Published online before print September 19, 2007
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RI signals
,#,1
,,3
* Department of Pathology, Stanford University School of Medicine, Stanford, California, USA;
# Department of Dental Anesthesiology, Tokyo Dental College, Chiba, Japan;
Department of Allergy and Immunology, National Research Institute for Child Health and Development, Tokyo, Japan;
Division of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University Graduate School of Medical Sciences, Tokyo, Japan;
Atopy Research Center, Juntendo University, Tokyo, Japan;
|| Department of Respiratory Medicine, National Disaster Medical Center of Japan, Tokyo, Japan; and
¶ Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
2Correspondence: Department of Pathology, L-235, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5324, USA. E-mail: sgalli{at}stanford.edu
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RI-stimulated mouse mast cells. Another IL-1-related molecule, IL-33, was identified recently as a ligand for T1/ST2. Although mouse mast cells constitutively express ST2, the effects of IL-33 on mast cell function are poorly understood. We found that IL-33, but not IL-1β or IL-18, induced IL-13 and IL-6 production by mouse bone marrow-derived, cultured mast cells (BMCMCs) independently of IgE. In BMCMCs incubated with the potently cytokinergic SPE-7 IgE without specific antigen, IL-33, IL-1β, and IL-18 each promoted IL-13 and IL-6 production, but the effects of IL-33 were more potent than those of IL-1β or IL-18. IL-33 promoted cytokine production via a MyD88-dependent but Toll/IL-1R domain-containing adaptor-inducing IFN-β-independent pathway. By contrast, IL-33 neither induced nor enhanced mast cell degranulation. At 200 ng/ml, IL-33 prolonged mast cell survival in the absence of IgE and impaired survival in the presence of SPE-7 IgE, whereas at 100 ng/ml, IL-33 had no effect on mast cell survival in the absence of IgE and reduced mast cell survival in the presence of IgE. These observations suggest potential roles for IL-33 in mast cell- and Th2 cytokine-associated immune responses and disorders.
Key Words: cytokines • Fc receptors • mast cells/basophils • allergy
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IL-17 [1
2
3
]. It is notable that IL-1 or IL-18 also can enhance production of Th2 cytokines, such as IL-9 or IL-13, by mouse mast cells in the presence of an additional signal, such as those induced by IgE/antigen-FcRI, stem cell factor (SCF), or IL-3 [4
5
6
]. IL-33 (also called IL-1F11 [7
], NF-HEV [8
], or DVS27 [9
]), a recently identified member of the IL-1 family of molecules, has biological activities that are mediated in part by its interactions with the receptor ST2 (also called T1, DER-4, Fit-1, or IL-1R4) [7
]. Like IL-1
[10
], some of the biological activities of IL-33 may also reflect the ability of a precursor/intracellular form of the cytokine to function as an intracrine chromatin-associated nuclear factor, which can regulate transcription [11
]. ST2 is preferentially expressed on Th2 cells in humans [12 ] and mice [13 ] and on mouse mast cells [14 ], suggesting possible roles for ST2-IL-33 interactions in Th2 cell-associated immune responses. Additional evidence for such roles includes increased serum concentrations of soluble ST2 in patients with exacerbations of asthma [15 ], increased expression of ST2 in an asthma model in mice [16 ], attenuated development of Th2 cell- and eosinophil-associated airway inflammation in mice after blockade of interactions between ST2 and its ligand [13 , 17 ], and increased phosphorylation of MAPKs such as JNK, p42/p44, and p38, as well as increased Th2-cytokine production in CD4+ T cells after cross-linking of ST2 [18 , 19 ]. Moreover, ST2-deficient mice exhibit impaired pulmonary granuloma formation, eosinophilia, and Th2 cytokine production after injection of Schistosoma mansoni eggs [20 ] and decreased IL-5 production by lymph node cells during Nippostrongylus brasiliensis infection [21 ]. Finally, in addition to enhancing IL-5 and IL-13 production by mouse Th2 cells in vitro [7 ], recombinant IL-33 (rIL-33), when administrated tomice in vivo, can induce the development of splenomegaly and eosinophilia in the lungs and gut, as well as elevated numbers of blood eosinophils and increased amounts of serum IgA, IgE, IL-5, and IL-13 [7 ].
The binding of IL-33 to ST2 activates a signaling pathway, which involves the recruitment of MyD88, IL-1R-associated kinase 1 (IRAK1), IRAK4, and TNFR-associated factor 6 (TRAF6), leading to the activation of MAPK and NF-
B [7
]. IL-33 also can enhance MAPK phosphorylation in mouse mast cells [7
], and ST2-deficient mast cells have been reported to exhibit normal histamine release after IgE/antigen stimulation [22
]. Apart from that, little is known about the effects of IL-33 on mouse mast cells.
