Abstract:
Provided herein are methods of modulating IL-33 activity, e.g., for the purpose of treating immune diseases and conditions, as well as methods of screening for compounds capable antagonizing IL-33 signaling.

Description:
This application is a Division of U.S. patent application Ser. No. 11/779,755, filed on Jul. 18, 2007, now U.S. Pat. No. 7,560,530, issued Jul. 14, 2009, which claims benefit of U.S. Provisional Patent Application Nos. 60/832,256 (filed Jul. 20, 2006), 60/835,250 (filed Aug. 3, 2006), and 60/887,257 (filed Jan. 30, 2007), each of which is hereby incorporated by reference in their entireties. 
    
    
     The Sequence Listing filed electronically herewith is also hereby incorporated by reference in its entirety (File Name: BP06497US02_SeqListing.txt; Date Created: Jul. 10, 2009; File Size: 20.0 KB.) 
     FIELD OF THE INVENTION 
     The present invention relates to methods for modulating IL-33 activity. 
     BACKGROUND OF THE INVENTION 
     The immune system protects individuals from infective agents, e.g., bacteria, multi-cellular organisms, as well as cancers. This system includes several types of lymphoid and myeloid cells such as monocytes, macrophages, dendritic cells (DCs), eosinophils, T cells, B cells, and neutrophils. These lymphoid and myeloid cells often produce signaling proteins known as cytokines. Immune response includes inflammation, i.e., the accumulation of immune cells systemically or in a particular location of the body. In response to an infective agent or foreign substance, immune cells secrete cytokines which, in turn, modulate immune cell proliferation, development, differentiation, or migration. Immune response sometimes results in pathological consequences, that is, inflammatory disorders. These inflammatory disorders, which involve immune cells and cytokines, include, e.g., psoriasis, rheumatoid arthritis (RA), Crohn&#39;s disease (CD), multiple sclerosis (MS), and atherosclerosis. 
     The interleukin-1 (IL-1) family of cytokines contributes to the pathology of inflammatory disorders and proliferative conditions, e.g., arthritis and cancer. There are 11 members of the IL-1 cytokine family. IL-1 cytokines bind to members of the IL-1 cytokine receptor family. There are ten members in the IL-1 receptor family, and IL-1 ligands typically require participation of two different IL-1 receptors to make up the cell surface complex. For example, IL-1α and IL-1β bind first to the cell surface receptor IL-1R1, and this IL-1/IL-1R1 complex subsequently binds to a second cell surface IL-1R family member, IL-1 Receptor accessory chain protein (IL-1RAcP) Binding of IL-1α or IL-1β to both receptor components is necessary to transduce the IL-1 signal. 
     IL-1 family members play a role in inflammatory conditions, e.g., rheumatoid arthritis, psoriasis, asthma, chronic obstructive pulmonary disorder (COPD), sepsis, and inflammatory bowel disorder (IBD). Rheumatoid arthritis (RA) is a common chronic inflammatory disorder characterized by degradation of joints, e.g., the synovial membrane, cartilage, and bone. IL-1 stimulates a number of cells involved in arthritic inflammation, e.g., fibroblasts, osteoclasts, chondrocytes, and neutrophils, which may show abnormal proliferation and release enzymes causing joint destruction. 
     Proliferative disorders are the second most common cause of death in the United States. Cytokines of the IL-1 family have been implicated in the control and pathology of proliferative disorders, i.e., cancer. IL-1 modulates progression through the cell cycle, e.g., by changing expression of cyclin-dependent kinases and cyclin-dependent kinase inhibitors. High doses of IL-1β promote tumor invasiveness, while low doses can promote immune eradication of tumors. 
     IL-33 exerts its biological effects via the receptor protein ST2 and induces T Helper Type 2-associated cytokines (Schmitz, J. et al.,  Immunity  23: 479-490 (2005)). In cells stimulated with IL-33, the signaling components MyD88, IRAK, IRAK4 and TRAF6 are recruited to ST2. In addition, IL-33 mediated signaling involves phosphorylation of IκBα and phosphorylation of the MAP kinases Erk1/2, p38 and JNK. T H 2 cells, but not T H 1 cells, respond to IL-33 stimulation with increased production of IL-5 and IL-13. IL-33 administration results in splenomegaly with significantly higher numbers of spleen eosinophils, mononuclear cells, and plasma cells, but not neutrophils. IL-33 administration also results in increased levels of blood eosinophils, lymphocytes and neutrophils. IL-33 administration also leads to induction of IL-4, IL-5 and IL-13 gene and protein expression in vivo. IL-33 administration also results in airway epithelial lining hypertrophy and mucous production, epithelial hyperplasia in the esophagus, and inflammatory infiltrates of eosinophils, neutrophils and mononuclear cells in the esophageal epithelium. In addition, IL-33 administration results in increased levels of serum IgE and IgA. 
     There remains an unmet need to treat inflammatory and immune disorders. Specifically, the need exists for improved compositions and methods of treatment for immune disorders related to IL-33 signaling in which reduction of IL-33 activity would provide therapeutic benefit. 
     SUMMARY OF THE INVENTION 
     We have previously shown that the orphan receptor ST2 is one of the components of the IL-33 receptor complex (Schmitz, J. et al.,  Immunity  23: 479-490 (2005)). It was thought that the receptor complex that binds IL-33 is a complex of ST2 and SIGIRR. See WO 2005/079844. Here, we explain that the second component of the IL-33 receptor complex is not SIGIRR, but is instead IL-1RAcP. Although no receptor promiscuity has been observed for the major IL-1 ligands (IL-1α and IL-1β exclusively bind to IL-1R1 and IL-1RAcP, and IL-18 binds exclusively to IL-18Rα and IL-18Rβ), we focused on IL-1RAcP after we were unsuccessful in finding the second IL-33 receptor among the orphan IL-1 receptors. Accordingly, the present invention provides that IL-33 signal transduction occurs through ST2 and IL-1RAcP. 
