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Innate basophil IL-4 responses against allergens, endotoxin, and cytokines require the Fc receptor γ-chain

2015; Elsevier BV; Volume: 137; Issue: 5 Linguagem: Inglês

10.1016/j.jaci.2015.10.037

1097-6825

Seiji Kamijo, Satoshi Nunomura, Chisei Ra, Yasuhiko Kanaguchi, Yusuke Suzuki, Hideoki Ogawa, Ko Okumura, Toshiro Takai,

Asthma and respiratory diseases

Basophils are regarded as important effector cells that contribute to allergic inflammation and induction of TH2 responses because they generate large amounts of IL-4 in IgE-dependent and IgE-independent manners.1Schroeder J.T. Basophils: emerging roles in the pathogenesis of allergic disease.Immunol Rev. 2011; 242: 144-160Crossref PubMed Scopus (99) Google Scholar, 2Sokol C.L. Barton G.M. Farr A.G. Medzhitov R. A mechanism for the initiation of allergen-induced T helper type 2 responses.Nat Immunol. 2008; 9: 310-318Crossref PubMed Scopus (760) Google Scholar Although the IgE-dependent high-affinity IgE receptor (FcεRI)–mediated mechanisms of basophil and mast cell activation, in which the Fc receptor γ-chain (FcRγ) and spleen tyrosine kinase (Syk) play critical roles in intracellular signaling, have been examined extensively and characterized,3Siraganian R.P. de Castro R.O. Barbu E.A. Zhang J. Mast cell signaling: the role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants.FEBS Lett. 2010; 584: 4933-4940Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar information on the molecular mechanisms responsible for basophil IL-4 responses with various innate stimuli is very limited. Herein we demonstrate basophil IL-4 responses with an innate stimulation using allergenic pollen grains. Furthermore, we found that the basophil IL-4 responses induced by IgE-independent stimuli with allergen proteases, pollen, endotoxin, and cytokines all required FcRγ. Bone marrow–derived (BM) basophils were prepared by culturing bone marrow cells from naive mice in the presence of IL-3 and purified (for further information, see the Methods section in this article's Online Repository at www.jacionline.org). As demonstrated in our previous study,4Kamijo S. Takeda H. Tokura T. Suzuki M. Inui K. Hara M. et al.IL-33-mediated innate response and adaptive immune cells contribute to maximum responses of protease allergen-induced allergic airway inflammation.J Immunol. 2013; 190: 4489-4499Crossref PubMed Scopus (100) Google Scholar BM basophils from wild-type mice released IL-4 when stimulated with papain (Fig 1, A, left), a recombinant house dust mite (HDM) protease allergen (rDer f 1; Fig 1, B, left), or the IgE crosslink. IL-4 was also released by basophils stimulated with allergenic pollen grains (Fig 1, C, left). Because the signaling pathway for protease- or pollen-induced IL-4 release might differ from that for IgE crosslink–induced IL-4 release, which needs to be mediated by FcRγ, we examined IL-4 responses in BM basophils from FcRγ−/− mice. FcRγ−/− basophils did not release IL-4 when stimulated by the allergen proteases, pollen grains, or IgE crosslink (Fig 1, A-C, right panels). A stimulation in the presence of IL-3 increased IL-4 release by wild-type basophils but not by FcRγ−/− basophils (see Fig E1 in this article's Online Repository at www.jacionline.org). Endotoxin, also known as LPS, functions as a TH2-inducing adjuvant.5Eisenbarth S.C. Piggott D.A. Huleatt J.W. Visintin I. Herrick C.A. Bottomly K. Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen.J Exp Med. 2002; 196: 1645-1651Crossref PubMed Scopus (987) Google Scholar The IL-1 family cytokines IL-18 and IL-334Kamijo S. Takeda H. Tokura T. Suzuki M. Inui K. Hara M. et al.IL-33-mediated innate response and adaptive immune cells contribute to maximum responses of protease allergen-induced allergic airway inflammation.J Immunol. 2013; 190: 4489-4499Crossref PubMed Scopus (100) Google Scholar have been shown to play roles in allergic inflammation and induction of TH2 responses. As reported previously by other researchers,6Yoshimoto T. Yasuda K. Tanaka H. Nakahira M. Imai Y. Fujimori Y. et al.Basophils contribute to TH2-IgE responses in vivo via IL-4 production and presentation of peptide-MHC class II complexes to CD4+ T cells.Nat Immunol. 