Allergy suppressants and degranulation suppressants

Abstract

To focus on Ly6G being neutrophil specifically-expressed protein to clarify a mast cell response regulatory mechanism of Ly6G via interaction between a mast cell and a neutrophil.SOLUTION: Ly6G being neutrophil-derived protein has a function as a mast cell response regulatory factor, and controls a degranulation amount being a secretion amount of inflammatory mediator.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an allergy suppressant and a degranulation suppressant. [Background technology]

[0002] (Onset of allergic diseases) Mast cells play an important role in the development of allergic diseases such as hay fever and atopic dermatitis. Mast cells express IgE receptors (FcεRI) on the cell membrane, and are activated by crosslinking FcεRI with antigens such as pollen and food via antigen-specific IgE. As a result, intracellular signals are activated, and a secretory reaction is induced in which inflammatory mediators present in intracellular granules are released outside the cell (see Non-Patent Document 1). In addition, mediators secreted by mast cells include histamine, proteases, lipid mediators, cytokines, and chemokines (see Non-Patent Documents 2 and 3). These actions increase vascular permeability, causing infiltration of immune cells such as neutrophils, basophils, eosinophils, and macrophages from blood vessels into inflamed tissues, and it is believed that the complex allergic reaction is regulated by their interaction (see Non-Patent Document 4).

[0003] Through research using a mouse model of passive cutaneous anaphylaxis, the present inventors have revealed that mast cells specifically activated by antigens in allergic inflammatory tissues and neutrophils that have infiltrated into the allergic inflammatory tissues are present in close proximity to each other and interact with each other (see non-patent document 5). Neutrophils are a type of white blood cell that usually resides within blood vessels. When immune cells in tissues recognize pathogens and release inflammatory mediators, they infiltrate into inflamed tissues with increased vascular permeability. Neutrophils that infiltrate into tissues play an important role in host defense by recognizing and killing pathogens, such as by phagocytosis and release of antibacterial proteins (see Non-Patent Documents 4 and 6). However, many aspects of the detailed molecular mechanisms of the interaction between mast cells and neutrophils in allergic diseases remain unclear. [Prior art documents]

Non-licensed literature

[0004]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

[0005] We focused on Ly6G, a protein that is specifically expressed in neutrophils, and aimed to clarify the mechanism by which Ly6G regulates mast cell responses via interactions between mast cells and neutrophils. [Means for solving the problem]

[0006] The present inventors discovered that Ly6G, a neutrophil-derived protein, functions as a mast cell response regulator and thereby controls the amount of degranulation, which is the secretion amount of inflammatory mediators, and thus completed the present invention.