     In one aspect, the present invention provides methods of modulating (e.g. preventing, reducing or ameliorating) an immune disorder or condition, comprising inhibiting IL-33 signal transduction through ST2 and IL-1RAcP by administering to a subject in need thereof an effective amount of: (a) an antagonist of IL-33 binding to a complex of ST2 and IL-1RAcP, and/or (b) an antagonist of IL-1RAcP binding to a complex of IL-33 and ST2. 
     The present invention also provides methods of modulating blood cell counts, comprising inhibiting IL-33 signal transduction through ST2 and IL-1RAcP by administering to a subject in need thereof an effective amount of: (a) an antagonist of IL-33 binding to a complex of ST2 and IL-1RAcP, and/or (b) an antagonist of IL-1RAcP binding to a complex of IL-33 and ST2. Such modulation, e.g. may include increasing the count of platelets and/or decreasing the counts of one or more of total white blood cells, neutrophils, lymphocytes and eosinophils. 
     In various embodiments the antagonist comprises an antibody, or antigen-binding fragment thereof, that binds to IL-33, ST2, or a complex of IL-33 bound to ST2. 
     In other embodiments, the antagonist comprises an antibody, or antigen-binding fragment thereof, that binds to IL-1RAcP, a complex of IL-1RAcP and ST2, or a complex of IL-1RAcP, ST2 and IL-33. In some embodiments the antibody or fragment does not bind to IL-33 alone and/or does not bind to ST2 alone. 
     In yet another embodiment, the antibody or fragment does not bind to IL-1RAcP alone. 
     In one embodiment, the antibody or fragment thereof is a monoclonal, humanized, or fully human antibody. In another embodiment, the antibody or fragment is an Fab, an Fv fragment, or an F(ab′) 2  fragment. 
     In some embodiments, the immune disorder or condition is selected from the group consisting of an innate response, asthma, an allergy, multiple sclerosis, inflammatory bowel disorder, arthritis, an infection, cancer, and a tumor. Exemplary arthritic conditions may be selected from the group consisting of rheumatoid arthritis, osteoarthritis, and psoriatic arthritis. Exemplary infections may be selected from the group consisting of an intracellular pathogen, a bacterium, a parasite and a virus. Exemplary intracellular pathogens may be selected from the group consisting of  Leishmania  sp.,  Mycobacterium  sp.,  Listeria  sp.,  Toxoplasma  sp.,  Schistosoma  sp. 
     In other embodiments, the immune disorder or condition is a T H 1-type response or a T H 2-type response. 
     In another aspect, the present invention provides methods and kits for the diagnosis of an immune condition or disorder. In one embodiment, the diagnostic method comprises determining the presence or level of one or more of IL-33, ST2, IL-1RAcP, a complex of IL-33 and ST2, a complex of ST2 and IL-1RAcP and a complex of ST2, IL-1RAcP and IL-33 in a sample from a subject, and comparing that level to the levels in non-diseased tissues, subjects that are known to have the immune condition or disorder, and/or subjects that are known not to have the immune condition or disorder. In one embodiment, the presence or level of a complex of IL-33 and ST2 is detected. In one embodiment the detection involves use of a binding compound comprising a detectable label, e.g. an antibody or antigen-binding fragment thereof. In another embodiment, the invention involves kits to perform the diagnostic methods of the invention, comprising: a compartment; a detectably-labeled molecule, such as an antibody, that binds one or more of IL-33, ST2, IL-1RAcP, a complex of IL-33 and ST2, a complex of ST2 and IL-1RAcP and a complex of ST2, IL-1RAcP and IL-33; and, optionally, instructions for use. In one embodiment, the detectably-labeled molecule binds to a complex of IL-33 and ST2. 
     In another aspect, the present invention provides an isolated and purified complex of ST2 and IL-1RAcP, optionally further comprising IL-33, and sequence variants thereof. In one embodiment the ST2, IL-1RAcP and IL-33 are human proteins. In other embodiments, the invention provides such complexes comprising polypeptide having 70%, 80%, 90%, 95%, 98% or 100% sequence identity with a naturally occurring human ST2, IL-1RAcP or IL-33 protein. In one aspect, the invention provides methods of making these isolated and purified complexes, comprising mixing two or more of the proteins and allowing said complex to form. 
     In yet another aspect, the present invention provides in vitro methods of determining whether a test compound is an antagonist of IL-33 binding to a complex of ST2 and IL-1RAcP, and/or IL-1RAcP binding to a complex of IL-33 and ST2, comprising an assay selected from the group consisting of a) an NF-κB-dependent reporter gene expression assay, b) an MyD88 IRAK, IRAK4 or TRAF6 recruitment assay, c) an Erk1/2, p38, IκBα or JNK phosphorylation assay, d) an NF-κB, Erk1/2 or p38 phosphorylation assay in cells that naturally express ST2, e) an IL-13, IL-6 or IL-5 expression assay, and f) an IL-6 production assay in mouse mast cell line WTMC. In some embodiments, the test compound is determined to be an antagonist if it reduces the activity of IL-33 in the assay when compared to an assay run without the test compound. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows that IL-33 administration increases the percentage of eosinophils found in the blood of wild-type (WT) mice, but not IL-1RAcP deficient (KO) mice. Individual data bars represent separate experimental animals, with one experiment involving five animals ( FIG. 1A ) and another involving three animals ( FIG. 1B ). 
         FIG. 2  shows that IL-33 administration increases IL-5 production in serum ( FIG. 2A ) and bronchoalveolar lavage (BAL) ( FIG. 2B ) in wild-type (WT) mice, but not in IL-1RAcP deficient (KO) mice. 
         FIG. 3  shows that IL-33 administration increases serum IgE in wild-type (WT) mice, but not in IL-1RAcP deficient (KO) mice. 
         FIGS. 4A-4F  show that, unlike wild-type (WT) mice, IL-1RAcP deficient (KO) mice do not upregulate T H 2 cytokine or cytokine receptor genes in response to IL-33 administration 
         FIG. 5  shows that IL-33 induces IL-6 production from WTMC cells in a dose dependent manner. 