2009; 10: 706-712Crossref PubMed Scopus (437) Google Scholar, 7Kroeger K.M. Sullivan B.M. Locksley R.M. IL-18 and IL-33 elicit Th2 cytokines from basophils via a MyD88- and p38alpha-dependent pathway.J Leukoc Biol. 2009; 86: 769-778Crossref PubMed Scopus (127) Google Scholar basophils released IL-4 when stimulated with endotoxin, IL-18, and IL-33 or each of these plus IL-3 (Fig 1, D, left panel). Endotoxin- or cytokine-induced IL-4 release was dependent on FcRγ (Fig 1, D, right panel). The FcεRI β-chain, which is the other signal-transducing subunit of the FcεRI complex and is necessary for cell-surface expression of murine but not human FcεRI α-chain, was essential for IgE crosslink−induced IL-4 release but was dispensable for cytokine-induced IL-4 release (Fig 2, B). The immunoreceptor tyrosine-based activation motif contained in FcRγ acts as a scaffold for the binding and activation of Syk.3Siraganian R.P. de Castro R.O. Barbu E.A. Zhang J. Mast cell signaling: the role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants.FEBS Lett. 2010; 584: 4933-4940Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Piceatannol, an inhibitor of Syk, inhibited papain-induced IL-4 release by wild-type basophils (Fig 2, A), as well as cytokine-induced IL-4 release by FcεRIβ−/− basophils (Fig 2, B). In the present study IL-4 release by FcRγ-deficient basophils was not induced by the IgE crosslink, allergen proteases (papain and HDM group 1 allergens), pollen grains, endotoxin, or cytokines (IL-18, IL-33, and IL-3) (Fig 1 and see Fig E1). The Syk inhibitor inhibited release of IL-4 induced by the IgE crosslink, papain, and cytokines (Fig 2). Hida et al8Hida S. Yamasaki S. Sakamoto Y. Takamoto M. Obata K. Takai T. et al.Fc receptor gamma-chain, a constitutive component of the IL-3 receptor, is required for IL-3-induced IL-4 production in basophils.Nat Immunol. 2009; 10: 214-222Crossref PubMed Scopus (74) Google Scholar previously reported the dependency of the IL-3−induced IL-4 response on the FcRγ-Syk axis, whereas Rosenstein et al9Rosenstein R.K. Bezbradica J.S. Yu S. Medzhitov R. Signaling pathways activated by a protease allergen in basophils.Proc Natl Acad Sci U S A. 2014; 111: E4963-E4971Crossref PubMed Scopus (27) Google Scholar very recently showed the dependency of papain- or IL-33−induced basophil IL-4 responses on FcRγ. Therefore the novel results of the present study are the pollen-induced release of IL-4; the dependency of IL-4 responses to HDM group 1 allergen proteases, pollen, endotoxin, and IL-18 on FcRγ; and the inhibition of papain-, IL-18–, or IL-33–induced IL-4 release by basophils by using the Syk inhibitor. These results suggested that FcRγ or the FcRγ-Syk axis is a required common adaptor or platform for various adaptive (IgE) or innate (allergen proteases, pollen, endotoxin, IL-18, or IL-33) stimuli caused by encounters with allergen sources but not for all IL-4–inducing stimuli because phorbol 12-myristate 13-acetate (PMA) plus ionomycin–induced (Fig 1) and anti-CD200R3 mAb–induced (data not shown)9Rosenstein R.K. Bezbradica J.S. Yu S. Medzhitov R. Signaling pathways activated by a protease allergen in basophils.Proc Natl Acad Sci U S A. 2014; 111: E4963-E4971Crossref PubMed Scopus (27) Google Scholar IL-4 release do not require FcRγ. Rosenstein et al9Rosenstein R.K. Bezbradica J.S. Yu S. Medzhitov R. Signaling pathways activated by a protease allergen in basophils.Proc Natl Acad Sci U S A. 2014; 111: E4963-E4971Crossref PubMed Scopus (27) Google Scholar recently reported that papain-induced basophil IL-4 responses required the calcium-dependent activation of calcineurin and nuclear factor of activated T cells proteins (similar to IgE-dependent or PMA/ionomycin-induced IL-4 release), activation of phosphoinositide 3-kinase but not extracellular signal-regulated kinase (similar to IgE-dependent IL-4 release), and the FcRγ immunoreceptor tyrosine-based activation motif (similar to IgE-dependent IL-4 release). However, papain did not induce phosphorylation of FcRγ or Syk, at least within 30 minutes of stimulation, whereas the rapid and distinct phosphorylation of FcRγ and Syk was detected within 5 minutes of IgE crosslink stimulation.9Rosenstein R.K. Bezbradica J.S. Yu