[0007] That is, the present invention is as follows. 1. Allergy suppressants containing Ly6 / uPAR family proteins. 2. The allergy suppressant according to the preceding paragraph 1, wherein the Ly6 / uPAR family is Ly6G. 3. The allergy suppressant according to the preceding item 1, wherein the Ly6 / uPAR family is human CD59, human CD177, human Ly6D, human Ly6E, human Ly6H, human Ly6K, human Ly6L, mouse Ly6G, mouse Ly6A, mouse Ly6C1, mouse Ly6C2, mouse Ly6D, mouse Ly6E, mouse Ly6F, mouse Ly6H, mouse Ly6I, mouse Ly6K, mouse Ly6L or mouse Ly6M. 4. The allergy suppressant according to the preceding item 1, wherein the Ly6 / uPAR family protein is any one of the following polypeptides: 1) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4 2) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10 3) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1 4) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2 5) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3 6) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5 7) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:6 8) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:7 9) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8 10) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:9 11) A polypeptide comprising an amino acid sequence having one or two amino acids substituted, deleted, inserted and / or added in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 10, and having substantially the same allergy suppressing activity as the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 10. 12) A polypeptide having an amino acid sequence which has 95% or more homology with any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 10 and has substantially the same allergy suppressing effect as any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 10. 5. Degranulation inhibitors including Ly6 / uPAR family proteins. 6. A degranulation inhibitor according to the preceding paragraph 5, wherein the Ly6 / uPAR family is Ly6G. 7.Ly6 / uPARファミリーは、human CD59、human CD177、human Ly6D、human Ly6E、human Ly6H、human Ly6K、human Ly6L、human-ACRV1、human-GML、human-GPIHBP1、human-LY6G5B、human-LY6G5C、human-Y6G6C、human-LY6G6D、human-LY6G6F、human-LYNX1、human-LYPD1、human-LYPD2、human-LYPD3、human-LYPD4、human-LYPD5、human-LYPD6、human-LYPD6B、human-LYPD8、human-PATE1、human-PATE2、human-PATE3、human-PATE4、human-PINLYP、human-PLAUR、human-PSCA、human-SLURP1、human-SLURP2、human-SPACA4、human-TEX101、mouse Ly6G、mouse Ly6A、mouse Ly6C1、mouse Ly6C2、mouse Ly6D、mouse Ly6E、mouse Ly6F、mouse Ly6H、mouse Ly6I、mouse Ly6K、mouseLy6L, mouse-Acrv1, mouse-Cd177, mouse-Cd59a, mouse-Cd59b, mouse-Gml, mouse-Gml2, mouse-Gpihbp1, mouse-Ly6a2, mouse-Ly6g2, mouse-Ly6g5b, mouse-Ly6g5c, mouse-Ly6g6c, mou se-Ly6g6d, mouse-Ly6g6e, mouse-Ly6g6f, mouse-Ly6g6g, mouse-Lynx1, mouse-Lypd1, mouse-Lypd2, mouse-Lypd3, mouse-Lypd4, mouse-Lypd5, mouse-Lypd6, mouse-Lypd6b, mouse-Ly 6. The degranulation inhibitor according to the preceding item 5, which is mouse-Pate1, mouse-Pate2, mouse-Pate3, mouse-Pate4, mouse-Pate5, mouse-Pate6, mouse-Pate7, mouse-Pate8, mouse-Pate9, mouse-Pate10, mouse-Pate11, mouse-Pate12, mouse-Pate13, mouse-Pate14, mouse-Pinlyp, mouse-Plaur, mouse-Psca, mouse-Slurp1, mouse-Slurp2, mouse-Spaca4, mouse-Tex101 or mouse Ly6M. 8. The degranulation inhibitor according to the preceding item 5, wherein the Ly6 / uPAR family protein is any one of the following polypeptides: 1) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4 2) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10 3) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1 4) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2 5) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3 6) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5 7) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:6 8) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:7 9) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8 10) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:9 11) A polypeptide having an amino acid sequence in which one or two amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 10, and which has substantially the same degranulation inhibitory activity as the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 10. 12) A polypeptide having an amino acid sequence which has 95% or more homology with any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 10 and has substantially the same degranulation inhibitory effect as any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 10. Effect of the Invention

[0008] The present invention provides an allergy suppressant and a degranulation suppressant. [Brief description of the drawings]