We wished to address two questions concerning the possible effects of IL-33 on mouse mast cells. First, can incubation of mast cells with IL-33 induce effects that are independent of the cells’ interactions with IgE? Second, can IL-33 influence the responses of mast cells to IgE? It is well-known that cross-linking of IgE, bound to high-affinity IgE receptors (FcRI) on mast cells, e.g., with multivalent antigens, initiates mast cell activation, leading to FcRI aggregation and the subsequent release of various proinflammatory mediators and cytokines [23
24
25
26
27
28
29
]. Mast cell activation mediated by IgE/antigen-FcRI signals can contribute to host defense, e.g., during infections with certain nematodes, whereas pathological mast cell activation can contribute to the development of various allergic and/or autoimmune diseases [23
24
25
26
27
28
29
30
31
32
]. Recently, it has been reported that certain IgE antibodies, even in the absence of specific antigens, can enhance the survival, migration, and mediator production of mouse mast cells [33
34
35
36
] or chemokine production by human mast cells [37
]. Moreover, in the mouse, certain IgE antibodies are more potent in inducing these "cytokinergic" effects than are others [35
, 38
]. We therefore investigated the effects of IL-1, IL-18, and IL-33 on mouse mast cells incubated in the presence of IgE, with or without specific antigen.
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RIhigh by flow cytometry analysis using FITC anti-mouse FcRI (MAR-1, eBioscience, San Diego, CA, USA) and allophycocyanin (APC) anti-mouse c-kit/CD117 (2B8, BD PharMingen, San Diego, CA, USA). Wild-type, MyD88–/–, and TRIF–/– BMCMCs were similar in appearance by light microscopy and exhibited similar amounts of surface expression of c-kit/CD117, FcRI, and ST2 by FACS (data not shown). All experiments were performed in compliance with the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press (revised 1996) and with the permission of Stanford University Committee on Animal Welfare (Stanford, CA, USA).
FACS analysis
For analysis of cell surface molecules, BMCMCs were incubated with various concentrations of anti-DNP IgE, H1--26 [41
] (as a dilution of ascites from mice bearing the H1--26 hypbridoma cell line), or SPE-7 (Sigma Chemical Co., St. Louis, MO, USA; as specified in Results and in the figure legends) in RPMI-1640 media + 10% FCS for 0–72 h. Cells were incubated with anti-CD16/CD32 mAb (93, eBioscience) for 15 min on ice, then incubated with APC anti-mouse c-kit/CD117 (2B8, eBioscience), and FITC or PE anti-mouse FcRI (MAR-1, eBioscience), plus PE anti-mouse IL-1R1 (35F5, BD PharMingen), biotin anti-mouse IL-18R (BAF856, R&D Systems, Minneapolis, MN, USA), or FITC or Alexa 647 anti-mouse T1/ST2 (DJ8, MD Biosciences, St. Paul, MN, USA) for 45 min on ice. For detection of IL-18R using biotin-conjugated antibody staining, PE-streptavidin (BD PharMingen) was used as described [3
, 42
]. The expression of cell surface markers on 7-aminoactinomycin-negative c-kit/CD117+ FcRI+ cells was determined by FACSCalibur and CellQuest software (Becton Dickinson, San Jose, CA, USA). For detection of MAPK, BMCMCs were cultured with or without 100 ng/ml recombinant human (rh)IL-33 (PeproTech, Rocky Hill, NJ, USA) or 5 µg/ml SPE-7 anti-DNP mAb for 5 min. Cells were then incubated with anti-CD16/CD32 mAb (93, eBioscience) for 15 min on ice, incubated with APC anti-mouse c-kit/CD117 and biotin anti-mouse FcRI (eBioscience) for 45 min on ice, and then incubated with PE-Cy5-streptavidin for 30 min on ice. After washing, cells were fixed in BD PhosFlow Fix Buffer I (BD Biosciences, San Jose, CA, USA) for 15 min at room temperature. Cells then were washed with HBSS buffer containing 2% FCS and 0.1% saponin (Sigma Chemical Co.) and incubated with PE antiphospho-ERK1/2 (T202/Y204; 20a, BD Biosciences), Alexa Fluor-647 antiphospho-p38 MAPK (T180/Y182; 36, BD Biosciences), or PE- or Alexa Fluor-647 mouse IgG1 (BD Biosciences) as an isotype-matched control Ig. The expression of phosphorylated MAPKs in c-kit/CD117+FcRI+ cells was measured by flow cytometry as described above.