S. Medzhitov R. Signaling pathways activated by a protease allergen in basophils.Proc Natl Acad Sci U S A. 2014; 111: E4963-E4971Crossref PubMed Scopus (27) Google Scholar On the other hand, the Syk inhibitors piceatannol (Fig 2) and R406 (data not shown) successfully inhibited papain-induced IL-4 release. This suggests the involvement of Syk, which might have occurred in a different manner or in another time course after stimulation with papain that was different from IgE-dependent FcεRI-mediated activation and/or involvement of the activation of other pathways that the inhibitors might also have affected. The mechanisms by which basophils sense allergen proteases upstream of FcRγ before releasing IL-4 currently remain unknown. Although previous studies described the contribution of the protease–activated receptor (PAR2, also known as F2RL2), Rosenstein et al9Rosenstein R.K. Bezbradica J.S. Yu S. Medzhitov R. Signaling pathways activated by a protease allergen in basophils.Proc Natl Acad Sci U S A. 2014; 111: E4963-E4971Crossref PubMed Scopus (27) Google Scholar reported that, although pertussis toxin inhibited papain-induced IL-4 release, responsiveness to papain was intact in BM basophils prepared from PAR2−/− mice or mice with other deficiencies. They suggested that basophils stimulated directly with papain but not indirectly with soluble proteolytic fragments derived from papain-treated serum or basophils induce the IL-4 response. Very recently, we reported that papain and bromelain cleaved the α-subunit of the murine IL-3 receptor.10Nishikado H. Fujimura T. Taka H. Mineki R. Ogawa H. Okumura K. et al.Cysteine protease antigens cleave CD123, the alpha subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion.Biochem Biophys Res Commun. 2015; 460: 261-266Crossref PubMed Scopus (5) Google Scholar Although cleavage of the IL-3 receptor α-subunit by papain suppressed the subsequent IL-3–mediated proliferation of basophils, whether the cleavage is related to papain-induced IL-4 release has yet to be investigated. In conclusion, the results obtained in the present study suggest that a signaling pathway mediated by FcRγ or the FcRγ-Syk axis is commonly required for innate basophil IL-4 responses under conditions mimicking encounters with allergen sources. Further investigations are needed to elucidate the molecular mechanisms responsible for the basophil IL-4 responses induced after stimulation of FcRγ or the FcRγ-Syk axis with various innate stimuli and also to determine how basophils sense allergen proteases and pollen upstream of FcRγ. We thank Michiyo Matsumoto for her secretarial assistance. Recombinant mouse IL-3 (Wako, Osaka, Japan) was used to generate mouse BM basophils. Reagents used to stimulate basophils were allergen proteases, including papain (Calbiochem, San Diego, Calif) and recombinant HDM group 1 allergens (rDer f 1/Der f 1-N53Q and rDer p 1/Der p 1-N52Q),E1Takai T. Mineki R. Nakazawa T. Takaoka M. Yasueda H. Murayama K. et al.Maturation of the activities of recombinant mite allergens Der p 1 and Der f 1, and its implication in the blockade of proteolytic activity.FEBS Lett. 2002; 531: 265-272Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, E2Takai T. Kato T. Yasueda H. Okumura K. Ogawa H. Analysis of the structure and allergenicity of recombinant pro- and mature Der p 1 and Der f 1: major conformational IgE epitopes blocked by prodomains.J Allergy Clin Immunol. 2005; 115: 555-563Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar the pollen grains of short ragweed (Ambrosia artemisiifolia; Polysciences, Warrington, Pa), Kentucky bluegrass (Poa pratensis; Sigma, St Louis, Mo), Japanese cypress (Chamaecyparis obtusa), and Japanese cedar (Cryptomeria japonica, Wako),E3Gunawan H. Takai T. Ikeda S. Okumura K. Ogawa H. Protease activity of allergenic pollen of cedar, cypress, juniper, birch and ragweed.Allergol Int. 2008; 57: 83-91Abstract Full Text PDF PubMed Scopus (65) Google Scholar, E4Gunawan H. Takai T. Kamijo S. Wang X.L. Ikeda S. Okumura K. et al.Characterization of proteases, proteins, and eicosanoid-like substances in soluble extracts from allergenic pollen grains.Int Arch Allergy Immunol. 