IL-33, a member of the IL-1 family of cytokines, provokes Th2-type inflammation accompanied by accumulation of eosinophils through IL-33R, which consists of ST2 and IL-1RAcP. We previously demonstrated that macrophages produce IL-33 in response to LPS. Some immune responses were shown to differ between ST2-deficient mice and soluble ST2-Fc fusion protein-treated mice. Even in anti-ST2 antibody (Ab)-treated mice, the phenotypes differed between distinct Ab clones, because the characterization of such Abs (i.e., depletion, agonistic or blocking Abs) was unclear in some cases.
To elucidate the precise role of IL-33, we newly generated neutralizing monoclonal Abs for IL-33. Exogenous IL-33 potentiated LPS-mediated cytokine production by macrophages. That LPS-mediated cytokine production by macrophages was suppressed by inhibition of endogenous IL-33 by the anti-IL-33 neutralizing mAbs.
Our findings suggest that LPS-mediated macrophage activation is accelerated by macrophage-derived paracrine IL-33 stimulation.
Citation: Ohno T, Oboki K, Morita H, Kajiwara N, Arae K, et al. (2011) Paracrine IL-33 Stimulation Enhances Lipopolysaccharide-Mediated Macrophage Activation. PLoS ONE 6(4): e18404. doi:10.1371/journal.pone.0018404
Editor: Lena Alexopoulou, Centre d'Immunologie de Marseille-Luminy, CNRS-Inserm, France
Received: September 29, 2010; Accepted: March 7, 2011; Published: April 11, 2011
Copyright: © 2011 Ohno et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Japan Chemical Industry Association, Grants-in-Aid for Young Scientists (B) (T.O., K. Oboki. and S.N.), the Program for Improvement of Research Environment for Young Researchers, The Special Coordination Funds for Promoting Science and Technology (S.N.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan and a grant from the National Institute of Biomedical Innovation (H.S.). S.T. and M.I., who are the researchers in Medical & Biological Laboratories Co., Ltd., generated the anti-IL-33 mAb in the submitted manuscript. The other funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
IL-33 (also called IL-1F11, DVS27 and NF-HEV), which is a member of the IL-1 family of cytokines that includes IL-1 and IL-18, was identified as a ligand for ST2 (also called T1, DER-4, Fit-1 and IL-1R4) , , , . IL-33 is considered to be a cytokine that potently induces production of such Th2-cytokines as IL-5 and IL-13 by ST2-expressing immune cells such as Th2 cells , , , mast cells , , , , , eosinophils , , , basophils , ,  and macrophages , , and by stem-cell-like cells such as CD34+ hematopoietic stem cells , natural helper cells  and nuocytes . IL-33 is thereby thought to contribute to the development of Th2-cytokine-associated immune responses, including host defense against nematode infection and allergic diseases , , .
Indeed, administration of IL-33 to mice resulted in increased serum levels of Th2-cytokines such as IL-4, IL-5 and IL-13, as well as IgG1 and IgE, and development of inflammation accompanied by accumulation of eosinophils in the lung and gut . Moreover, polymorphism of the ST2 and/or IL-33 genes was found in patients with asthma , , , atopic dermatitis , rhinitis  and rhinosinusitis . The mRNA and/or protein levels of ST2, soluble ST2, which acts as a decoy receptor for IL-33, and IL-33 are increased in specimens from patients with allergic diseases such as asthma , , , , , , conjunctivitis , rhinitis  and atopic dermatitis . Therefore, these observations strongly suggest the importance of IL-33 and ST2 for the development of Th2-cytokine-associated allergic disorders.
However, based on the results of a study using mice treated with anti-ST2 Ab or soluble ST2-Fc fusion proteins and/or deficient in ST2, the roles of IL-33 and ST2 in the pathogenesis of certain immune diseases, including allergic airway inflammation, remain controversial . Studies using ST2-deficient mice found that ovalbumin (OVA)-induced airway inflammation developed normally in ST2-deficient mice sensitized twice with OVA emulsified with alum , , , whereas it was attenuated in the case of a single sensitization . On the other hand, mice treated with anti-ST2 mAb clone “3E10,” which induced Th2 cell activation as an agonistic Ab, at least in vitro , without depleting ST2-expressing cells in vivo , and mice treated with soluble ST2 showed reduced development of OVA-induced airway inflammation, even though they were sensitized twice with OVA with alum , . Unlike in ST2-deficient mice , , , the development of OVA-induced airway inflammation was aggravated in mice injected with ST2-deficient OVA-specific TCR (DO11.10)-expressing Th2 cells in comparison with those injected with wild-type DO11.10 Th2 cells after OVA challenge . That finding suggests that ST2 plays a negative role in Th2 cells, at least in that setting. On the other hand, it was shown that administration of anti-ST2 mAb “3E10” and soluble ST2-Fc fusion proteins to mice injected with DO11.10 Th2 cells resulted in attenuation of OVA-induced airway inflammation , . These seemingly contradictory observations could be explained on the basis of different roles for IL-33 and ST2 in distinct ST2-expressing cells. In support of that concept, IL-33 is able to enhance IFN-γ production by NK cells and iNKT cells , which are also involved in the pathogenesis of allergic airway inflammation , . Therefore, the precise roles of IL-33 and ST2 in different types of cells need to be elucidated.
We and others have demonstrated that IL-33 is able to enhance cytokine secretion by mast cells ,  and macrophages . We also reported that both mast cells and macrophages can produce IL-33 after stimulation with IgE and LPS, respectively . These observations suggest that IL-33 may be involved in the activation of these cells by autocrine/paracrine IL-33 release after such stimulation. In the present study, we used newly generated anti-IL-33 mAbs and demonstrated that activation of macrophages, but not mast cells, was modulated by paracrine IL-33 stimulation.