Cell survival and apoptosis
BMCMCs (1x106/ml) were cultured with 10 ng/ml recombinant mouse (rm)IL-3 (R&D Systems) or with 10 µg/ml H1--26 anti-DNP mAb or 5 µg/ml SPE-7 anti-DNP mAb in the presence or absence of 100 ng/ml rmIL-1β (PeproTech), 100 ng/ml rmIL-18 (R&D Systems), or 100 or 200 ng/ml rhIL-33 (Alexis, San Diego, CA, USA; or PeproTech) for 0–6 days (for assays of cell survival and apoptosis) or 0, 1, 10, or 100 ng/ml recombinant cytokines for 1 h (for β-hexosaminidase release assay). We assessed cell viability by trypan blue staining or by the Annexin V-FITC Apoptosis Detection Kit I (BD PharMingen).
β-Hexosaminidase release
BMCMC suspensions (8x106 cells/ml) were prepared in Tyrodes buffer [containing 10 mM HEPES, pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 0.1% glucose, and 0.1% BSA (fraction V, Sigma Chemical Co.)]. Concentration (4x; 12.5 µl) of anti-DNP IgE mAb (H1--26 or SPE-7, final 10 µg/ml) and 12.5 µl 4x concentration of cytokine [final 0, 1, 10, or 100 ng/ml rmIL-1β (PeproTech), rmIL-18 (R&D Systems), or rhIL-33 (Alexis)] were added into wells of 96-well V-bottom plates (Costar, Corning, NY, USA), and then 25 µl of 8 x 106 cells/ml IgE-sensitized BMCMCs (10 µg/ml H1--26 anti-DNP mAb or 5 µg/ml SPE-7 anti-DNP mAb, overnight) were added and incubated at 37°C for 1 h. As a positive control, IgE-sensitized BMCMCs (37°C, overnight) were stimulated with 10 ng/ml 2,4-DNP-conjugated human serum albumin (DNP-HSA; Sigma Chemical Co.) or 0.1 µg/ml PMA (Sigma Chemical Co.) + 1 µg/ml A23187 calcium ionophore (Sigma Chemical Co.). After centrifugation, supernatants were collected. Nonantigen-stimulated, IgE-sensitized BMCMCs were treated with 50 µl 0.5% (v/v) Triton X-100 (Sigma Chemical Co.), and supernatants were collected. β-Hexosaminidase release in supernatants was measured by enzyme immunoassay with p-nitrophenyl-N-acetyl-β-D-glucosamine (Sigma Chemical Co.) as a substrate. In brief, 10 µl culture supernatants were added into wells of 96-well flat-bottom plates, then 50 µl 1.3 mg/ml p-nitrophenyl-N-acetyl-β-D-glucosamine solution in 100 mM sodium citrate (pH 4.5) was added to each well, and the plate was incubated at room temperature for 15–30 min. Glycine (140 µl 200 mM, pH 7.0) was added to each well to stop the reaction. Enzymic activities (OD405) were measured by a plate reader. Data show the percent release of β-hexosaminidase under various conditions of stimulation relative to the total amount of β-hexosaminidase in the cells, as measured in the supernatants of Triton X-100-treated cells.
Cytokine ELISA
BMCMCs (1x106/ml) were cultured with or without 10 µg/ml H1--26 anti-DNP mAb or 1 or 5 µg/ml SPE-7 anti-DNP mAb in the presence or absence of 0–100 ng/ml rmIL-1β, rmIL-18, or rhIL-33 for 6 h (with 5 µg/ml SPE-7) for 24, 48, or 72 h (with 1 µg/ml SPE-7) or in the presence or absence of 100 ng/ml recombinant cytokines [rmG-CSF, GM-CSF, IL-1, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-13, IL-28B/IFN-
3, M-CSF, SCF, and TNF; rhIL-6, IL-13, IL-16, IL-28A/IFN-2, and TGF-β1 (PeproTech); rmIFN-, IL-15, IL-17, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27, lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells (LIGHT), lymphotoxin (LT)2β1, LT1β2, and thymic stromal lymphopoietin (TSLP); rhIL-24, IL-26 dimer, IL-29/IFN-1, and IL-33 (R&D Systems); and mIL-31 and SF20 (Antigenix America, Inc., Huntington Station, NY, USA; and Cell Sciences, Canton, MA, USA), respectively]. Amounts of IL-4, IL-6, or IL-13 in culture supernatants were measured by mouse IL-4 and IL-6 BD OptEIATM ELISA sets (BD PharMingen) or DuoSet mouse IL-13 (R&D Systems).