         FIG. 6  shows that blocking ST2 prevents IL-33-induced IL-6 production in WTMC cells. 
         FIG. 7  shows that IL-1RAcP is necessary for IL-33-enhanced IL-13 ( FIG. 7A ), IL-6 ( FIG. 7B ), and IL-5 ( FIG. 7C ) production from T H 2 cells 
         FIG. 8  shows the detection of the ST2/IL-33/IL-1RAcP complex, in which ST2 is immobilized on the plate in  FIG. 8A  and IL-1R1 is immobilized on the plate in  FIG. 8B . 
         FIG. 9  shows that dominant negative IL-1RAcP inhibits IL-33 signaling. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise. 
     “Administering” refers to the delivery to an animal, human, subject, cell, tissue, organ, or biological fluid of an exogenous composition, pharmaceutical, therapeutic, or diagnostic agent, e.g. an agent containing an antagonist of IL-33 signal transduction through ST2 and IL-1RAcP. In one embodiment, “administering” refers to delivery to a subject. In another embodiment, “administering” refers to delivery to a human subject. 
     An “antagonist of IL-33 binding to a complex of ST2 and IL-1RAcP” is a molecule that inhibits IL-33 binding to a complex of ST2 and IL-1RAcP. An “antagonist of IL-1RAcP binding to a complex of IL-33 and ST2” is a molecule that inhibits binding of IL-1RAcP to a complex of IL-33 and ST2. In one embodiment, the antagonist is an antibody, such as a polyclonal antibody, a monoclonal antibody, a humanized antibody, or a human antibody. In another embodiment, the antagonist is an antibody fragment, such as a Fab, an Fv fragment, or a F(ab′) 2  fragment. 
     In another embodiment, the antibody or fragment thereof specifically binds to (a) IL-33, (b) IL-1RAcP, (c) ST2, (d) a complex of ST2 bound to IL-1RAcP, (e) a complex of IL-33 bound to ST2, or (f) a complex of IL-33, ST2 and IL-1RAcP. 
     In one embodiment, an “effective amount” of the antagonist of IL-33 signal transduction through ST2 and IL-1RAcP means an amount sufficient to ameliorate a symptom or sign of a disorder or physiological condition, or an amount sufficient to permit or facilitate the diagnosis of a disorder or physiological condition. An effective amount for a human or veterinary subject may vary depending on factors such as the condition being treated, the overall health of the subject, the method route and dose of administration and the severity of side affects. In one embodiment, an effective amount is the maximal dose or dosing protocol that avoids significant side effects or toxic effects. The effect will result in an improvement of a diagnostic measure, parameter, or detectable signal by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, where 100% is defined as the diagnostic parameter shown by a normal subject (see, e.g., Maynard, et al.,  A Handbook of SOPs for Good Clinical Practice , Interpharm Press, Boca Raton, Fla. (1996); Dent,  Good Laboratory and Good Clinical Practice , Urch Publ., London, UK (2001)). 
     In another embodiment, an “effective amount” of the antagonist of IL-33 signal transduction through ST2 and IL-1RAcP means an amount sufficient to inhibit IL-33 signal transduction through ST2 and IL-1RAcP. 
     “Inhibiting IL-33 signal transduction through ST2 and IL-1RAcP” means that the degree to which IL-33 stimulates signal transduction through ST2 and IL-1RAcP is diminished in the presence of an antagonist, relative to the degree to which IL-33 stimulates signal transduction through ST2 and IL-1RAcP in the absence of the antagonist. 
     To examine the extent of inhibition, a sample is treated with a potential inhibitor and is compared to a control sample without the inhibitor. Control samples, i.e., not treated with antagonist, are assigned a relative activity value of 100%. Inhibition is achieved when the activity value relative to the control is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% or less. 
     An endpoint in inhibition may comprise a predetermined quantity or percentage of, e.g., an indicia of inflammation, oncogenicity, or cell degranulation or secretion, such as the release of a cytokine. An endpoint of inhibition is generally 75%, 50%, 25%, or 10% of the control or less. 
     Inhibition of IL-33 signal transduction through ST2 and IL-1RAcP can be determined by assaying for IL-33 signal transduction in an in vitro assay. One in vitro assay that can be used is an NF-κB-dependent reporter assay (Schmitz, J. et al.,  Immunity  23: 479-490 (2005)). In this assay, cells, e.g., HEK293FT cells, are transfected with either a mock expression vector or with an expression vector encoding ST2 in conjunction with an NF-κB-driven reporter gene expression product, e.g., green fluorescent protein (GFP). Cells are incubated with IL-33 alone, or with IL-33 and a test molecule that is being tested for antagonistic activity. Cells are then analyzed for reporter gene expression product, e.g., by fluorescence activated cell sorting (FACS®). If IL-33 treated cells exhibit reduced reporter gene expression after incubation with the test molecule, the test molecule is an antagonist of IL-33 binding to the IL-1RAcP receptor complex. 
     Another reporter gene expression product that can be used is luciferase. In this assay, cellular extracts are assayed for NF-κB-driven luciferase expression in response to incubation with IL-33, and with or without a test molecule, i.e. a putative inhibitor of IL-33 signal transduction. 
     Another in vitro assay that can be performed is to assay for MyD88 IRAK, IRAK4, or TRAF6 recruitment (Schmitz, J. et al.,  Immunity  23: 479-490 (2005)). In this assay, cells, e.g., HEK293FT cells, are transfected with either a mock expression vector or with an expression vector encoding ST2, and incubated with IL-33. In the control experiment, no test molecule is added to the incubation. In the test experiment, the molecule to be tested for its ability to inhibit IL-33 signal transduction is added. Cellular extracts are immunoprecipitated with an anti-ST2 antibody, followed by Western analysis with anti-MyD88, IRAK, IRAK4, or TRAF6 antibodies. A decrease in expression of MyD88, IRAK, IRAK4, or TRAF6 in extracts from IL-33 treated cells when incubated with the test molecule indicates that the test molecule is an antagonist of IL-33 binding to the IL-1RAcP receptor complex. 