Materials and Methods
BALB/cA (BALB) mice, C57BL/6J (B6J) mice and C57BL/6N (B6N) mice were purchased from CLEA Japan and Sankyo Lab, respectively. B6J-TLR4−/− mice  and BALB-ST2−/− mice  were kindly provided by Drs. Tsuneyasu Kaisho (RIKEN, Japan) and Andrew N.J. McKenzie (MRC, Cambridge, UK), respectively. B6J-TRAF6−/− mice  and B6N-IL-33−/− mice  were generated as described elsewhere. All mice were housed under specific-pathogen-free conditions in our institutes (National Research Institute for Child Health and Development or The Institute of Medical Science, The University of Tokyo), and the animal protocols were approved by the Institutional Review Board of the National Research Institute for Child Health and Development (#06-10) and The Institute of Medical Science, The University of Tokyo (#A09-10).
Anti-mouse ST2 Abs
Anti-mouse ST2 mAb (clone 3E10) had been generated as described elsewhere . FITC-conjugated and non-conjugated anti-mouse ST2 mAbs (clones DJ8 , , 245707 and 245714) were obtained from MD Bioscience and R&D Systems, respectively.
Anti-human/mouse IL-33 mAb (Nessy-1, Alexis), anti-mouse IL-33 mAb (518017, R&D Systems) and anti-mouse IL-33 polyAb (AF3626, R&D Systems) were used.
Generation of anti-mouse IL-33 mAbs
Anti-mouse IL-33 mAbs were generated and provided by Medical & Biological Laboratories Co., Ltd. (Nagano, Japan). cDNA encoding the mouse IL-33 corresponding to amino acids 109–266 was expressed in E. coli as an N-terminal tagged fusion protein. After purification of the fusion protein, the tagged sequence was cleaved enzymatically and removed by affinity purification. Five-week-old female C3H mice (Japan SLC, Hamamatsu) were immunized with the purified protein emulsified with Freund's complete adjuvant (Sigma-Aldrich) by injection into the footpads 5 times at 1-week intervals]. Three days after the final immunization, cells from the lymph nodes of the immunized mice were fused with P3-U1 mouse myeloma cells in the presence of 50% (w/v) polyethylene glycol (PEG4000) (Wako). Hybridomas were screened by ELISA and immunoblotting to identify those generating mAbs. Positive clones were subcloned two times by limiting dilution and rescreened by ELISA and immunoblotting. The mAbs were purified from the culture supernatant using Protein A-Sepharose (GE Healthcare). The eluted antibodies were analyzed by SDS-PAGE.
Bone marrow cell-derived and fetal liver cell-derived cultured mast cells
Mouse femoral bone marrow cell-derived cultured mast cells (BMCMCs) were generated as described elsewhere . For generation of fetal liver cell-derived cultured mast cells (FLCMCs), livers were harvested from newborn TRAF6+/+ and TRAF6−/− mice, and liver single-cell suspensions were prepared by grinding the tissues through a 70-µm nylon cell strainer (BD Falcon) with the plunger of a 5-ml disposable syringe. Bone marrow cells and fetal liver cells were cultured in the presence of 10 ng/ml rmIL-3 (PeproTech) for 6–8 weeks, at which time flow cytometry showed the cells to be a >98% c-kit+ FcεRIα+ population. Before using the cells, rmIL-3 was removed by washing. MCs (2×105 cells/well in 96-well flat-bottom plates) were cultured with 1 µg/ml IgE (SPE-7, Sigma), 30 or 100 ng/ml rmIL-33 (R&D Systems) and a combination of 1 µg/ml SPE-7 plus 100 ng/ml rmIL-33 in the presence and absence of 40 or 80 µg/ml anti-mouse ST2 mAb, anti-IL-33 Ab or isotype-matched control IgG for 24 h.
Thioglycolate (TGC)-induced macrophages
For collection of thioglycolate (TGC)-induced mouse peritoneal macrophages (TGC-macrophages), mice were injected intraperitoneally with 5 ml of 2% TGC (Nissui). Three days later, peritoneal exudate cells (PECs) were collected. TGC-macrophages (2×105 cells/well in 96-well flat-bottom plates) were incubated with 0–100 ng/ml LPS (Salmonella enterica serotype typhimurium; SIGMA) in the presence and absence of 40 µg/ml anti-ST2 mAb, anti-IL-33 mAb or isotype-matched control IgG for 24 or 48 h.
BMCMCs were incubated with anti-CD16/CD32 mAb (93, eBioscience; or 2.4G2, BD Biosciences) for 15 min on ice. The cells were then incubated with PE-conjugated anti-mouse FcεRIα (MAR-1, eBioscience), APC-conjugated anti-mouse c-Kit (2B8, eBioscience) and FITC-conjugated or non-conjugated anti-mouse ST2 mAb (DJ8, 3E10, 245707 or 245714) for 45 min on ice. After washing, the cells were incubated with mFITC-conjugated anti-rat IgG2b (RG7/11.1, BD Biosciences) or anti-rat IgG2a (RG7/1.30, BD Biosciences) as the second antibody for non-conjugated anti-mouse ST2 mAbs for 45 min on ice. The expression of ST2 on 7-amino actinomycin D-negative FcεRIα+ c-Kit+ BMCMCs was analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using CellQuest software (Becton Dickinson).