Statistics
The unpaired Student’s t-test, two-tailed, or ANOVA, as appropriate, was used for statistical evaluation of the results.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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expression on BMCMCs [4
]. Although we detected IL-1R1 and IL-18R proteins by Western blotting in lysates of BMCMCs (data not shown), naïve BMCMCs did not detectably express IL-1R1 or IL-18R on their cell surface (Fig. 1)
. By contrast, we found that peritoneal mast cells of C57BL/6J mice expressed IL-1R1 (data not shown).
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Figure 1. Cell surface expression of members of the IL-1R family on mouse mast cells. Naïve BMCMCs were incubated with various concentration of H1--26 IgE or SPE-7 IgE for 16 h. Cell surface expression of members of the IL-1R family on mast cells (i.e., c-kit+, FcRI+ cells) was analyzed by flow cytometry. (A) Shaded areas, Isotype-matched control IgG mAb; bold lines, specific mAb. (B) Data are representative of similar results, which were obtained in the three experiments we performed, each with a different batch of BMCMCs (A), or show the average ± SD (n=3 different batches of BMCMCs) of values from the three experiments. *, P < 0.05, versus corresponding values for no IgE (0 µg/ml) by Student’s t-test; , P < 0.05, versus corresponding values for control IgG staining by ANOVA. MFI, Mean fluorescence intensity.
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-26 or SPE-7, for 16 h resulted in marked up-regulation of surface expression of IL-18R, but not IL-1R1 (Fig. 1)
. The less cytokinergic of these mAb, H1--26, had a greater effect on IL-18R surface expression under these conditions than did SPE-7 (Fig. 1)
. Moreover, even the lowest concentrations of IgE tested were sufficient to enhance IL-18R expression. The expression of T1/ST2 was also increased by exposure to SPE-7, but not by H1--26 (Fig. 1)
.
By Western blotting, we detected IL-18Rβ as well as IL-18R proteins in whole cell lysates of naïve BMCMCs (data not shown). However, by FACS, we did not observe IL-18Rβ expression on the surface of such cells using any of three antibodies for IL-18Rβ [BAF1520 (R&D Systems), IMG-5353A (Imgenex, San Diego, CA, USA), or TC30-28E3 (BD PharMingen); data not shown].
We next examined the kinetics of the effects of IgE on surface expression of IL-1R1, T1/ST2, and IL-18R, using 5 µg/ml SPE-7 or 10 µg/ml H1--26, the concentrations which induced approximately maximal levels of IL-18R expression (Fig. 1B)
. Increased surface expression of T1/ST2 was observed 3 h after SPE-7 exposure, but expression declined by 48 h (Fig. 2A
and 2B
). Long-term (72 h) exposure to SPE-7 resulted in progressively enhanced IL-18R expression (Fig. 2A
and 2B)
. Exposure to H1--26 promoted IL-18R surface expression to a greater extent than did exposure to SPE-7 but down-regulated T1/ST2 expression time-dependently (Fig. 2A
and 2B)
. IL-1R1 expression was unaffected by H1--26 or SPE-7 exposure at any time-point examined (Fig. 2A
and 2B)
.
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Figure 2. Changes in cell surface expression of members of the IL-1R family on mouse mast cells upon exposure to IgE antibodies. (A) Naïve BMCMCs were incubated with 10 µg/ml H1--26 IgE or 5 µg/ml SPE-7 IgE for the indicated times. Cell surface expression of members of the IL-1R family on mast cells (c-kit+, FcRI+ cells) was analyzed by flow cytometry. Shaded areas, Isotype-matched control IgG mAb; bold lines, specific mAb. (B) MFI of BMCMCs is analyzed as in A. (C) Surface expression of c-kit/CD117 in the cells is shown in A. (D) Amounts of IL-6 in the culture supernatants of cells are treated as those shown in A. Data are representative of results obtained from the three experiments we performed, each with different batches of BMCMCs, all of which gave similar results (A and C) or show the average ± SD from the three experiments (n=3; B and D). *, P < 0.05 vs. corresponding values for 0 h by Student’s t-test (B and D); P < 0.05 vs. corresponding values for control IgG staining (B) or for H1--26 treatment (D) by ANOVA.
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-26 IgE down-regulated kit expression on BMCMCs between 3 and 48 h after exposure, and kit levels returned to baseline by 72 h (Fig. 2C)
. Consistent with previous reports [34
, 35
], exposure to SPE-7, but not to H1--26, IgE induced substantial IL-6 production by BMCMCs (Fig. 2D)
.
These observations are consistent with prior reports indicating that different IgE mAb can have quite different effects on some aspects of mast cell function and that SPE-7 is a much more highly cytokinergic IgE mAb than is H1--26 [33
34
35
].