     Another in vitro assay that can be performed is to assay for phosphorylation of Erk1/2, p38, IκBα, or JNK (Schmitz, J. et al.,  Immunity  23: 479-490 (2005)). In this assay, cells, e.g., HEK293FT cells, are transfected with either a mock expression vector or with an expression vector encoding ST2, and incubated with IL-33. In the control experiment, no test molecule is added to the incubation. In the test experiment, the molecule to be tested for its ability to inhibit IL-33 signal transduction is added. Cellular extracts are prepared and blotted with anti-phospho-Erk1/2, anti-phospho-p38, anti-phospho-IκBα, or anti-phospho-JNK antibodies. A decrease in the level of phosphorylated Erk1/2, p38, IκBα, or JNK in extracts from IL-33 treated cells when incubated with the test molecule indicates that the test molecule is an antagonist of IL-33 binding to the IL-1RAcP receptor complex. 
     Another in vitro assay that can be performed is to assay for phosphorylation of NF-κB, Erk1/2 or p38 in cells that naturally express ST2, e.g., mouse mast cells (WTMC) (Schmitz, J. et al.,  Immunity  23: 479-490 (2005)). In the control experiment, WTMC cells are incubated with IL-33 but no test molecule is added to the incubation. In the test experiment, cells are incubated with IL-33 and the molecule to be tested for its ability to inhibit IL-33 signal transduction. Cell lysates are separated by SDS-PAGE and electroblotted. Cellular extracts are blotted with anti-NF-κB, anti-phospho-Erk1/2, or anti-phospho-p38 antibodies. A decrease in the level of phosphorylated NF-κB, Erk1/2, or p38 in extracts from IL-33 treated cells when incubated with the test molecule indicates that the test molecule is an antagonist of IL-33 binding to the IL-1RAcP receptor complex. 
     Another in vitro assay that can be performed is to assay expression of IL-5 or IL-13 in T H 2 cells (Schmitz, J. et al.,  Immunity  23: 479-490 (2005)). In the control experiment, T H 2 cells are incubated with IL-33 but no test molecule is added to the incubation. In the test experiment, T H 2 cells are incubated with IL-33 and the molecule to be tested for its ability to inhibit IL-33 signal transduction. Supernatants are analyzed for IL-5 and IL-13 production. A decrease in the level of IL-5 or IL-13 in supernatant from IL-33 treated cells when incubated with the test molecule indicates that the test molecule is an antagonist of IL-33 binding to the IL-1RAcP receptor complex. 
     Another in vitro assay that can be used to determine whether a molecule is an antagonist of IL-33 binding is to determine the effect of a test molecule on IL-33-induced production of IL-6 in the mouse mast cell line WTMC. A decrease in the level of IL-6 production by IL-33 treated cells when incubated with the test molecule indicates that the test molecule is an antagonist of IL-33 binding to the IL-1RAcP receptor complex. 
     In vivo assays that can be used to determine whether a molecule is an antagonist of IL-33 binding to the IL-1RAcP receptor complex is to determine the effect of a test molecule on the influence of IL-33 administration on spleen size, spleen cells (e.g., mononuclear cells, eosinophils, and plasma cells), blood cell count (e.g., eosinophils, lymphocytes or neutrophils) (Schmitz, J. et al.,  Immunity  23: 479-490 (2005)). A decrease in the effect of IL-33 after administration of IL-33 and the test molecule on an in vivo endpoint, relative to the effect of IL-33 alone, indicates that the test molecule is an antagonist of IL-33 binding to the IL-1RAcP receptor complex. 
     In a one embodiment, the antagonist that is assayed for in the above-described assays is an antibody. 
     “Expression” refers to a measure of mRNA or polypeptide encoded by a specific gene. Units of expression may be a measure of, e.g., the number of molecules of mRNA or polypeptide/mg protein, the number of molecules of mRNA or polypeptide/cell, in measurements of expression by cell, tissue, cell extract, or tissue extract. The units of expression may be relative, e.g., a comparison of signal from control and experimental mammals or a comparison of signals with a reagent that is specific for the mRNA or polypeptide versus with a reagent that is non-specific. 
     “Immune condition” or “immune disorder” encompasses, e.g., pathological inflammation, an inflammatory disorder, and an autoimmune disorder or disease. “Immune condition” also refers to infections, persistent infections, and proliferative conditions, such as cancer, tumors, and angiogenesis. 
     In one embodiment, the immune disorder or immune condition is selected from the group consisting of an innate response, asthma, allergy, multiple sclerosis, inflammatory bowel disorder, arthritis, an infection, cancer, and a tumor. In another embodiment, the arthritis is selected from the group consisting of rheumatoid arthritis, osteoarthritis, and psoriatic arthritis. In another embodiment, the immune disorder or condition comprises a T H 1-type response or a T H 2-type response. 
     “Infection” refers to a disorder resulting from a pathogen, microbe, bacterium, parasite, virus, and the like. In one embodiment, the infection is caused by an organism selected from the group consisting of a bacterium, a parasite, and a virus. In another embodiment, the infection is caused by an organism selected from the group consisting of  Leishmania  sp.,  Mycobacterium  sp.,  Listeria  sp.,  Toxoplasma  sp.,  Schistosoma  sp., and a respiratory virus. 
     “Inflammatory disorder” means a disorder or pathological condition where the pathology results, in whole or in part, from, e.g., a change in number, change in rate of migration, or change in activation, of cells of the immune system. Cells of the immune system include, e.g., T cells, B cells, monocytes or macrophages, antigen presenting cells (APCs), dendritic cells, microglia, NK cells, NKT cells, neutrophils, eosinophils, mast cells, or any other cell specifically associated with the immunology, for example, cytokine-producing endothelial or epithelial cells. 