TGC-induced peritoneal macrophages (1×106 cells/ml for FACS analysis and 2.5×105 cells/ml for lactate dehydrogenase [LDH] release assay, respectively) were cultured in the presence and absence of 100 ng/ml LPS for 0–48 h. Cell viability was assessed using a MEBCYTO-Apoptosis kit (MBL) or LDH assay kit (CytoTox 96; Promega) as described previously .
The levels of IL-6, IL-13 and TNF in culture supernatants were measured with mouse IL-6, IL-13 and TNF ELISA sets (eBioscience).
The number of IL-33-secreting cells by ELISPOT assay was performed as described elsewhere . Breifly, MultiScreen-IP plates (MAIPS4510; Millipore) were coated with anti-mouse IL-33 polyclonal Ab (R&D Systems; 2 µg/ml in PBS) as a capture Ab at 4°C overnight. After blocking with PBS containing 10% FCS, TGC-induced peritoneal macrophages (2×104/200 µl) were cultured in the presence or absence of 100 mg/ml LPS or 0.1 µg/ml PMA plus 1 µg/ml ionomycin at 37°C for 24 h or 48 h. After washing the wells, biotinylated anti-mouse/human IL-33 mAb (Nessy-1; Alexis Biochemicals, 400 ng/ml in PBS containing 10% FCS) as a detection Ab was applied and incubated at r.t. for 1 h. Then, after washing the wells, HRP-conjugated streptavidin (BD Biosciences) was added to the wells at r.t. for 1 h. AEC (Sigma) were used as substrates. Positive spots on Ab-coated plates were analyzed with NIH Image software.
An unpaired Student's t-test, 2-tailed, was used for statistical evaluation of the results.
Effects of anti-ST2 mAbs on cytokine production by BMCMCs
Several mAbs against mouse ST2, i.e., clones DJ8 , , 3E10 , 245707 and 245714, have been generated to study the role(s) of ST2 in immune responses. It was recently demonstrated in vitro that IL-33-mediated cytokine production by macrophages was inhibited by addition of DJ8 , suggesting that DJ8 acts as a neutralizing Ab for IL-33 bioactivity. The crosslinking of ST2 by 3E10 enhanced Th2 cytokine production by Th2 cells in vitro , while the administration of 3E10 in mice resulted in the suppression of Th2 cell/cytokine-mediated allergic or viral airway inflammation , ,  without depletion of ST2-expressing cells . However, the effects of the other mAbs on IL-33-mediated immune cell activation remain unknown.
Recombinant mouse IL-33 (rmIL-33) can induce cytokine secretion by mouse bone marrow cell-derived cultured mast cells (BMCMCs) (Fig. 1A) dependent on MyD88, which is an essential adapter molecule for signal transduction of the TLR/IL-1R (TIR) superfamily . As in the case of MyD88−/− BMCMCs  and ST2−/− BMCMCs (data not shown), IL-6, IL-13 and TNF production by FLCMCs deficient in TRAF6, which is a downstream molecule of MyD88, was impaired by rmIL-33 (derived from E. coli) (Fig. 1B). On the other hand, rmIL-33-mediated secretion of these cytokines was observed to be comparable in wild-type (WT) and TLR4−/− BMCMCs (Fig. 1), indicating that the biological activity of rmIL-33 was not influenced by contamination with endotoxin.
Figure 1. IL-33 induces TRAF6-dependent cytokine production by mast cells.
BMCMCs obtained from B6J-WT mice (A) and B6J-WT and -TLR4−/− mice (B; left panels) and FLCMCs obtained from B6J-WT and -TRAF6−/− mice (B; right panels) were cultured in the presence of various concentration of rmIL-33 (A) or in the presence and absence of 100 ng/ml rmIL-33 for 6 h (for TNF measurement) and 24 h (for IL-6 and IL-13 measurement). The levels of IL-6, IL-13 and/or TNF in the culture supernatants were determined by ELISA. Data show the mean + SD (n = 3). *p<0.05 vs. WT.doi:10.1371/journal.pone.0018404.g001
We next examined the effects of the anti-mouse ST2 mAbs on cytokine production by BMCMCs after IL-33 stimulation. Cytokine secretion by BMCMCs in response to 3–30 or 100 ng/mL rmIL-33 was profoundly or partially (nearly half maximum) inhibited in the presence of 40 µg/mL anti-ST2 mAb (DJ8), respectively (Fig. 2A). Therefore, we used 30 or 100 ng/mL rmIL-33 in the other neutralization studies. IL-33-mediated IL-6 and IL-13 production by WT BMCMCs was inhibited by addition of 245707 as well as DJ8, but not 3E10 or 245714 (Fig. 2B). Like rIL-33, it has been reported that crosslinking of ST2 by 3E10 promoted cytokine secretion by Th2 cells in vitro as an agonistic Ab . On the other hand, 3E10 alone could not enhance IL-6 or IL-13 production by WT BMCMCs (Fig. 2B), although 3E10 as well as DJ8 and 245707, but not 245714, bound to ST2 on the cell surface of BMCMCs (Fig. 2C). We also found that crosslinking of ST2 by 3E10 and anti-rat IgG did not induce IL-6 or TNF production by BMCMCs (data not shown). These observations suggest that DJ8 and 245707, but not 3E10 or 245714, have neutralizing activity for IL-33-mediated mast cell activation, at least in vitro. Moreover, these observations indicate that the effect of 3E10 differs between Th2 cells  and mast cells.
Figure 2. Effects of anti-ST2 mAbs on cytokine production by IL-33-stimulated BMCMCs.