Effects of IL-1β, IL-18, or IL-33 on BMCMC degranulation and survival
When tested individually at 100 ng/ml, without IgE or in the presence of H1--26 or SPE-7 IgE, none of the cytokines examined promoted BMCMC degranulation (β-hexosaminidase release; Fig. 3A
).
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Figure 3. Effects of IL-1β, IL-18, or IL-33 on mouse mast cell degranulation, survival, and apoptosis. (A) Naïve BMCMCs were cultured in the presence or absence of 10 µg/ml H1--26 IgE or 5 µg/ml SPE-7 IgE, with or without 100 ng/ml IL-1β, IL-18, or IL-33 for 1 h. As positive controls, naïve BMCMCs were stimulated with PMA + ionomycin (P+I), or BMCMCs sensitized with anti-DNP IgE (H1--26 or SPE-7) overnight were cultured with DNP-HSA for 1 h. Extent of mast cell degranulation was assessed as percent β-hexosaminidase release. Data are the average ± SD of triplicate determinations in one of three experiments we performed, each using different batches of BMCMCs and each of which gave similar results. *, P < 0.05, versus the corresponding values for BMCMCs incubated without IgE (by Student’s t-test, two-tailed). (B and C) Naïve BMCMCs were cultured for the indicated number of days with medium alone, 10 ng/ml rmIL-3, 10 µg/ml H1--26 IgE, or 5 µg/ml SPE-7 IgE; or 100 ng/ml IL-1β, IL-18, or IL-33 in the presence or absence of 10 µg/ml H1--26 IgE or 5 µg/ml SPE-7 IgE. (B) The numbers of living cells, assessed by trypan blue staining. (C) The percent of propidium iodide-negative (PI–) Annexin V+ BMCMCs. Data, which are the average ± SD from triplicate determinations, are representative of similar results, which were obtained from the three different batches of BMCMCs tested. (B and C) *, P < 0.05, versus the corresponding values at that time-point for BMCMCs + SPE-7;  versus  ,  , or • at 2 h; all later intervals, P < 0.05 (by ANOVA).
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RI can enhance mouse mast cell survival and cytokine production without promoting degranulation [35
, 36
]. When tested with either type of IgE, neither IL-1β nor IL-18 had any significant effects on BMCMC survival or apoptosis (Fig. 3B
and 3C)
. By contrast, at 1 or 10 ng/ml (data not shown; n=3) or at 100 ng/ml (Fig. 3B
and 3C)
, IL-33 reduced BMCMC survival, slightly, albeit significantly, and enhanced apoptosis in the presence of SPE-7 IgE but not H1--26 IgE.
These observations indicate that IL-33 (at concentrations from 1 to 100 ng/ml), but neither IL-1β nor IL-18, can negatively regulate BMCMC survival in the presence of SPE-7 IgE. By contrast, none of these cytokines promotes mast cell degranulation in the presence or absence of SPE-7 or H1--26 IgE.
IL-33 induces BMCMC IL-13 production without IgE, and this requires MyD88
It is notable that we found that IL-33, unlike IL-1β or IL-18, induced production of IL-6 and IL-13 by naïve BMCMCs in the absence of IgE (Fig. 4
). Except for IL-3 and SCF, which are growth factors for mast cells, of all the cytokines tested (including rmIFN-, IL-1/β, -2, -3, -4, -6, -10, -12, -13, -15, -17A-F (IL-17A is also known as IL-17), -18, -19, -20, -21, -22, -23, -27, -28B, and -31, TNF, LIGHT, LT2β1, LT1β2, G-CSF, GM-CSF, M-CSF, SCF, SF20, and TSLP; and rhIL-6, -13, -16, -24, -26, -28A, -29, and -33 and TGF-β1), only IL-33 induced IL-6 or IL-13 production directly by naïve BMCMCs (Figs. 4
and 5
).
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Figure 4. IL-33 promotes mouse mast cell cytokine production with or without IgE. Naïve BMCMCs were cultured in the presence or absence of 10 µg/ml H1--26 IgE or 5 µg/ml SPE-7 IgE plus various concentrations of IL-1β, IL-18, or IL-33 for 6 h. IL-4, IL-6, and IL-13 levels in culture supernatants of BMCMCs were measured by ELISA. Data are the average ± SD (n=3 different batches of BMCMCs). *, P < 0.05, versus the corresponding values for BMCMCs incubated without cytokine (indicated as "0") by Student’s t-test; , P < 0.05, versus the corresponding values for BMCMCs incubated with IL-1β or IL-18; , P < 0.05, versus the corresponding values for BMCMCs incubated with H1--26 or without IgE by ANOVA, respectively.