     “IL-1 RAcP” means the Interleukin-1 Receptor Accessory Protein. IL-1 RAcP is further described at online Mendelian Inheritance in Man (OMIM) entry 602626 and Gene ID No. 3556, and exemplary naturally occurring sequences for human IL-1 RAcP are found at GenBank Accession Nos. NP — 608273 (SEQ ID NO: 1) and NP — 002173 (SEQ ID NO: 2), all of which are available through the National Center for Biotechnology Information (NCBI) website. 
     “IL-33 receptor complex” refers to the specific binding of ST2 to IL-1RAcP to form a protein complex that itself specifically binds to IL-33 and transduces the IL-33 signal from the cell surface to the cell interior. 
     “IL-33/ST2 complex” refers to the specific binding of IL-33 and ST2 to form a protein complex that itself specifically binds to IL-1 RAcP. IL-33 is further described at OMIM entry 608678 and Gene ID No. 90865, and an exemplary naturally occurring sequence for human IL-33 (SEQ ID NO: 3) is found at GenBank Accession No. AY905581, all of which are available through the NCBI website. See also Schmitz et al. (2005) Immunity 23: 479-490. 
     An “isolated and purified” complex, such as an isolated and purified complex of ST2 and IL-1RAcP, optionally further comprising IL-33, is an in vitro complex that is isolated and purified from its natural source. Specifically, an isolated and purified complex does not encompass complexes as they form in nature. Such complexes may be formed, e.g., by mixture of two or more purified proteins, or by mixture of one purified protein with a cell extract or biological sample, or by the purification of a complex from a natural source. 
     “IL-33 signal transduction through ST2 and IL-1RAcP” refers to the molecular events that occur when IL-33, ST2 and IL-1RAcP form a protein complex. In one embodiment, the IL-33 signal transduction refers to recruitment of one or more of MyD88, IRAK, IRAK4, and TRAF6 to ST2 in response to the formation of a complex of IL-33, ST2, and IL-1RAcP. In another embodiment, IL-33 signal transduction refers to phosphorylation of one or more of IκBκ, Erk1/2, p38 and JNK in response to the formation of a complex of IL-33, ST2, and IL-1RAcP. In another embodiment, IL-33 signal transduction refers to IL-5 or IL-13 secretion by T H 2 cells. 
     Specificity of binding refers to a binding interaction between a predetermined ligand and a predetermined receptor that enables one to distinguish between the predetermined ligand and other ligands, or between the predetermined receptor and other receptors. In one embodiment, the ligand is IL-33, and the receptor is ST2. In another embodiment, the ligand is IL-33, and the receptor is a protein complex of ST2 and IL-1RAcP. In another embodiment, the ligand is a protein complex of IL-33 and ST2, and the receptor is IL-1RAcP. In another embodiment, the ligand is an antibody or a fragment thereof that specifically binds to IL-33, IL-1RAcP, ST2, a complex of ST2 bound to IL-1RAcP, a complex of IL-33 bound to ST2, or a complex of IL-33, ST2 and IL-1RAcP. 
     A “pharmaceutical composition” comprises the active drug ingredient and one or more pharmaceutically acceptable excipients, carriers and/or diluents. 
     “Specifically binds” means a binding reaction that is determinative of the presence of a protein in a heterogeneous population of proteins and other biologics. For example, under designated conditions, one protein binds to another protein and does not bind in a significant amount to other proteins present in the sample. In various embodiments, an antibody or fragment thereof binds to its antigen with an affinity that is at least two fold greater, at least ten times greater, at least 20-times greater, or at least 100-times greater than the affinity to any other protein. 
     In various embodiments, an antibody, or fragment thereof, that specifically binds to IL-33, IL-1RAcP, ST2, a complex of ST2 bound to IL-1RAcP, a complex of IL-33 bound to ST2, or a complex of IL-33, ST2 and IL-1RAcP will bind with a K D  of at least about 10 −6  M, 10 −7  M, 10 −8  M, 10 −9  M, 10 −10  M, or 10 −11  M or better (lower K D ). 
     “ST2” as used herein is synonymous with the T1/ST2 protein (also known as IL-1RL1). ST2 is further described at OMIM entry 601203 and Gene ID No. 9173, and exemplary naturally occurring sequences for human ST2 are found at GenBank Accession Nos. NP — 003847 (SEQ ID NO: 4) and NP — 057316 (SEQ ID NO: 5), all of which are available through the NCBI website. 
     “ST2/IL-1RAcP complex” refers to the specific binding of ST2 and IL-1RAcP to form a protein complex that itself specifically binds IL-33. 
     Purification of antigen is not necessary for the generation of antibodies. Immunization can be performed by DNA vector immunization, see, e.g., Wang, et al.  Virology  228:278-284 (1997). Alternatively, animals can be immunized with cells bearing the antigen of interest. Splenocytes can then be isolated from the immunized animals, and the splenocytes can fused with a myeloma cell line to produce a hybridoma (Meyaard et al.,  Immunity  7:283-290 (1997); Wright et al.,  Immunity  13:233-242 (2000); Preston et al.,  Eur. J. Immunol.  27:1911-1918 (1997)). Resultant hybridomas can be screened for production of the desired antibody by functional assays or biological assays, that is, assays not dependent on possession of the purified antigen. Immunization with cells may prove superior for antibody generation than immunization with purified antigen (Kaithamana et al.,  J. Immunol.  163:5157-5164 (1999)). 
     Any suitable method can be used to elicit an antagonist antibody with the desired biologic properties. It may be desirable to prepare monoclonal antibodies (mAbs) from mammalian hosts such as mice, rodents, primates, humans, etc. Techniques for preparing such monoclonal antibodies may be found in, e.g., Stites et al. (eds.) B ASIC AND  C LINICAL  I MMUNOLOGY  (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Harlow and Lane (1988) A NTIBODIES : A L ABORATORY  M ANUAL  CSH Press; Goding (1986) M ONOCLONAL  A NTIBODIES : P RINCIPLES AND  P RACTICE  (2d ed.) Academic Press, New York, N.Y. Thus, monoclonal antibodies may be obtained by a variety of techniques familiar to researchers skilled in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell. See Kohler and Milstein (1976)  Eur. J. Immunol.  6:511-519. Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. See, e.g., Doyle et al. (eds. 1994 and periodic supplements) C ELL AND  T ISSUE  C ULTURE : L ABORATORY  P ROCEDURES , John Wiley and Sons, New York, N.Y. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a antigen binding fragment thereof by screening a DNA library from human B cells according, e.g., to the general protocol outlined by Huse et al. (1989)  Science  246:1275-1281. 