B6J-WT BMCMCs were stimulated with 0–1,000 ng/ml (A) or 100 ng/ml (B) rmIL-33 in the presence of 40 µg/ml of several anti-ST2 mAbs or isotype control rat IgG for 24 h. The levels of IL-6 and IL-13 in the culture supernatants were determined by ELISA. Data show the mean + SEM (n = 3). *p<0.05 vs. rat IgG+IL-33. The expression of ST2 on the cell surface of BALB-WT and ST2−/− BMCMCs was determined using several distinct anti-ST2 mAb clones. Representative data by flow cytometry are shown (C). Shaded area indicates isotype-matched control IgG staining, and bold line indicates anti-ST2 mAb staining.doi:10.1371/journal.pone.0018404.g002
Effects of anti-IL-33 mAb on cytokine production by BMCMCs
It was shown that ST2-expressing cells were depleted by anti-ST2 polyclonal Ab in vitro . Therefore, anti-IL-33 Ab(s) rather than anti-ST2 Ab(s) would be useful for elucidating the role(s) of the IL-33-ST2 pathway in vitro and in vivo. Accordingly, we next examined the effects of anti-IL-33 mAbs (Nessy-1 and 518017) and polyclonal Ab (AF3626) on cytokine production by BMCMCs in response to rmIL-33. Nessy-1, but not 518017 or AF3626, inhibited IL-33-mediated IL-13 production by BMCMCs (Fig. 3A). However, the inhibitory effect of Nessy-1 was weak in comparison with that of the DJ8 anti-ST2 mAb, as shown in Figure 2A. Therefore, we newly generated anti-IL-33 mAbs (which were confirmed by western blot analysis to recognize rmIL-33; data not shown) and investigated their effects on IL-33-mediated cytokine production by BMCMCs. Ten (1D2, 1F11, 2A2, 2E6, 2C7, 4A3, 4D4, 4G4, 5F1 and 5D11) of 100 tested anti-IL-33 mAbs were able to inhibit IL-33-mediated IL-13 production (Fig. 3B). Like DJ8 (Fig. 2A), some of those mAbs (i.e., 2A2, 2E6 and so on) strongly inhibited IL-33 activity (Fig. 3B).
Figure 3. Effects of anti-IL-33 Abs on cytokine production by IL-33-stimulated BMCMCs.
B6J-WT BMCMCs were stimulated with 30-ng/ml rmIL-33 in the presence and absence of commercially available anti-IL-33 Abs (A), our newly generated anti-IL-33 mAbs (B) or control IgG (A, B) for 24 h. The levels of IL-13 in the culture supernatants were determined by ELISA. Data show the mean + SEM (n = 3). *p<0.05 vs. control IgG+IL-33.doi:10.1371/journal.pone.0018404.g003
Effects of anti-IL-33 mAbs on cytokine production by TGC-induced macrophages and BMCMCs
It was recently reported that recombinant IL-33 enhanced LPS-mediated cytokine production by macrophages . Consistent with this, we found that IL-33 augmented IL-6 production by TGC-induced peritoneal macrophages in response to LPS (Fig. 4A). We reported that TGC-induced peritoneal macrophages produced IL-33 in response to LPS . In addition, it is thought that IL-33 is released by necrotic cells after stimulation , . The proportion of annexin V-negative and propidium iodide (PI)-positive necrotic macrophages, the levels of LDH release in the culture supernatants and the number of IL-33-secreting macrophages were significantly increased at 48 h after LPS stimulation (Fig. 4B–D). Consistent with previous reports , we could not detect IL-33 proteins in the culture supernatants and cell lysates by ELISA and western blot analysis, respectively (data not shown). These observations suggest that necrotic macrophage-derived IL-33 may paracrinely promote cytokine production by viable macrophages after LPS stimulation. In support of this, IL-6 production by IL-33−/− macrophages was reduced in comparison with WT macrophages at 24 and 48 h after LPS stimulation (Fig. 4E). To more fully elucidate this, we examined the effects of endogenous IL-33 on cytokine production by LPS-stimulated TGC-induced macrophages in the presence of anti-ST2 mAbs and anti-IL-33 mAbs. The LPS-mediated IL-6 production by TGC-induced macrophages was inhibited by addition of anti-ST2 mAbs DJ8 and 245707, but not 3E10 or 245714 at 48 h, but not 24 h, after LPS stimulation (Fig. 5A). These responses by TGC-induced macrophages were also inhibited by addition of anti-IL-33 mAbs 2C7 and 1F11, but not other mAbs including 5D11, 1D2, 2A2 and 2E6, at 48 h, but not 24 h, after LPS stimulation (Fig. 5B and data not shown). We previously demonstrated that IL-33 mRNA expression was increased in BMCMCs after stimulation with highly cytokinergic IgE , FcεRI-crosslinking by IgE and antigens, and PMA+ionomycin, but not LPS . However, the expression level of IL-33 protein by BMCMCs was less than that by TGC-induced macrophages after stimulation . In accordance with this, IL-13 production by BMCMCs was not influenced by addition of any of the anti-IL-33 mAbs at 48 h after IgE stimulation (anti-DNP IgE; SPE-7) (Fig. 5C). These observations suggest that macrophages, rather than mast cells, are potential producers of IL-33, and that macrophage-derived IL-33 can activate macrophages in a paracrine manner after LPS stimulation.
Figure 4. IL-33 enhances LPS-mediated cytokine production by macrophages.