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Figure 5. IL-33 induces IL-6 and IL-13 production directly by naïve BMCMCs, which were cultured in the presence or absence of 100 ng/ml recombinant mouse or human cytokines, as indicated, for 6 h. IL-6 and IL-13 levels in culture supernatants of BMCMCs were measured by ELISA. Data are the average + SD (n=3 different batches of BMCMCs). *, P < 0.05, versus the corresponding values for BMCMCs incubated without cytokine (indicated as "Medium") by Student’s t-test. ND, Not determined.
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-26 (10 µg/ml), IgE for 6 h induced IL-4, IL-6, and IL-13 production by naïve BMCMCs (Fig. 4)
. Each cytokine tested (IL-1β, IL-18, or IL-33) enhanced SPE-7 IgE-induced production of IL-6 and IL-13, but not IL-4, by BMCMCs (Fig. 4)
. However, IL-33 enhanced IL-6 and especially, IL-13 cytokine production much more strongly than did IL-1β or IL-18. For example, the IL-13 production induced by 100 ng/ml IL-33 in the presence of SPE-7 (
350 ng/ml) was approximately sevenfold that (50 ng/ml) induced by 100 ng/ml IL-1β or IL-18 (Fig. 4)
. IL-33 also enhanced the production of IL-6 and IL-13 by mast cells incubated for up to 72 h in the presence or absence of a low concentration of SPE-7 IgE (1 µg/ml) (Fig. 6
).
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Figure 6. Dose- and time-dependence of IL-33-induced mouse mast cell cytokine production, with or without IgE. Naïve BMCMCs were cultured in the presence or absence of 1 µg/ml SPE-7 IgE plus various concentrations of rhIL-33 for 24, 48, or 72 h. IL-6 and IL-13 levels in culture supernatants of BMCMCs were measured by ELISA. Data are the average ± SD (n=4 different batches of BMCMCs). *, P < 0.05, versus the corresponding values for BMCMCs incubated without cytokine (indicated as 0) by Student’s t-test.
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Figure 7. MyD88 is required for IL-33- but not IgE-induced MAPK phosphorylation. (A, B) Naïve BMCMCs were incubated with or without 100 ng/ml rhIL-33 or 5 µg/ml SPE-7 IgE for 5 min. Then, phosphorylated p38 (p-p38) and phosphorylated ERK1/2 (p-ERK1/2) expression was measured by FACS. (A) Shaded areas, Isotype-matched control IgG mAb; bold lines, specific mAb. (B) MFI. Data are representative of similar results, which were obtained from the three experiments we performed, each with a different batch of BMCMCs (A), or show the average + SD of values from the three experiments (n=3). *, P < 0.05, versus corresponding values for control IgG staining (Cont. IgG); , P < 0.05, versus corresponding values for MyD88+/+ or TRIF+/+ BMCMCs by Student’s t-test.
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Figure 8. IL-33 mediates effects on mast cell cytokine production and survival via a MyD88-dependent but TRIF-independent pathway. (A) Naïve BMCMCs were cultured in the presence or absence of 5 µg/ml SPE-7 IgE or 200 ng/ml rhIL-33 for 2 days. The percent of PI– Annexin V-negative, live BMCMCs was measured by FACS. Data are the average ± SD from one experiment testing control BMCMCs (MyD88+/+ and TRIF+/+; n=8 batches of BMCMCs from different mice), MyD88–/– BMCMCs (n=3 batches of BMCMCs from different mice), and TRIF–/– BMCMCs (n=3 batches of BMCMCs from different mice). Similar results were obtained in another experiment testing six, three, and three batches of BMCMCs, respectively. *, P < 0.05, versus the corresponding values for medium; , P < 0.05, versus the corresponding values for MyD88–/– BMCMCs, by Student’s t-test. (B) IL-6 and IL-13 levels in culture supernatants of control BMCMCs (MyD88+/+ and TRIF+/+) or MyD88–/– or TRIF–/– BMCMCs were measured by ELISA 6 h after exposure to IL-33, with or without IgE. Data are the average ± SD of triplicate determinations in one of three experiments, each using two batches of BMCMCs and each of which gave similar results. , P < 0.05, versus the corresponding values for MyD88–/– BMCMCs; , P < 0.05, versus the corresponding values for BMCMCs incubated without IgE by ANOVA.