     Other suitable techniques involve selection of libraries of antibodies in phage or similar vectors. See, e.g., Huse et al. supra; and Ward et al. (1989)  Nature  341:544-546. The polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly U.S. Pat. No. 4,816,567; and Queen et al. (1989)  Proc. Nat&#39;l Acad. Sci. USA  86:10029-10033; or made in transgenic mice, see Mendez et al. (1997)  Nature Genetics  15:146-156. See also Abgenix and Medarex technologies. 
     Any suitable non-human antibody can be used as a source for the hypervariable region of a humanized antibody. Sources for non-human antibodies include, but are not limited to, murine, Lagomorphs (including rabbits), bovine, and primates. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance of the desired biological activity. For further details, see Jones et al. (1986)  Nature  321:522-525; Reichmann et al. (1988)  Nature  332:323-329; and Presta (1992)  Curr. Op. Struct. Biol.  2:593-596. 
     Methods for recombinantly engineering antibodies have been described, e.g., by Boss et al. (U.S. Pat. No. 4,816,397), Cabilly et al. (U.S. Pat. No. 4,816,567), Law et al. (European Patent Application Publication No. 438310) and Winter (European Patent Application Publication No. 239400). 
     In one embodiment, the antagonist of the present invention is administered in a pharmaceutical composition. In another embodiment, the antagonist of the present invention is administered in a sterile composition. 
     To prepare pharmaceutical or sterile compositions, an active drug ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients and/or diluents. A Pharmaceutical formulation can be prepared in the form of, e.g., a lyophilized powder, an aqueous solution, or a suspension. See, e.g., Hardman, et al.,  Goodman and Gilman&#39;s The Pharmacological Basis of Therapeutics , McGraw-Hill, New York, N.Y. (2001); Gennaro,  Remington: The Science and Practice of Pharmacy , Lippincott, Williams, and Wilkins, New York, N.Y. (2000); Avis, et al. (eds.),  Pharmaceutical Dosage Forms: Parenteral Medications , Marcel Dekker, NY (1993);  Excipient Toxicity and Safety , Marcel Dekker, Inc., New York, N.Y. (2000). 
     Selecting an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. Preferably, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies is available. See, e.g., Wawrzynczak,  Antibody Therapy , Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.),  Monoclonal Antibodies, Cytokines and Arthritis , Marcel Dekker, New York, N.Y. (1991); Bach (ed.),  Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases , Marcel Dekker, New York, N.Y. (1993). 
     An antibody or fragment thereof can be provided by continuous infusion, or by doses at intervals. An antibody or fragment thereof may be administered intravenously, intramuscularly, or subcutaneously. 
     A preferred dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. In various embodiments, the total weekly dose is 0.05 μg/kg, 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg or more. 
     An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side affects (see, e.g., Maynard, et al.,  A Handbook of SOPs for Good Clinical Practice , Interpharm Press, Boca Raton, Fla. (1996); Dent,  Good Laboratory and Good Clinical Practice , Urch Publ., London, UK (2001)). 
     Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced. Preferably, a biologic that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing a humoral response to the reagent. 
     The present invention also provides a method of modulating blood cell counts, the method comprising administering an effective amount of an antagonist of IL-33 signal transduction through ST2 and IL-1RAcP. In one embodiment, the antagonist increases the count of platelets. In another embodiment, the antagonist decreases the counts of total white blood cells, neutrophils, lymphocytes, and/or eosinophils. 
     The present invention also provides methods and kits for the diagnosis of an immune condition or disorder. A method of diagnosis can comprise contacting a sample from a subject, e.g., a test subject, with an antibody or fragment thereof that specifically binds to (a) IL-33, (b) IL-1RAcP, (c) ST2, (d) a complex of ST2 and IL-1RAcP, (e) a complex of IL-33 and ST2, or (f) a complex of IL-33, ST2 and IL-1RAcP. The method can further comprise contacting a sample from a control subject, normal subject, or normal tissue or fluid from the test subject, with an antibody or fragment thereof. Moreover, the method can additionally comprise comparing the specific binding of the antibody or fragment thereof to the test subject with the specific binding of the composition to the normal subject, control subject, or normal tissue or fluid from the test subject. Expression or activity of a test sample or test subject can be compared with that from a control sample or control subject. A control sample can comprise, e.g., a sample of non-affected or non-inflamed tissue in a patient suffering from an immune disorder. Expression or activity from a control subject or control sample can be provided as a predetermined value, e.g., acquired from a statistically appropriate group of control subjects. 
     Kits for the diagnosis of an immune condition or disorder may comprise a compartment and a binding compound, e.g. an antibody or antigen-binding fragment thereof, that specifically binds to one or more components of IL-33 signal transduction through ST2 and IL-1RAcP. Such kits may optionally include instructions for use. In some embodiments the binding compound is detectably labeled. 
     The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to specific embodiments. 
     Many modifications and variations of this invention, as will be apparent to one of ordinary skill in the art can be made to adapt to a particular situation, material, composition of matter, process, process step or steps, to preserve the objective, spirit and scope of the invention. All such modifications are intended to be within the scope of the claims appended hereto without departing from the spirit and scope of the invention. The specific embodiments described herein are offered by way of example only, and the invention includes the full scope of equivalents to which such claims are entitled; and the invention is not to be limited by the specific embodiments that have been presented herein. 
     All citations herein are incorporated herein by reference to the same extent as if each individual publication, patent application, patent, database entry or other document was specifically and individually indicated to be incorporated by reference including all figures and drawings. 