TGC-induced peritoneal macrophages derived from B6J-WT mice (A–D) and B6N-WT and -IL-33−/− mice (E) were cultured in the presence and absence of 100 ng/ml LPS, with and without 100 ng/ml IL-33, for 9, 24 and/or 48 h. (A, E) The levels of IL-6 in the culture supernatants by ELISA. (B) The percentage of PI-positive cells by flow cytometry. (C) LDH levels in the culture supernatants. (D) The number of IL-33-secreting cells by ELISPOT. Data show the mean +/± SEM (n = 3 [A] or 4 [B–E]). *p<0.05 vs. the indicated group (A) or Medium (B–E), and †p<0.05 vs. 24 h (C, D) or WT (E). P+I = PMA+ionomycin.doi:10.1371/journal.pone.0018404.g004
Figure 5. Inhibitory effects of anti-IL-33 mAbs on LPS-mediated macrophage activation by paracrine IL-33 stimulation.
(A, B) TGC-induced peritoneal macrophages derived from B6J-WT mice were cultured in the presence of 100 ng/ml LPS, with and without 40 µg/ml of several anti-ST2 mAbs (A), several anti-IL-33 mAbs (B) or control IgG (A, B) for 24 and 48 h. (C) B6J-WT BMCMCs were cultured in the presence of 1 µg/ml anti-DNP IgE (SPE-7), with and without 40 µg/ml of several anti-IL-33 mAbs or control IgG for 24 and 48 h. The levels of IL-6 or IL-13 in the culture supernatants were measured by ELISA. Data show the mean + SEM ([A] n = 7, [B] n = 8 [C] n = 4). *p<0.05 vs. Rat IgG (A) or Mouse IgG (B).doi:10.1371/journal.pone.0018404.g005
Like ST2−/− mice  and mice treated with a soluble ST2-Fc fusion protein , mice treated with a certain anti-ST2 mAb (generated by Amgen) showed attenuated development of collagen-induced arthritis . Since that ST2 mAb (Amgen) inhibited IL-33-mediated immune responses in vitro and in vivo, it is considered to act as a blocking Ab for binding of IL-33 to ST2. Conversely, mice treated with an anti-ST2 polyclonal Ab showed aggravated development of collagen-induced arthritis . Since that polyclonal Ab lysed ST2-expressing cells in vitro, its in vivo administration may have depleted certain ST2-expressing regulatory cells such as Tr1 cells  as well as ST2-expressing effector cells such as mast cells , thereby causing aggravation, rather than attenuation, of the arthritis. However, the precise activities (i.e., depletion, agonism, blocking, etc.) of the other ST2 Abs were poorly characterized in the previous studies, because many of which were performed before the identification of IL-33.
It is well known that the biological activities of the IL-1 family of cytokines are elaborately regulated by decoy/soluble receptors, binding proteins and/or receptor antagonists , . For example, the activities of IL-1α and IL-1β are mediated by IL-1R (IL-1R1 and IL-1RAcP), but blocked by IL-1R2, the soluble form IL-1Rs and IL-1 receptor antagonist (IL-1Ra) , . The activities of IL-18 are mediated by IL-18R, but inhibited by IL-18-binding protein , . On the other hand, inconsistent results were reported between a ligand- and its receptor-deficient mice even on the same genetic background. For example, experimental autoimmune encephalomyelitis developed normally in IL-18−/− mice, but not in IL-18Rα−/− mice . These observations suggest involvement of another ligand(s) besides IL-18, i.e., IL-1F7 , in the development of the disease. Moreover, IL-1F10, in addition to IL-1α, IL-1β and IL-1Ra, also can bind to IL-1R1, although its binding affinity is low compared with IL-1β and IL-1Ra . Therefore, like IL-18Rα and IL-1R1, ST2 may be a component of receptors for another ligand(s) besides IL-33. As another possibility, IL-33 may bind to other receptors besides ST2, SIGIRR/Tir8  and c-Kit . Thus, it was surmised that, for elucidation of the precise roles of IL-33 in vivo and in vitro, it would be more advantageous to use neutralizing Abs for IL-33 rather than for ST2. Therefore, in the present study, we newly generated anti-IL-33 mAbs and characterized their functions as well as the functions of anti-ST2 Abs.
We and the others have shown that macrophages can release IL-33 after LPS stimulation , and IL-33 can enhance LPS-mediated TNF and IL-6 production by macrophages (Fig. 4A) . We also found that IL-33−/− macrophages showed reduced IL-6 production in response to LPS (Fig. 4C). Likewise, LPS-mediated IL-6 production by macrophages was inhibited by treatment with anti-IL-33 mAbs (2C7 and 1F11, but not other mAbs) (Fig. 5B and data not shown), a soluble ST2-Fc fusion protein  or anti-ST2 mAbs (DJ8 and 245707, but not 3E10 and 245714) (Fig. 5A). It was reported that IL-1, IL-6, IL-12 and TNF production by macrophages from ST2−/− mice on the BALB/c background was increased  or comparable  with those from wild-type mice at 12, 24 or 48 h after LPS stimulation. The apparent discrepancy between ST2−/− macrophages and IL-33−/−/anti-ST2 mAb-treated/soluble ST2-Fc fusion protein-treated macrophages may be accounted for as described elsewhere . Nonetheless, these observations (except the study using ST2−/− macrophages ) suggest that macrophages produce IL-33 in response to LPS, and that that IL-33 then additively promotes LPS-mediated macrophage activation.
The inhibitory levels of cytokine production by macrophages treated with anti-IL-33 neutralizing Ab was lesser than those by IL-33−/− macrophages after LPS stimulation. It is considered that IL-33 has dual roles as a cytokine and a nuclear factor , , . The function of both secreted and nuclear IL-33 was abrogated in IL-33-deficient cells. On the other hand, the neutralizing antibody for IL-33 and/or ST2 can inhibit the effect of secreted IL-33, but not that of nuclear IL-33. Thus, the difference between anti-IL-33 neutralizing antibody-treated and IL-33-deficient macrophages may be due to the potential role of IL-33 in the nucleus.