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Taken together, these observations indicate that MyD88, but not TRIF, is required for IL-33-mediated signal transduction and for the ability of IL-33 to influence survival and promote cytokine production in mouse BMCMCs.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-26 (data not shown). Our findings offer a new perspective about previously published findings concerning the effects of IL-33 in vivo. Schmitz et al. [7 ] demonstrated that treatment of mice with rIL-33 resulted in inflammation of the lungs and the gastrointestinal tract, which was associated with eosinophil recruitment, and resulted in elevations of serum concentrations of IgA, IgE, IL-5, and IL-13. Moreover, they showed that IL-33-mediated inflammation was abolished in IL-13-deficient mice [7 ]. As mast cells are present in the lungs and gut, and mast cells represent one potential source of IL-13, our in vitro findings raise the possibility that IL-33 can induce IL-13-dependent inflammation in vivo in part by inducing IL-13 production by mast cells.
In contrast to its strong effects on mouse mast cell cytokine production, IL-33 neither induced mouse mast cell degranulation in vitro, as assessed in our study by β-hexosaminidase release, nor is required for the induction of mast cell degranulation by IgE-FcRI-dependent signaling. For example, mast cells derived from ST2-deficient mice exhibited no abnormalities in histamine release after IgE/antigen stimulation or in IL-4 production after treatment with PMA + ionomycin [22
].
Although IL-33 clearly can have dramatic effects on mouse mast cell cytokine production in vitro, the influence of IL-33/ST2 interactions on mast cell function in vivo remains to be determined. It was reported that ST2-deficient mice, which had been sensitized with OVA admixed with the adjuvant aluminium hydroxide (alum), exhibited no impairment in the development of OVA-induced allergic airway inflammation [22
]. This protocol for OVA sensitization and challenge, which results in the development of airway hyperreactivity (AHR) and Th2-dominant and eosinophil-rich pulmonary inflammation, is now widely used as a model for asthma [43
]. However, mast cells [44
, 45
], B cells [46
], IgE/FcRI interactions [47
], IL-1 [48
, 49
], and TNF [50
] are not essential for the development of OVA-induced AHR and/or pulmonary inflammation induced in mice sensitized by injection of OVA admixed with alum. By contrast, such cells and mediators are crucial for the development of OVA-induced AHR and/or pulmonary inflammation induced in mice sensitized by injection of OVA without alum [45
, 47
48
49
50
]. Therefore, we think that it is more likely that IL-33/ST2-dependent effects (on mast cells or other cell types) may influence models of Th2-associated inflammation which are induced in mice having been sensitized without alum than in those sensitized with standard protocols using that adjuvant.
In CD4+ T cells, cross-linking of ST2 induced the phosphorylation of MAPKs such as JNK, p42/p44, and p38 [18
, 19
]. Moreover, IL-33-dependent signaling through ST2 involves the recruitment of MyD88, IRAK1, IRAK4, and TRAF6 and results in the activation of MAPK and NF-B [7
]. Schmitz et al. [7
] reported that IL-33 can enhance MAPK phosphorylation in mouse mast cells. We confirmed that IL-33 can activate MAPK (ERK1/2 and p38) phosphorylation in mouse mast cells and showed that this process, as well as IL-33-induced production of IL-6 and IL-13, depends, importantly, on a pathway involving MyD88 but not TRIF.
The biological consequences of IL-33/ST2 interactions appear to be quite complex, depending on the type of ST2-expressing cell, the concentration of IL-33, and other factors. For example, macrophages treated with soluble ST2 or deficient in ST2 exhibited increased proinflammatory cytokine production by LPS [51 52 53 ], suggesting that ST2 functions as an inhibitory receptor in such settings. By contrast, we have established that IL-33 has activating or, with IgE, coactivating functions in mouse mast cell cytokine production. Moreover, we have observed that IL-6 and TNF production by LPS-stimulated BMCMCs was enhanced significantly by the addition of rhIL-33 (unpublished observation). Thus, in mouse mast cells (our data) and in human mast cells [54 ], IL-33 can initiate or enhance cell activation pathways leading to cytokine secretion.
However, depending on its concentration, IL-33 appears to be able to exert positive or negative effects on mouse mast cell survival. We found that at concentrations of 1, 10, or 100 ng/ml, IL-33 significantly, albeit slightly, reduced the enhancement of BMCMC survival induced when the cells were incubated with the potently cytokinergic IgE mAb, SPE-7. By contrast, at 200 ng/ml, IL-33 significantly increased survival of BMCMCs in the absence of IgE but markedly decreased their survival when the cells were coincubated with SPE-7 IgE. We do not yet understand why, depending on the circumstances, IL-33 can exhibit such distinct effects on BMCMC survival in vitro nor do we know whether any of these effects can be detected in vivo.