[0009] [Figure 1] Confirmation of differentiation and purification rate of mast cells and neutrophils. (A) Bone marrow cells were collected from mouse bone marrow and cultured in the presence of growth factors (IL-3, SCF) for one month to differentiate, yielding bone marrow-derived mast cells. Bone marrow cells were collected from mouse bone marrow and purified by the negative selection method using a neutrophil isolation kit to obtain bone marrow-isolated neutrophils. (B) Analysis was performed using a flow cytometer with marker proteins (mast cells: FcεRIα, neutrophils: Ly6G) as indicators. [Diagram 2]Transfer of the neutrophil-derived protein Ly6G to mast cells. (A) Mast cells and neutrophils were co-cultured for 24 hours on a dish coated with Matrigel. (B) Confocal laser microscope images of mast cells (MC) and neutrophils (N) when specifically stimulated with antigen after sensitization with IgE are shown. Staining was performed with anti-Ly6G antibody and phalloidin. At the area where mast cells (MC) and neutrophils (N) adhered, the fluorescence of Ly6G expressed on neutrophils was observed transferring to mast cells (top). After antigen stimulation, Ly6G fluorescence was observed inside mast cells (bottom). [Diagram 3] FIG. 1 is a schematic diagram of the translocation mechanism of Ly6G during the interaction between mast cells and neutrophils. [Figure 4] Changes in the amount of Ly6G in the supernatant of cocultured mast cells and neutrophils. Mast cells and neutrophils were cocultured, and the culture supernatants unstimulated (0 hour) or after antigen stimulation (0.5, 1, 3, and 6 hours) were detected by Western blotting using an anti-Ly6G antibody (clone:1A8) (mean±SEM, *p<0.05, ***p<0.001). [Diagram 5] Purification of recombinant Ly6G and binding to mast cells. (A) E. coli lysates and recombinant Ly6G before and after the addition of IPTG, a protein expression inducer, were detected by Western blotting using anti-Ly6G antibody (clone: ​​1A8) and anti-Histag antibody (clone: ​​OGHis). (B) Mast cells after IgE sensitization were treated with Ly6G, left unstimulated or specifically stimulated with antigen, and stained with anti-Ly6G antibody (clone: ​​1A8). Mast cells with Ly6G binding (Ly6G+) were detected using a flow cytometer, and the percentage was calculated. [Figure 6] Analysis of the amount of mast cell degranulation induced by Ly6G. (A, B) Mast cells after IgE sensitization were treated with recombinant Ly6G (A: 2.5 μM, B: 0.25, 0.5, 2.5, 5 μM, control: PBS) and then stimulated with antigen for 30 minutes. The degranulation reaction was evaluated by quantifying the amount of β-hexosaminidase in the mast cell culture supernatant (mean ± SEM, **p < 0.01). [Figure 7]Quantitative analysis of TNFα from mast cells using Ly6G. Mast cells after IgE sensitization were treated with Ly6G (control: PBS) and stimulated with antigen for 8 hours. The amount of TNFα secreted in the mast cell culture supernatant was quantified using ELISA (mean ± SEM, ***p <0.001). [Figure 8] Analysis of calcium ion concentration in mast cells using Ly6G. (A) Mast cells treated with IgE, calcium fluorescent indicators (Fluo4, Fura red), and recombinant Ly6G (control: PBS) were photographed at 5-second intervals using a confocal laser microscope, and antigen stimulation was performed 1 minute after the start of imaging. Images were then taken for 9 minutes (total of 10 minutes). The graph shows the ratio of fluorescence intensity between Fluo 4 and Fura red (Fluo 4 / Fura red), and the fluorescence intensity ratio (Ft) at each time point was corrected by the value at the start of imaging (F0) (mean ± SEM, *p <0.05). (B) Analysis was performed on the fluorescence intensity ratio 1 minute (2 minutes after the start of imaging) and 9 minutes (10 minutes after the start of imaging) after antigen stimulation of mast cells (mean ± SEM, ****p <0.0001). [Figure 9] Analysis of activation of mast cell signaling molecules by Ly6G. Mast cells after IgE sensitization were treated with recombinant Ly6G (control: PBS), and the activation state of each protein was analyzed after no stimulation (0 min) or antigen stimulation (2, 5, 15, 30 min). Cell lysates were subjected to SDS-PAGE, and detection was performed by Western blotting using site-specific anti-phosphorylation antibodies for each protein. [Figure 10] Control of mast cell degranulation by Ly6G peptide. Mast cells after IgE sensitization were treated with Ly6G peptides #1 to #9 (10 μM, control: 10% DMSO / PBS) and stimulated with antigen for 30 minutes. Degranulation was evaluated by quantifying the amount of β-hexosaminidase in the mast cell culture supernatant (mean ± SEM, *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001). [Figure 11]Control of TNFα secretion from mast cells by Ly6G fragment peptides. Mast cells after IgE sensitization were treated with Ly6G peptides #1 to #9 (10 μM, control: 10% DMSO / PBS) and stimulated with antigen for 3 hours. The amount of TNFα secreted in the mast cell culture supernatant was quantified by ELISA (mean ± SEM, *p <0.05, **p <0.01). [Figure 12] Overview of Ly6G transfer mechanisms and regulation of mast cell responses via mast cell–neutrophil interactions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] (Subject of the present invention) The present invention relates to an allergy suppressant, a degranulation inhibitor, and a method for screening an allergy suppressant or a degranulation inhibitor.

[0011] (Ly6G) Ly6G belongs to the Ly6 / uPAR family and is a low molecular weight protein of about 25 kDa (including the glycosylation site) that is specifically expressed on the cell membrane of neutrophils via a GPI anchor (see J LeukocBiol. 94(4):585-94. (2013)). Ly6 family proteins are proteins expressed in various immune cells including neutrophils and share a common site called the Ly6 / uPAR domain. More than 60 types have been identified in mice to date, but the physiological functions of most of them are unknown. Ly6B, Ly6C, Ly6G, etc. are expressed in neutrophils, and Ly6G is commonly used as a marker protein for neutrophils because it is a neutrophil-specific protein (J Leukoc Biol. 94(4):585-94. (2013)). Ly6G is a mouse-specific protein that is highly expressed mainly in mouse neutrophils. However, it is known that CD177, which has a common function as a mouse Ly6G homologue, is also present in human neutrophils (Hum Genomics. 21;10:10. (2016), Blood.120(7):1352-3. (2012)).