Anti-IL-33 mAb 5D11 inhibited recombinant IL-33109–266-dependent IL-13 production by BMCMCs (Fig. 3B). Conversely, it did not inhibit the activity of endogenous IL-33 (probably full-length IL-33) in LPS-mediated IL-6 production by macrophages (Fig. 5B). Like anti-IL-33 mAb Nessy-1, our anti-IL-33 mAbs were generated against IL-33109–266, not full-length IL-33, and it seemed that some of them including 1D2, 2A2, and 2E6 could not inhibit the activities of full-length IL-33 even though they neutralized recombinant IL-33109–266 (Fig. 3B and data not shown).
In conclusion, our findings suggest that IL-33-neutralizing mAbs, which we newly generated, will be useful tools for the understanding the pathophysiological function(s) of IL-33 in vitro and presumably in vivo. They also have potential for aiding in the development of new therapeutics for certain IL-33-mediated disorders.
We thank Drs. Tsuneyasu Kaisyo and (RIKEN, Japan) and Andrew N.J. McKenzie (MRC, Cambridge, UK) for providing B6J-TLR4−/− mice and BALB-ST2−/− mice, and Michiko Yamada, Hiromi Wakita, Shuhei Fukuda and Noriko Hashimoto for their excellent technical assistance.
Analyzed the data: TO K. Oboki KA HM NK. Contributed reagents/materials/analysis tools: M. Iikura TA JI KM KS MA K. Okumura TK HS. Wrote the paper: TO K. Oboki SN. Designed the study: TO K. Oboki SN. Performed the research: TO K. Oboki KA HM NK. Generation of anti-IL-33 mAbs: ST M. Ikeda.
- 1. Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, et al. (2005) IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 23: 479–490.
- 2. Smith DE (2010) IL-33: a tissue derived cytokine pathway involved in allergic inflammation and asthma. Clin Exp Allergy 40: 200–208.
- 3. Liew FY, Pitman NI, McInnes IB (2010) Disease-associated functions of IL-33: the new kid in the IL-1 family. Nat Rev Immunol 10: 103–110.
- 4. Oboki K, Ohno T, Kajiwara N, Saito H, Nakae S (2010) IL-33 and IL-33 receptors in host defense and diseases. Allergol Int 59: 143–160.
- 5. Chackerian AA, Oldham ER, Murphy EE, Schmitz J, Pflanz S, et al. (2007) IL-1 receptor accessory protein and ST2 comprise the IL-33 receptor complex. J Immunol 179: 2551–2555.
- 6. Cherry WB, Yoon J, Bartemes KR, Iijima K, Kita H (2008) A novel IL-1 family cytokine, IL-33, potently activates human eosinophils. J Allergy Clin Immunol 121: 1484–1490.
- 7. Ho LH, Ohno T, Oboki K, Kajiwara N, Suto H, et al. (2007) IL-33 induces IL-13 production by mouse mast cells independently of IgE-FcepsilonRI signals. J Leukoc Biol 82: 1481–1490.
- 8. Moulin D, Donze O, Talabot-Ayer D, Mezin F, Palmer G, et al. (2007) Interleukin (IL)-33 induces the release of pro-inflammatory mediators by mast cells. Cytokine 40: 216–225.
- 9. Iikura M, Suto H, Kajiwara N, Oboki K, Ohno T, et al. (2007) IL-33 can promote survival, adhesion and cytokine production in human mast cells. Lab Invest 87: 971–978.
- 10. Allakhverdi Z, Smith DE, Comeau MR, Delespesse G (2007) The ST2 ligand IL-33 potently activates and drives maturation of human mast cells. J Immunol 179: 2051–2054.
- 11. Ali S, Huber M, Kollewe C, Bischoff SC, Falk W, et al. (2007) IL-1 receptor accessory protein is essential for IL-33-induced activation of T lymphocytes and mast cells. Proc Natl Acad Sci U S A 104: 18660–18665.
- 12. Suzukawa M, Iikura M, Koketsu R, Nagase H, Tamura C, et al. (2008) An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor. J Immunol 181: 5981–5989.
- 13. Pecaric-Petkovic T, Didichenko SA, Kaempfer S, Spiegl N, Dahinden CA (2009) Human basophils and eosinophils are the direct target leukocytes of the novel IL-1 family member IL-33. Blood 113: 1526–1534.
- 14. Kondo Y, Yoshimoto T, Yasuda K, Futatsugi-Yumikura S, Morimoto M, et al. (2008) Administration of IL-33 induces airway hyperresponsiveness and goblet cell hyperplasia in the lungs in the absence of adaptive immune system. Int Immunol 20: 791–800.
- 15. Sweet MJ, Leung BP, Kang D, Sogaard M, Schulz K, et al. (2001) A novel pathway regulating lipopolysaccharide-induced shock by ST2/T1 via inhibition of Toll-like receptor 4 expression. J Immunol 166: 6633–6639.
- 16. Brint EK, Xu D, Liu H, Dunne A, McKenzie AN, et al. (2004) ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance. Nat Immunol 5: 373–379.
- 17. Allakhverdi Z, Comeau MR, Smith DE, Toy D, Endam LM, et al. (2009) CD34+ hemopoietic progenitor cells are potent effectors of allergic inflammation. J Allergy Clin Immunol 123: 472–478.
- 18. Moro K, Yamada T, Tanabe M, Takeuchi T, Ikawa T, et al. (2010) Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells. Nature 463: 540–544.
- 19. Neill DR, Wong SH, Bellosi A, Flynn RJ, Daly M, et al. (2010) Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 464: 1367–1370.
