb, suggesting that FcRIII mediates the stimulation with IgE as well as IgG (Fig. 2A). Furthermore, IFN- production upon stimulation with either IgG or IgE was completely abrogated when NK cells from FcR-deficient mice were used (Fig. 2B). Together with the previous findings that NK cells from FcR-deficient mice did not induce IgG-mediated ADCC through FcRIII due to the lack of the cell surface FcRIII expression (16, 18), these data indicate that IgE-induced NK cell activation is also mediated through FcRIII and FcR. These results clearly demonstrate that NK cells were activated with IgE through the FcRIII complex composed of FcRIII and the FcR.
IgE-induced production of cytokines and a chemokine
NK cells are known to promptly produce a large amount of cytokines, such as IFN-, TNF-, and GM-CSF upon stimulation through FcRIII with IgG (19, 20). Therefore, we analyzed whether NK cells produce these cytokines or chemokines after stimulation with IgE by using real-time quantitative RT-PCR. In addition to IFN- production, NK cells produced significant levels of TNF- and GM-CSF upon stimulation with IgE or IgG, particularly at the early stage of the stimulation (Fig. 3). The secretion of a chemokine MIP-1 was also induced after stimulation with IgE or IgG. Because the maximum concentration of IgG and IgE (50 µg/ml, Fig. 2) was used to stimulate NK cells in this experiment, the amounts of these cytokines/chemokines by IgE stimulation were comparable to those by IgG stimulation. Because TNF-, GM-CSF, and MIP-1 are important to anaphylaxis, NK cells seem to play a role in anaphylaxis by producing these factors and chemokines with IgE stimulation.
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Finally, we addressed the question of whether the IgE-mediated NK cell activation induces cytotoxicity against IgE-coated target cells. To compare the ADCC activity induced by IgG and that by IgE, we used two pairs of IgG and IgE mAbs that share the same Ag specificity: anti-DNP IgG and IgE mAbs or anti-dansyl IgG and IgE mAbs. P815 cells, an NK-resistant cell line, were coated with these haptens (DNP or dansyl) by surface biotinylation followed by addition of DNP-streptavidin or dansyl-streptavidin. These targets were mixed with NK cells in the presence of anti-hapten IgG or IgE mAb (anti-DNP IgG/IgE for Fig. 4 or anti-dansyl IgG/IgE for Fig. 5). NK cells induced strong ADCC with IgG and also showed significant cytotoxicity against IgE-coated target cells. In both cases with anti-DNP Abs (Fig. 4) and anti-dansyl Abs (Fig. 5), IgE induced significant ADCC, although with less efficiency than IgG. In contrast, NK cells from FcR-/- mice, which lack the expression of the cell surface FcRIII, completely failed to exhibit ADCC against either IgE- or IgG-coated target cells (Fig. 5). These results suggest that the enhancement of IgE-mediated ADCC is specifically mediated through FcRIII. IgE-mediated ADCC of NK cells was also observed using other target cells including T cell hybridoma cells (data not shown). These results revealed that ADCC was induced upon activation of NK cells through binding of IgE to FcRIII. It is noteworthy that IgE-mediated but not IgG-mediated ADCC requires continuous presence of Ab during the killing assay for 4 h (data not shown), suggesting that IgE has lower affinity-binding to FcRIII (5) and requires a higher dose of Ab for ADCC as compared with IgG.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI is expressed mainly on mast cells, basophils, and eosinophils. It has been thought to be necessary and sufficient for IgE-mediated anaphylaxis, based on the analysis of FcRI-deficient mice (1). However, further analyses using genetic mutant or gene-targeted mice have revealed that FcRI and IgE are not the only initiators of anaphylactic reactions. Indeed, in the late 1960s, Vaz and Ovary (2) showed that IgG induced cutaneous anaphylaxis. Recent studies using both FcR-/- and FcRI-/- mice confirmed that OVA-induced systemic anaphylaxis largely depends on IgG1 binding to FcRIII (3). Furthermore, Ujike et al. (6) showed that IgE-mediated systemic anaphylaxis was reduced in FcRIII-deficient mice, suggesting a contribution of FcRIII not only to IgG- but also to IgE-mediated passive anaphylactic response.
In contrast, W/W mutant mice bearing a c-kit gene mutation and mast cell deficiency were shown to induce active anaphylactic responses associated with physiological changes and mortality rates that are similar to those observed in the Ag-sensitized normal mice (7, 8, 9). From these studies, some cells other than mast cells might be involved in allergic responses to certain Ags. In fact, mast cells may not be involved in systemic anaphylaxis induced by penicillin V, although the anaphylaxis is well correlated with serum IgE level (10). Collectively, all these data suggest the involvement of cells other than mast cells at the cellular level and FcRIII at the molecular level in IgE-mediated systemic anaphylaxis in vivo, although the cells responsible for these IgE-mediated FcRIII-dependent responses remained to be identified.
We have demonstrated in the present study that IgE-stimulated NK cells produce cytokines and chemokines that are related to anaphylaxis, and they exhibit IgE-mediated ADCC through FcRIII. Takizawa et al. (5) previously reported that IgE binds to FcRIII on FcRIII-transfected COS cells, as well as to M and mast cell lines. These authors also showed serotonin release upon stimulation of mast cells with aggregated IgE through FcRIII. FcRIII on NK cells is composed of only FcRIII and the FcR dimer, while that on mast cells is made up of FcRIII, FcR, and the FcR dimer. Therefore, our study demonstrated for the first time that IgE binding to the FcRIII2 complex induces activation and effector function of NK cells.