In addition to analyzing effects of IL-33 on mast cells, we investigated the effects of IL-1β and IL-18. It has been reported that IL-1β or IL-18 can enhance IL-9 or IL-13 production by BMCMCs which have been stimulated with IgE and specific antigen or IgE and anti-IgE [4
5
6
]. We showed that in the absence of specific antigens recognized by IgE, IL-1β or IL-18 enhanced IL-6 and IL-13 production by BMCMCs which had been sensitized with the SPE-7 IgE mAb but not with the less potently cytokinergic IgE mAb, H1--26. These effects of IL-1β or IL-18 on BMCMC cytokine production occurred without detectably inducing mast cell degranulation.
The effect of IL-1β on BMCMC cytokine production is particularly notable, given that IL-1R1 expression was hardly detectable on naive BMCMCs or on cells sensitized with SPE-7 or H1--26 IgE. Despite our inability to detect IL-1R1 surface expression on mouse BMCMCs under the conditions tested, we (this study) and others [5
, 6
] have detected effects of IL-1 on mouse mast cell function. These results are reminiscent of findings in T cells, in that IL-1 can promote Th1 or Th17 cell differentiation and IFN- or IL-17 production by Th1 or Th17 cells, respectively [1
, 3
], although expression of IL-1Rs is hardly detectable by FACS analysis on the surface of such T cells [3
, 55
]. Similarly, TNF, another potent proinflammatory cytokine, can also promote Th1/Th17 cell differentiation and IFN- or IL-17 production by Th1 or Th17 cells, respectively [3
], although such cells exhibit little or no detectable surface expression of TNFR by FACS analysis [3
].
Sensitization with H1--26 IgE enhanced IL-18R surface expression more strongly on BMCMCs than did sensitization with SPE-7 IgE, but IL-18 did not detectably promote cytokine production in H1--26-sensitized BMCMCs. However, when we maintained H1--26- or SPE-7-sensitized BMCMCs in the presence of specific antigen (DNP-HSA) for 6 h, surface expression of IL-18R, but not IL-1R1, was up-regulated to levels higher than those observed in BMCMCs which had been incubated with the same mAb but in the absence of antigen (data not shown). Moreover, IL-1β or IL-18 enhanced IL-6 and IL-13 production by BMCMCs sensitized with H1--26 or SPE-7 when the cells were maintained for 6 h in the presence of the specific antigen DNP-HSA (data not shown).
Taken together, our observations provide additional evidence that IL-1β or IL-18 can enhance mast cell cytokine production in the presence of IgE-FcRI-derived signals.
In summary, we found that IL-33 can induce naïve mouse mast cells to secrete IL-6 or IL-13 in vitro and can enhance production of these cytokines in mast cells which are also stimulated via IgE or IgE and specific antigens. We also found that at 200 ng/ml, IL-33 enhanced mouse mast cell survival significantly in the absence of IgE or exogenous growth factors. Thus, the effects of IL-33 in mouse mast cells are similar to those reported recently for IL-33 in human umbilical cord blood-derived mast cells (HUCBMCs). Specifically, we found recently that IL-1β, IL-18, or IL-33 induced phosphorylation of Erk, p38, and JNK in naive HUCBMCs and that IL-33 or IL-1β also induced IL-8 and IL-13 production in naïve HUCBMCs and enhanced production of these cytokines in HUCBMCs stimulated with IgE and anti-IgE, without enhancing secretion of PGD2 or histamine [54 ]. IL-33 or IL-1β, but not IL-18, also enhanced the survival of naive HUCBMCs [54 ]. Based on the effects of IL-33 in mouse or human mast cells in vitro, it is tempting to speculate that IL-33 may contribute to the development or perpetuation of mast cell-dependent immune responses or disease processes in vivo.
-26 anti-DNP IgE mAb, Drs. Tsuneyasu Kaisho (Riken, Research Center for Allergy and Immunology, Kanagawa, Japan) and Yoichiro Iwakura (University of Tokyo, Japan) for MyD88–/– BM cells, Dr. Janet Kalesnikoff (Stanford University, Stanford, CA, USA) for helpful discussions, and Michiko Yamada (National Research Institute for Child Health and Development, Tokyo, Japan) for technical assistance. None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. 
3 Correspondence: Department of Allergy and Immunology, National Research Institute for Child Health & Development, Tokyo 157-8535, Japan. E-mail: snakae{at}nch.go.jp
Received April 2, 2007; revised August 20, 2007; accepted August 29, 2007.
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and TNF- are required for IL-12-induced development of Th1 cells producing high levels of IFN- in BALB/c but not C57BL/6 mice J. Immunol. 160,1708-1716 production from human T cells Int. Immunol. 12,151-160 is an intracrine proinflammatory activator of transcription Proc. Natl. Acad. Sci. USA 101,2434-2439RI Nature 402,B24-B30[CrossRef][Medline]RI Proc. Natl. Acad. Sci. USA 100,12911-12916This article has been cited by other articles:
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