- 20. Gudbjartsson DF, Bjornsdottir US, Halapi E, Helgadottir A, Sulem P, et al. (2009) Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction. Nat Genet 41: 342–347.
- 21. Reijmerink NE, Postma DS, Bruinenberg M, Nolte IM, Meyers DA, et al. (2008) Association of IL1RL1, IL18R1, and IL18RAP gene cluster polymorphisms with asthma and atopy. J Allergy Clin Immunol 122: 651–654 e658.
- 22. Ali M, Zhang G, Thomas WR, McLean CJ, Bizzintino JA, et al. (2009) Investigations into the role of ST2 in acute asthma in children. Tissue Antigens 73: 206–212.
- 23. Shimizu M, Matsuda A, Yanagisawa K, Hirota T, Akahoshi M, et al. (2005) Functional SNPs in the distal promoter of the ST2 gene are associated with atopic dermatitis. Hum Mol Genet 14: 2919–2927.
- 24. Sakashita M, Yoshimoto T, Hirota T, Harada M, Okubo K, et al. (2008) Association of serum interleukin-33 level and the interleukin-33 genetic variant with Japanese cedar pollinosis. Clin Exp Allergy 38: 1875–1881.
- 25. Castano R, Bosse Y, Endam LM, Desrosiers M (2009) Evidence of association of interleukin-1 receptor-like 1 gene polymorphisms with chronic rhinosinusitis. Am J Rhinol Allergy 23: 377–384.
- 26. Smithgall MD, Comeau MR, Yoon BR, Kaufman D, Armitage R, et al. (2008) IL-33 amplifies both Th1- and Th2-type responses through its activity on human basophils, allergen-reactive Th2 cells, iNKT and NK cells. Int Immunol 20: 1019–1030.
- 27. Kurowska-Stolarska M, Stolarski B, Kewin P, Murphy G, Corrigan CJ, et al. (2009) IL-33 amplifies the polarization of alternatively activated macrophages that contribute to airway inflammation. J Immunol 183: 6469–6477.
- 28. Kuroiwa K, Li H, Tago K, Iwahana H, Yanagisawa K, et al. (2000) Construction of ELISA system to quantify human ST2 protein in sera of patients. Hybridoma 19: 151–159.
- 29. Oshikawa K, Kuroiwa K, Tago K, Iwahana H, Yanagisawa K, et al. (2001) Elevated soluble ST2 protein levels in sera of patients with asthma with an acute exacerbation. Am J Respir Crit Care Med 164: 277–281.
- 30. Prefontaine D, Lajoie-Kadoch S, Foley S, Audusseau S, Olivenstein R, et al. (2009) Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells. J Immunol 183: 5094–5103.
- 31. Matsuda A, Okayama Y, Terai N, Yokoi N, Ebihara N, et al. (2009) The role of interleukin-33 in chronic allergic conjunctivitis. Invest Ophthalmol Vis Sci 50: 4646–4652.
- 32. Pushparaj PN, Tay HK, H'Ng SC, Pitman N, Xu D, et al. (2009) The cytokine interleukin-33 mediates anaphylactic shock. Proc Natl Acad Sci U S A 106: 9773–9778.
- 33. Hoshino K, Kashiwamura S, Kuribayashi K, Kodama T, Tsujimura T, et al. (1999) The absence of interleukin 1 receptor-related T1/ST2 does not affect T helper cell type 2 development and its effector function. J Exp Med 190: 1541–1548.
- 34. Mangan NE, Dasvarma A, McKenzie AN, Fallon PG (2007) T1/ST2 expression on Th2 cells negatively regulates allergic pulmonary inflammation. Eur J Immunol 37: 1302–1312.
- 35. Kurowska-Stolarska M, Kewin P, Murphy G, Russo RC, Stolarski B, et al. (2008) IL-33 induces antigen-specific IL-5+ T cells and promotes allergic-induced airway inflammation independent of IL-4. J Immunol 181: 4780–4790.
- 36. Meisel C, Bonhagen K, Lohning M, Coyle AJ, Gutierrez-Ramos JC, et al. (2001) Regulation and function of T1/ST2 expression on CD4+ T cells: induction of type 2 cytokine production by T1/ST2 cross-linking. J Immunol 166: 3143–3150.
- 37. Kearley J, Buckland KF, Mathie SA, Lloyd CM (2009) Resolution of allergic inflammation and airway hyperreactivity is dependent upon disruption of the T1/ST2-IL-33 pathway. Am J Respir Crit Care Med 179: 772–781.
- 38. Coyle AJ, Lloyd C, Tian J, Nguyen T, Erikkson C, et al. (1999) Crucial role of the interleukin 1 receptor family member T1/ST2 in T helper cell type 2-mediated lung mucosal immune responses. J Exp Med 190: 895–902.
- 39. Oshikawa K, Yanagisawa K, Tominaga S, Sugiyama Y (2002) Expression and function of the ST2 gene in a murine model of allergic airway inflammation. Clin Exp Allergy 32: 1520–1526.
- 40. Lohning M, Stroehmann A, Coyle AJ, Grogan JL, Lin S, et al. (1998) T1/ST2 is preferentially expressed on murine Th2 cells, independent of interleukin 4, interleukin 5, and interleukin 10, and important for Th2 effector function. Proc Natl Acad Sci U S A 95: 6930–6935.
- 41. Culley FJ (2009) Natural killer cells in infection and inflammation of the lung. Immunology 128: 151–163.
- 42. Umetsu DT, DeKruyff RH (2006) A role for natural killer T cells in asthma. Nat Rev Immunol 6: 953–958.