     EXAMPLE 1 
     Animal Experiments 
     In order to determine whether IL-1RAcP is the second member of the receptor complex, wild-type (WT) and IL-1RAcP deficient (KO) mice were used. The wild-type (WT) mice used were strain B6; 129SF2/J (Jackson Laboratories). The IL-1RAcP deficient (KO) mice used were strain B6; 129S1-Il1rap tm1/Rom1 /J (Jackson Laboratories). The IL-1RAcP deficient mice have been genetically altered and do not have a functional IL-1RAcP receptor protein. 
     Wild-type and IL-1RAcP deficient mice were given daily intraperitoneal injections of saline or 2 micrograms of human IL-33 for six days. On day seven, the mice were sacrificed and analyzed for IL-33 responsiveness by several endpoints, as discussed in the following Examples. Blood eosinophilia was measured in blood smears. Serum and bronchoalveolar lavage (BAL) fluid were collected to quantitate cytokine levels and serum immunoglobulin (Ig) E levels. Lung and spleen tissue was collected to quantitate messenger RNA of key cytokines Lung and small intestine tissue was analyzed for histological changes. Two replicate experiments were performed with either three or five mice per treatment group. 
     The following data show that IL-1RAcP deficient mice do not respond to IL-33 administration, indicating that IL-1RAcP is necessary to mediate the in vivo effects of IL-33. These results implicate IL-1RAcP as the second component of the IL-33 receptor. 
     EXAMPLE 2 
     Lack of Blood Eosinophilia in IL-1RAcP Deficient Mice 
     Tail blood was smeared onto glass slides, fixed in 95% ethanol, and stained by the Wright-Giemsa method. Cells were enumerated by microscope, and at least 300 cells were counted per mouse. The graphs in  FIGS. 1A and 1B  represent the two replicate experiments performed, involving five and three animals, respectively. IL-33 administration to wild-type mice dramatically increased the percentage of eosinophils found in the blood. However the percent of eosinophils in the blood of IL-33-treated IL-1RAcP deficient mice was not increased, and was similar to saline treated mice. 
     EXAMPLE 3 
     Lack of Goblet Cell Hyperplasia and Mucous Production in IL-1RAcP Deficient Mice 
     Formalin-fixed, paraffin-embedded small intestines were stained by Periodic acid-Schiff. Wile-type mice, but not IL-1RAcP deficient mice, responded to IL-33 with increased goblet cell mucous production in the small intestine. A total of eight mice were examined per treatment condition. A similar finding was noted in the upper airways of the lung. 
     EXAMPLE 4 
     IL-1RAcP Deficient Mice Fail to Respond to IL-33 with Increased Production of IL-5 
     Serum and BAL fluid were analyzed for cytokine content by multiplex immunoassay. In both the serum ( FIG. 2A ) and BAL fluid ( FIG. 2B ), IL-33 treatment dramatically increased the production of IL-5 in wild-type mice, but not in IL-1RAcP deficient mice. The data are representative of two experiments. 
     EXAMPLE 5 
     Serum Levels of IgE are Increased in Wild-Type but not IL-1RAcP Deficient Mice 
     Serum IgE levels were measured by ELISA. IL-33 increased serum IgE in wild-type, but not in IL-1RAcP deficient mice ( FIG. 3 ). The data are representative of two experiments. 
     EXAMPLE 6 
     IL-1RAcP Deficient Mice do not Upregulate T H 2Cytokine Genes in Response to IL-33 Administration 
     RT-PCR was performed on RNA obtained from snap-frozen spleen and lung tissue, and gene expression is shown in  FIG. 4 , relative to the levels of ubiquitin (ub) in the sample. IL-33 increased mRNA expression of the T helper 2 (T H 2) cytokines and cytokine receptors IL-4 ( FIG. 4A ), IL-5 ( FIG. 4C ), IL-6 ( FIG. 4B ), IL-13 ( FIG. 4D ), IL-17RB ( FIG. 4E ), and ST2 ( FIG. 4F ), in the lung and spleen of wild-type mice, but not in IL-1RAcP deficient (“KO”) mice. 
     EXAMPLE 7 
     IL-33 Induces IL-6 Production in WTMC Cells 
     The mouse mast cell line WTMC (Wright, G. J., et al.,  J. Immunol.  171: 3034-3046 (2003)), expresses ST2 and responds to both human and mouse IL-33 in a dose dependent manner by producing IL-6. IL-33 activity can be blocked by preincubating the cells with a blocking rat anti-mouse ST2 antibody (MD Biosciences). 
     1×10 5  WTMCs were plated per well (96-well plate format) in RPMI 1640 w/L-glutamine containing 10% fetal calf serum, 1 mM Na-Pyruvate, 0.05 mM 2-mercapto-ethanol, 0.2 mM L-glutamine, 1×MEM non-essential amino acids, 1× PenStrep, 2.5 ng/ml mouse IL-3, and 2.5 ng/ml mouse IL-4. Cells were incubated for 24 hours with varying concentrations of mouse or human IL-33. Supernatants were assessed for IL-6 production by ELISA. As shown in  FIG. 5 , IL-33 induced IL-6 production in a dose dependent manner. 
     As shown in  FIG. 6 , a blocking rat anti-mouse ST2 antibody (MD Biosciences) prevented IL-33 induced production in WTMCs. Mast cells were seeded with into 24 well plates at 1×10 6  cells/well. Some cells were preincubated with anti-ST2 antibody (10 μg/ml) prior to the addition of IL-33 (50 ng/ml). Supernatants were collected after 6 and 24 hours and analyzed for cytokine production by multiplex immunoassay. 