[0012] The Ly6 / uPAR family protein of the present invention includes the above-mentioned proteins. Particularly preferred proteins include human CD59, human CD177, human Ly6D, human Ly6E, human Ly6H, human Ly6K, human Ly6L, human-ACRV1, human-GML, human-GPIHBP1, human-LY6G5B, human-LY6G5C, human-Y6G6C, human-LY6G6D, human-LY6G6F, human-LYNX1, human-LYPD1, human-LYPD2, human-LYPD3, human-LYPD4, human-LYPD5, human-LYPD6, human-LYPD6B, human-LYPD8, human-PATE1, human-PATE2, human-PATE3, human-PATE4, human-PINLYP, human-PLAUR, human-PSCA, human-SLURP1, human-SLURP2, human-SPACA4, human-TEX101, mouse Ly6G, mouse Ly6A, mouse Ly6C1, mouse Ly6C2, mouse Ly6D, mouse Ly6E, mouse Ly6F, mouse Ly6H, mouse Ly6I, mouse Ly6K, mouseLy6L, mouse-Acrv1, mouse-Cd177, mouse-Cd59a, mouse-Cd59b, mouse-Gml, mouse-Gml2, mouse-Gpihbp1, mouse-Ly6a2, mouse-Ly6g2, mouse-Ly6g5b, mouse-Ly6g5c, mouse-Ly6g6c, mou se-Ly6g6d, mouse-Ly6g6e, mouse-Ly6g6f, mouse-Ly6g6g, mouse-Lynx1, mouse-Lypd1, mouse-Lypd2, mouse-Lypd3, mouse-Lypd4, mouse-Lypd5, mouse-Lypd6, mouse-Lypd6b, mouse-Ly pd8, mouse-Lypd9, mouse-Lypd10, mouse-Lypd11, mouse-Pate1, mouse-Pate2, mouse-Pate3, mouse-Pate4, mouse-Pate5, mouse-Pate6, mouse-Pate7, mouse-Pate8, mouse-Pate9, mouse e-Pate10, mouse-Pate11, mouse-Pate12, mouse-Pate13, mouse-Pate14, mouse-Pinlyp, mouse-Plaur, mouse-Psca, mouse-Slurp1, mouse-Slurp2, mouse-Spaca4, mouse-Tex101 and mouse Ly6M (Reference: Loughner et al. Human Genomics (2016) 10:10). In particular, the amino acid sequences of mouse Ly6A, mouse Ly6C1, mouse Ly6C2 and mouse Ly6M share approximately 80% or more homology with the amino acid sequence of Ly6G peptide #9 (SEQ ID NO: 9), and therefore these proteins have activity equivalent to Ly6G peptide #9 (SEQ ID NO: 9).

[0013] The Ly6 / uPAR family proteins of the present invention also include the following polypeptides. 1) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4 2) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10 3) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1 4) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2 5) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3 6) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5 7) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:6 8) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:7 9) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8 10) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:9 11) A polypeptide consisting of an amino acid sequence having one or two amino acids substituted, deleted, inserted and / or added in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 10, and having substantially the same anti-allergy activity or anti-degranulation activity as the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 10. 12) A polypeptide having an amino acid sequence which has a homology of 95% or more (particularly, 96% or more, 97% or more, 98% or more, or 99% or more is preferable) with the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 10 and has substantially the same anti-allergy effect or anti-degranulation effect as the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 10.

[0014] From the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide when introducing mutations into the peptide, for example, mutual substitutions between homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) can be easily envisioned. The term "an allergy suppressing effect or degranulation suppressing effect substantially the same as that of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 10" means that the level of the effect may be stronger or weaker than the allergy suppressing effect or degranulation suppressing effect of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 10. Examples of the effect include about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, and about 150% compared to the allergy suppressing effect or degranulation suppressing effect of the amino acid sequence set forth in SEQ ID NO: 10. Alternatively, identity can be calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, using default or initial setting parameters). As used herein, polypeptide includes proteins, polypeptides and oligopeptides, the minimum size of which is two amino acids. As used herein, the term "protein" includes its degradation products, fragmented peptides, etc. For example, Ly6 / uPAR family proteins include fragmented peptides of Ly6 / uPAR family proteins.