2008; 147: 276-288Crossref PubMed Scopus (41) Google Scholar, E5Wang X.L. Takai T. Kamijo S. Gunawan H. Ogawa H. Okumura K. NADPH oxidase activity in allergenic pollen grains of different plant species.Biochem Biophys Res Commun. 2009; 387: 430-434Crossref PubMed Scopus (38) Google Scholar, E6Kamijo S. Takai T. Kuhara T. Tokura T. Ushio H. Ota M. et al.Cupressaceae pollen grains modulate dendritic cell response and exhibit IgE-inducing adjuvant activity in vivo.J Immunol. 2009; 183: 6087-6094Crossref PubMed Scopus (33) Google Scholar LPS (List Biological Laboratories, Campbell, Calif), recombinant mouse IL-3 (Wako), recombinant mouse IL-18 (MBL, Nagoya, Japan), recombinant mouse IL-33 (R&D Systems, Minneapolis, Minn), a mouse IgE mAb specific to trinitrophenol (clone IgE-3; BD Biosciences, San Diego, Calif), rat mAb against mouse IgE (clone R35-72, BD Biosciences), PMA, ionomycin, and piceatannol (Sigma). Treatment with the irreversible inhibitor specific to cysteine proteases E64 (Peptide Institute, Osaka, Japan) was performed, as described previously.E7Kamijo S. Takeda H. Tokura T. Suzuki M. Inui K. Hara M. et al.IL-33-mediated innate response and adaptive immune cells contribute to maximum responses of protease allergen-induced allergic airway inflammation.J Immunol. 2013; 190: 4489-4499Crossref PubMed Scopus (133) Google Scholar Seven- to 10-week-old female C57BL/6 mice (Charles River Japan, Yokohama, Japan), FcRγ−/− mice,E8Park S.Y. Ueda S. Ohno H. Hamano Y. Tanaka M. Shiratori T. et al.Resistance of Fc receptor- deficient mice to fatal glomerulonephritis.J Clin Invest. 1998; 102: 1229-1238Crossref PubMed Scopus (236) Google Scholar and FcεRIβ−/− mice (C57BL/6 background)E9Hiraoka S. Furumoto Y. Koseki H. Takagaki Y. Taniguchi M. Okumura K. et al.Fc receptor beta subunit is required for full activation of mast cells through Fc receptor engagement.Int Immunol. 1999; 11: 199-207Crossref PubMed Scopus (35) Google Scholar were maintained in specific pathogen-free animal facilities at Juntendo University or Nihon University and used in accordance with the guidelines of the institutional committees on animal experiments. Mouse BM basophils were generated, as previously described.E7Kamijo S. Takeda H. Tokura T. Suzuki M. Inui K. Hara M. et al.IL-33-mediated innate response and adaptive immune cells contribute to maximum responses of protease allergen-induced allergic airway inflammation.J Immunol. 2013; 190: 4489-4499Crossref PubMed Scopus (133) Google Scholar In brief, BM cells prepared from the tibias and femurs of mice were cultured in RPMI 1640 medium (Sigma) supplemented with 30 ng/mL recombinant IL-3, 2 mmol/L l-glutamine, 10% (vol/vol) heat-inactivated FCS, 0.05 mmol/L 2-mercaptoethanol, and antibiotics at a density of 5 × 106 cells/mL (day 0). Every 3 to 4 days, cells were replated at a density of 1 × 106 cells/mL. On day 14, cells were collected and subjected to magnetic separation for the CD49b+ population by using anti-CD49b microbeads and autoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the separated cells was assessed by using flow cytometry. A total of 92% of the cells were CD49b+FcεRIα+ after magnetic separation. BM basophils and CD49b+ cells purified with MACS, as described above, and suspended in fresh medium containing 5% FCS were plated onto 96-well culture plates (3 × 105 cells/80 μL/well). Twenty microliters of the solution containing stimulants was added to each of the wells (total = 100 μL/well). The final concentrations of the stimulants are indicated in the figure legends, except for PMA (50 ng/mL) plus ionomycin (1 μmol/L). rDer f 1 and rDer p 1 were treated with l-cysteine before being used to stimulate basophils. In some experiments piceatannol, a Syk inhibitor, was simultaneously added with the stimulation or added to basophils 5 minutes before stimulation. CD49b+ cells were sensitized with 10 μg/mL monoclonal mouse IgE (clone IgE-3) for 1 hour, washed, and then suspended in fresh medium containing 5% FCS to crosslink FcεRI. Cells were stimulated with 10 μg/mL anti-mouse IgE mAb (clone R35-72). After stimulation of BM basophils for 24 hours, culture supernatants were recovered by means of centrifugation at 310g for 5 minutes. Cytokine concentrations were measured with ELISA kits (Quantikine or DuoSet, R&D Systems).