     EXAMPLE 8 
     IL-1RAcP is Necessary for IL-33-Enhanced IL-5, IL-6, and IL-13 Production from T H 2Cells 
     CD4+ T cells were isolated from wildtype (B6; 129SF2/J) and IL-1RAcP knock-out (B6; 129S1-Il1rap tm1/Rom1 /J) mouse spleen and lymph nodes using the CD4+ T cell isolation kit (Miltenyi Biotec). To polarize towards a T H 2 phenotype, cells were cultured on anti-CD3 coated plates in the presence of 5 ng/ml IL-2, 10 ng/ml IL-4, 10 μg/ml anti-IFNγ, and 1 μg/ml anti-CD28 in complete RPMI for 4 days. Cells were then washed, and rested for 3 days in IL-2 (5 ng/ml) alone. Cells were then washed and stimulated with media alone, human IL-33 (50 ng/ml), or mouse IL-1β (10 ng/ml). Some cells were preincubated with anti-ST2 blocking antibody prior to cytokine addition. Supernatants were harvested after 24 hours and cytokine levels were measured using a multiplexed immunoassay (22-plex mouse cytokine premixed beads, Linco), and analyzed by Luminex® immunoassay. 
     As shown in  FIG. 7 , both IL-1β and IL-33 enhanced the production of IL-13, IL-6, and IL-5 in wildtype, but not IL-1RAcP−/− T H 2 cells. The increased production of these cytokines in wildtype mice by IL-33 was blocked in the presence of anti-ST2 antibodies. Anti-ST2 did not block IL-1β-enhanced cytokine production. 
     EXAMPLE 9 
     Detection of the ST2/IL-33/IL-1RAcP Complex 
     Murine IL-1β or IL-33 was titrated into plates containing soluble his6-tagged murine IL-1RAcP and plate-bound ST2-Fc ( FIG. 8A ) or IL-1R1-Fc ( FIG. 8B ). Peroxidase-conjugated anti-his6 was used as a detection antibody. Data is representative of 2 experiments. Data represent the mean+/−SEM of duplicate wells. 
     Receptor-ligand complex ELISA: 3 μg/mlmurine ST2-Fc or IL-1R1-Fc fusion proteins (R&amp;D Systems) in PBS were coated overnight onto MaxiSorp plates (Nunc). After washing, plates were incubated with 3 μg/ml recombinant his6-tagged murine IL-1RAcP and increasing concentrations of murine IL-33 or IL-1β in PBS/1% bovine serum albumin/0.05% Tween-20® polyoxyethylene sorbitan monolaurate for 1.5 hours. Plates were washed and incubated with anti-his6-peroxidase (Roche Diagnostics) for 1 hour, washed again, developed with TMB Peroxidase substrate (KPL), stopped with H 2 PO 4 , and read on a plate reader at 450-570 nm. 
     Although IL-1RAcP is required to mediate the effects of IL-33 in vivo and on Th2 cells, the possibility exists that this is due to an indirect rather than a direct interaction of IL-33 and IL-1RAcP. Therefore, we wished to detect formation of the ligand-receptor complex in vitro. In the IL-1 receptor complex, IL-1RAcP cannot bind IL-1 directly, rather it is hypothesized to interact with the complex of IL-1 and its primary receptor, IL-1R1. We are also unable to see a direct interaction of IL-33 with IL-1RAcP (not shown). However, we can detect a specific ST2/IL-33/IL-1RAcP complex by ELISA. We titrated IL-33 or IL-1β into wells containing plate-bound ST2 and soluble his6-tagged IL-1RAcP ( FIG. 8A ). We detect IL-33 receptor complex formation with an anti-his6 antibody as the concentration of IL-33, but not IL-1β, increases. Conversely, if the plates are coated with IL-1R1 instead of ST2, we detect IL-1 receptor complex formation with increasing amounts of IL-1β but not IL-33 ( FIG. 8B ). This result demonstrates that ST2, IL-33, and IL-1RAcP can form a ligand/receptor complex. 
     EXAMPLE 10 
     Dominant Negative IL-1RAcP Inhibits IL-33 Signaling 
     Signal transduction by IL-1RAcP in the IL-1 signaling cascade is mediated by its cytoplasmic Toll/IL-1 receptor (TIR) domain and involves recruitment of the adaptor MyD88 and activation of NF-κB. IL-33 uses the same signaling components as IL-1, including IRAK, IRAK4, MyD88, and TRAF6, leading to the activation of NF-κB and MAP kinases. MyD88 is required for IL-33 signaling, as MyD88−/− mice do not respond to IL-33 administration (data not shown). In order to investigate the contribution of IL-1RAcP to IL-33 signal transduction, we used a dominant negative form of IL-1RAcP that contains a stop codon before the TIR domain. 
     As a positive control, HEK293FT cells, which endogenously express human IL-1R1 and IL-1RAcP, were transfected with 3 μg of a NF-κB-driven reporter gene construct (pNF-κB-hrGFP, Stratagene), and with increasing amounts of cMyc-tagged murine dominant negative (dn) IL-1RAcP construct. Cells were stimulated at 24 hours post-transfection with 20 ng/ml murine IL-1α (R&amp;D Systems) for 24 hours, and analyzed for GFP expression by FACS® cell sorting. This control tests the ability of the dn IL-1RAcP to interact with the endogenous IL-1R1 and block IL-1α mediated downstream signaling. This interaction is reflected by the diminution of GFP expression when increasing amounts of the dn IL1-RAcP are transfected into control cells ( FIG. 9 , bottom row). 
     For experimental samples, HEK293FT cells were transfected with 1 μg of a plasmid encoding murine ST2, 3 μg of the NF-KB-driven reporter gene construct, and with increasing amounts of cMyc-tagged murine dominant negative (dn) IL-1RAcP construct. Cells were stimulated at 24 hours post-transfection with 20 ng/ml murine IL-33 for 24 hours, and analyzed for GFP expression by FACS® cell sorting. Results of a typical experiment are provided at  FIG. 9  (top row). This experimental system tests the ability of the dn IL-1RAcP to interact with murine ST2 and block IL-33 signaling. The addition of the dn IL-1RAcP caused a roughly dose-responsive decrease in IL-33-induced reporter gene expression, suggesting that ST2 uses IL-1RAcP as a second receptor to mediate IL-33 signal transduction.