[0015] (Mast cells) Mast cell secretory granules contain histamine, cytokines, chemokines, and various proteases, and play an important role in the development of allergic diseases. In addition, inflammatory mediators contained in mast cell secretory granules have various physiological functions. Histamine has the effect of increasing vascular permeability, and in fact, it has been shown that when mast cells are activated by antigens, these inflammatory mediators are secreted, increasing vascular permeability, allowing neutrophils to infiltrate from the blood vessels into tissues, and that mast cells and neutrophils exist in close proximity to each other and interact with each other. Thus, in mast cell-induced inflammatory tissues, it is believed that the inflammatory response is regulated by exposure of cells in the vicinity of mast cells to various inflammatory mediators secreted by mast cells. The mast cells used in the screening method for allergy suppressors or degranulation suppressors of the present invention may be cells of any origin, but are preferably derived from humans, mice, or rats. Furthermore, commercially available mast cells can be used.

[0016] (allergy) The allergy of the present invention includes type I, II, III, and IV allergies. The allergic diseases of the present invention include, but are not limited to, hay fever, food allergies, atopic dermatitis, allergic rhinitis, allergic conjunctivitis, bronchial asthma, urticaria, anaphylaxis, drug allergies, mite allergies, metal allergies, house dust allergies, latex allergies, toxin allergies, and animal allergies.

[0017] (Allergy suppressant) The allergy suppressant of the present invention is intended for patients (including humans and non-human mammals. Preferred non-human mammals include pets and livestock) with any symptoms (mild, moderate, severe) of the allergic disease of the present invention. In particular, the allergy suppressant of the present invention is preferably intended for type I allergy. In addition, suppression includes treating, preventing, preventing recurrence, alleviating, curing, and the like of allergies. As shown in the following examples, the allergy suppressant of the present invention can not only suppress but also control the amount of inflammatory mediators released from mast cells.

[0018] (Degranulation inhibitor) The degranulation inhibitor of the present invention is also intended for patients (including humans and non-human mammals. Preferred non-human mammals include pets and livestock) with diseases associated with an increase in the amount of degranulation of mast cells (hay fever, food allergies, atopic dermatitis, allergic rhinitis, allergic conjunctivitis, bronchial asthma, urticaria, anaphylaxis, drug allergies, mite allergies, metal allergies, house dust allergies, latex allergies, toxin allergies, and animal allergies). In addition, suppression includes treatment, prevention, prevention of recurrence, alleviation, complete cure, etc. of diseases associated with an increase in the amount of degranulation. As shown in the following examples, the degranulation inhibitor of the present invention can not only inhibit but also control the amount of degranulation of mast cells.

[0019] (Administration method, dosage form) The route of administration of the inhibitor of the present invention is not particularly limited, but preferred routes of administration include intravenous, oral, transdermal, and transmucosal (oral, rectal, vaginal, etc.) routes. Examples of preparations for oral administration include tablets, capsules, granules, powders, syrups (dry syrups), oral jellies, etc. Examples of preparations for transdermal or transmucosal administration include patches, ointments, etc. Tablets, capsules, granules, powders, etc. can be made into enteric preparations. For example, tablets, granules, and powders are provided with an enteric coating. As the enteric coating agent, a poorly soluble enteric coating agent in the stomach can be used. In addition to the active ingredient, the inhibitor of the present invention can contain a pharmacologically acceptable carrier depending on the administration form. Examples of the pharmacologically acceptable carrier include excipients, disintegrants or disintegration assistants, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers or solubilization assistants, isotonicity agents, pH regulators, stabilizers, propellants, and adhesives.

[0020] The dosage and frequency of administration of the inhibitor of the present invention may vary as appropriate depending on the conditions of the subject, its age, body weight, sex, purpose (prevention or treatment, etc.), severity of symptoms, dosage form, administration route, etc. When administered to humans, the dosage of the Ly6 / uPAR family protein, which is the active ingredient, is, for example, about 0.0001 mg / kg body weight to 10 mg / kg body weight per day. The frequency of administration may be once or multiple times per day, or once every few days. For example, it may be 1 to 3 times, 1 to 2 times, or once per day. The inhibitor of the present invention can be made into medicines, quasi-drugs, medical devices, sanitary products, foods, beverages, and supplements.

[0021] (Treatment method) The present invention also relates to a method for preventing and / or treating allergies or diseases associated with increased degranulation levels, using a Ly6 / uPAR family protein as an active ingredient.

[0022] (Screening Method) The screening method for an allergy suppressant or degranulation suppressant of the present invention targets the following: - Determine substances that promote the uptake (binding) of Ly6 / uPAR family proteins (especially Ly6G) by mast cells. - Identify substances that increase the release of Ly6 / uPAR family proteins (especially Ly6G) from neutrophils.