The affinity constant of monomeric IgE toward FcRIII was calculated to be 4.8 x 105 M-1, whereas that of IgG to FcRIII was 6.7 x 106 M-1 (5). In our study, stimulation of FcRIII with IgE was always weaker than with IgG for both cytokine production and ADCC. The cytokine production by NK cells upon IgE stimulation despite very low affinity of IgE binding to FcRIII may be mediated by cooperation of specific adhesion molecules because adhesion molecules have been shown to be involved in cytokine production and cytotoxicity by NK cells (21, 22). An obvious question may occur whether IgE-mediated NK activation takes place under physiological conditions with such low affinity of IgE to FcRIII and requirement of higher concentration of IgE than IgG. It is noteworthy that the concentration of IgE in the plasma of some atopic patients reaches 10 µg/ml (23), and the serum IgE level of NC/Nga mice, a recently established mouse model for allergic dermatitis is 
80 µg/ml (24). We observed significant ADCC of NK cells upon stimulation with 5 µg/ml IgE. Thus, it is possible to activate NK cells with IgE under physiological conditions in vivo including some pathological situation. Considering that the tissue distribution of NK cells and mast cells is quite different, it is possible that NK cells may be involved in allergic responses at different sites from mast cells.
Recently, several reports have described the involvement of NK cells in allergy. In particular, NK cells were shown to regulate the development of allergen-induced eosinophilic airway inflammation in mice (12), although how NK cells contribute to the process was not defined. NK cells produced a large amount of cytokines and chemokines, including IFN-, TNF-, GM-CSF, and TGF-, upon stimulation through FcRIII (19, 20). Furthermore, we have observed that NK cells also produced the chemokines MIP-1 and MIP-1 after activation (our unpublished observation). Our present study showed that NK cells induced the production of IFN-, TNF-, GM-CSF, and MIP-1 upon IgE stimulation. TNF- and TGF- contribute to anaphylaxis in either induction or promotion of the response (25). MIP-1 and MIP-1 induce chemotaxis of T cells and eosinophils (26, 27). Through the production of these cytokines and chemokines during an anaphylactic response, NK cells may play an important role in allergy. Indeed, it is possible that the NK cell may be one of the cells responsible for the FcRI-independent allergic reactions by the production of a large amount of cytokines and chemokines.
Type II hypersensitivity is believed to be mediated solely by IgG-induced ADCC against autoantigens. However, several reports indicated that idiopathic thrombocytopenic purpura correlates with IgE levels (28). If IgE-mediated ADCC through FcRIII on NK cells functions in vivo, IgE might also be involved in type II hypersensitivity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Takashi Saito, Department of Molecular Genetics, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: saito{at}med.m.chiba-u.ac.jp
3 Abbreviations used in this paper: wt, wild type; ADCC, Ab-dependent cell cytotoxicity; M, macrophage; BMMC, bone marrow-derived mast cell; MIP, macrophage-inflammatory protein.
Received for publication November 28, 2001. Accepted for publication January 15, 2003.
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References |
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chain gene. Cell 75:969.[Medline]
-G antibodies. I. PCA and RPCA reactions with homologous and heterologous antibodies. J. Immunol. 100:169.RIII: assessment of the cardiopulmonary changes, mast cell degranulation, and death associated with active or IgE- or IgG1-dependent passive anaphylaxis. J. Clin. Invest. 99:901.[Medline]
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RII and FcRIII. J. Exp. Med. 176:469. subunit of the FcRI is associated with the FcRIII on mast cells. J. Exp. Med. 175:447. is a molecular amplifier of IgE- and IgG- mediated in vivo responses. Immunity 8:517.[Medline]
is essential for activation signal through NKR-P1 (CD161) in natural killer (NK) cells and NK1.1+ T cells. J. Exp. Med. 186:1957. chain deletion results in pleiotrophic effector cell defects. Cell 76:519.[Medline]
1 integrin-induced interleukin-8 production in human natural killer cells. Immunity 12:7.[Medline]
1 on the IgE-dependent anaphylaxis reaction. J. Immunol. 162:4960. induces significant recruitment of neutrophils and monocytes. J. Immunol. 164:3392.This article has been cited by other articles:
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  Y. Gu, Y Fujimiya, and N Kunugita Long-term exposure to gaseous formaldehyde promotes allergen-specific IgE-mediated immune responses in a murine model Human and Experimental Toxicology, January 1, 2008; 27(1): 37 - 43. [Abstract] [PDF]
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 J. M. Roda, R. Parihar, C. Magro, G. J. Nuovo, S. Tridandapani, and W. E. Carson III Natural Killer Cells Produce T Cell-Recruiting Chemokines in Response to Antibody-Coated Tumor Cells Cancer Res., January 1, 2006; 66(1): 517 - 526. [Abstract] [Full Text] [PDF]
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 X. Xie, H. He, M. Colonna, T. Seya, T. Takai, and B. A. Croy Pathways Participating in Activation of Mouse Uterine Natural Killer Cells During Pregnancy Biol Reprod, September 1, 2005; 73(3): 510 - 518. [Abstract] [Full Text] [PDF]
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 S. Dhanji, K. Tse, and H.-S. Teh The Low Affinity Fc Receptor for IgG Functions as an Effective Cytolytic Receptor for Self-Specific CD8 T Cells J. Immunol., February 1, 2005; 174(3): 1253 - 1258. [Abstract] [Full Text] [PDF]
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 S. Zompi, H. Gu, and F. Colucci The absence of Grb2-associated binder 2 (Gab2) does not disrupt NK cell development and functions J. Leukoc. Biol., October 1, 2004; 76(4): 896 - 903. [Abstract] [Full Text] [PDF]
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and 
) or IgE (
and •) in the presence (
and 
, Spontaneous cytotoxicity of target cells in the presence of NK cells without Ab against the DNP-coated same target cells. B, IgE (