- 43. Espinassous Q, Garcia-de-Paco E, Garcia-Verdugo I, Synguelakis M, von Aulock S, et al. (2009) IL-33 enhances lipopolysaccharide-induced inflammatory cytokine production from mouse macrophages by regulating lipopolysaccharide receptor complex. J Immunol 183: 1446–1455.
- 44. Ohno T, Oboki K, Kajiwara N, Morii E, Aozasa K, et al. (2009) Caspase-1, caspase-8, and calpain are dispensable for IL-33 release by macrophages. J Immunol 183: 7890–7897.
- 45. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, et al. (1999) Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 162: 3749–3752.
- 46. Townsend MJ, Fallon PG, Matthews DJ, Jolin HE, McKenzie AN (2000) T1/ST2-deficient mice demonstrate the importance of T1/ST2 in developing primary T helper cell type 2 responses. J Exp Med 191: 1069–1076.
- 47. Naito A, Azuma S, Tanaka S, Miyazaki T, Takaki S, et al. (1999) Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 4: 353–362.
- 48. Oboki K, Ohno T, Kajiwara N, Arae K K, Morita H, et al. (2010) IL-33 is a crucial amplifier of innate rather than acquired immunity. Proc Natl Acad Sci U S A 107: 18581–18586.
- 49. Moritz DR, Gheyselinck J, Klemenz R (1998) Expression analysis of the soluble and membrane-associated forms of the interleukin-1 receptor-related T1 protein in primary mast cells and fibroblasts. Hybridoma 17: 107–116.
- 50. Moritz DR, Rodewald HR, Gheyselinck J, Klemenz R (1998) The IL-1 receptor-related T1 antigen is expressed on immature and mature mast cells and on fetal blood mast cell progenitors. J Immunol 161: 4866–4874.
- 51. Walzl G, Matthews S, Kendall S, Gutierrez-Ramos JC, Coyle AJ, et al. (2001) Inhibition of T1/ST2 during respiratory syncytial virus infection prevents T helper cell type 2 (Th2)- but not Th1-driven immunopathology. J Exp Med 193: 785–792.
- 52. Xu D, Chan WL, Leung BP, Huang F, Wheeler R, et al. (1998) Selective expression of a stable cell surface molecule on type 2 but not type 1 helper T cells. J Exp Med 187: 787–794.
- 53. Luthi AU, Cullen SP, McNeela EA, Duriez PJ, Afonina IS, et al. (2009) Suppression of interleukin-33 bioactivity through proteolysis by apoptotic caspases. Immunity 31: 84–98.
- 54. Cayrol C, Girard JP (2009) The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1. Proc Natl Acad Sci U S A 106: 9021–9026.
- 55. Kawakami T, Kitaura J, Xiao W, Kawakami Y (2005) IgE regulation of mast cell survival and function. Novartis Found Symp 271: 100–107. discussion 108–114, 145–151.
- 56. Xu D, Jiang HR, Kewin P, Li Y, Mu R, et al. (2008) IL-33 exacerbates antigen-induced arthritis by activating mast cells. Proc Natl Acad Sci U S A 105: 10913–10918.
- 57. Leung BP, Xu D, Culshaw S, McInnes IB, Liew FY (2004) A novel therapy of murine collagen-induced arthritis with soluble T1/ST2. J Immunol 173: 145–150.
- 58. Palmer G, Talabot-Ayer D, Lamacchia C, Toy D, Seemayer CA, et al. (2009) Inhibition of interleukin-33 signaling attenuates the severity of experimental arthritis. Arthritis Rheum 60: 738–749.
- 59. McGuirk P, McCann C, Mills KH (2002) Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J Exp Med 195: 221–231.
- 60. Dinarello CA (2009) Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol 27: 519–550.
- 61. Sims JE, Smith DE (2010) The IL-1 family: regulators of immunity. Nat Rev Immunol 10: 89–102.
- 62. Gutcher I, Urich E, Wolter K, Prinz M, Becher B (2006) Interleukin 18-independent engagement of interleukin 18 receptor-α is required for autoimmune inflammation. Nat Immunol 7: 946–953.
- 63. Pan G, Risser P, Mao W, Baldwin DT, Zhong AW, et al. (2001) IL-1H, an interleukin 1-related protein that binds IL-18 receptor/IL-1Rrp. Cytokine 13: 1–7.
- 64. Lin H, Ho AS, Haley-Vicente D, Zhang J, Bernal-Fussell J, et al. (2001) Cloning and characterization of IL-1HY2, a novel interleukin-1 family member. J Biol Chem 276: 20597–20602.
- 65. Bulek K, Swaidani S, Qin J, Lu Y, Gulen MF, et al. (2009) The essential role of single Ig IL-1 receptor-related molecule/Toll IL-1R8 in regulation of Th2 immune response. J Immunol 182: 2601–2609.
- 66. Drube S, Heink S, Walter S, Lohn T, Grusser M, et al. (2010) The receptor tyrosine kinase c-Kit controls IL-33 receptor signaling in mast cells. Blood 115: 3899–3906.
- 67. Baekkevold ES, Roussigne M, Yamanaka T, Johansen FE, Jahnsen FL, et al. (2003) Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules. Am J Pathol 163: 69–79.
- 68. Carriere V, Roussel L, Ortega N, Lacorre DA, Americh L, et al. (2007) IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo. Proc Natl Acad Sci U S A 104: 282–287.
- 69. Roussel L, Erard M, Cayrol C, Girard JP (2008) Molecular mimicry between IL-33 and KSHV for attachment to chromatin through the H2A-H2B acidic pocket. EMBO Rep 9: 1006–1012.