[0023] "Determining a substance that promotes the uptake (binding) of Ly6 / uPAR family proteins (particularly Ly6G) by mast cells" may include, for example, any one or more of the following steps. (a-1) measuring the amount of Ly6 / uPAR family protein bound to mast cells in the presence of a test substance; and (a-2) A step of selecting a test substance that promotes the uptake (binding) of Ly6 / uPAR family proteins (particularly Ly6G) by mast cells based on the results of (a-1) above.

[0024] "Determining a substance that increases the amount of Ly6 / uPAR family protein (particularly Ly6G) released by neutrophils" may include, for example, any one or more of the following steps. (a-1) measuring the amount of Ly6 / uPAR family protein released from neutrophil mast cells in the presence of a test substance; and (a-2) A step of selecting a test substance that increases the amount of Ly6 / uPAR family proteins (particularly Ly6G) released from neutrophils based on the results of (a-1) above.

[0025] (Test substance) Any substance can be used as a test substance that is a candidate therapeutic substance used in the above screening. The type of test substance is not particularly limited, and may be an individual low molecular weight synthetic compound (e.g., siRNA), a compound present in a natural product extract, or a synthetic peptide. The test substance may be a chemical library, a genome editing library, a phage display library, or a combinatorial library. The construction of chemical libraries, phage display libraries, and combinatorial libraries is known to those skilled in the art, and commercially available chemical libraries can also be used. EXAMPLES

[0026] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0027] Materials and Methods The following materials and methods were used to carry out Examples 2-7.

[0028] (cell culture) Bone marrow-derived mast cells (BMMCs) were prepared by collecting bone marrow cells from the femurs and tibias of 6- to 12-week-old mice (C57BL / 6N) and culturing them in RPMI1640 (Nacalai Tesque) supplemented with 10% FBS (SIGMA), IL-3, SCF, MEM Non-Essential Amino Acids (Nacalai Tesque), Sodium pyruvate (Nacalai Tesque), Penicillin-Streptmycin (Nacalai Tesque), and HEPES (Nacalai Tesque) at 37°C and 5% CO. 2 The cells were cultured under these conditions for one month and allowed to differentiate. Bone marrow isolation Neutrophils were isolated from bone marrow cells collected from the femurs and tibias of 6- to 12-week-old mice by negative selection using a Neutrophil Isolation kit (MACS Miltenyi Biotec). A coculture system was prepared by culturing mast cells and neutrophils on a glass-bottom dish coated with Matrigel (Corning) for 24 hours.

[0029] (Analysis of differentiation and purification rates of bone marrow-derived mast cells and bone marrow-isolated neutrophils) 1.0×10 5 Mast cells or neutrophils were cultured in FACS Buffer (0.1% BSA, 0.02% NaN 3 The cells were suspended in 1 mM EDTA / PBS, and Fc block (5 μg / mL) was added, after which the antibody was reacted for 1 hour at 4°C. The cells were washed twice with FACS Buffer, passed through a nylon filter (41 μm), and detected with FACS Verse (BD). The specific antibodies used were FITC-labeled anti-mouse FcεRI antibody (clone: ​​MAR-1, eBioscience) and Alexa Fluor 647-labeled anti-mouse CD117 antibody (clone: ​​2B8, BioLegend), and the non-specific antibodies used were FITC-labeled Armenian Hamster IgG isotype control (eBioscience) and Alexa Fluor 647-labeled Rat IgG2b isotype control (BioLegend).

[0030] (Immunostaining of in vitro coculture of mast cells and neutrophils) 1.0×10 5 mast cells and 1.0 × 10 5A coculture system was prepared using neutrophils, and the neutrophils were sensitized with anti-DNP-IgE (clone: ​​H1-DNP-ε-26.82, 1 μg / mL) at 37°C for 1 hour. The neutrophils were then incubated in HEPES Tyrode Buffer (135 mM NaCl, 5 mM KCl, 20 mM HEPES, 5.6 mM Glucose, 1 mM MgCl 2 , 1.8 mM CaCl 2 The cells were then washed with 0.05% BSA (pH 7.4) and stimulated with DNP-HSA (50 ng / mL) to specifically activate mast cells. One hour after stimulation, the cells were fixed with 4% PFA and solubilized with 0.2% Triton X-100, after which they were incubated with specific antibodies at 4°C overnight. After washing with PBS, the cells were observed using a confocal laser microscope LSM710 (Zeiss). The specific antibodies used were FITC-labeled Anti-Mouse-Ly6G Antibody (BioLegend) and Alexa Fluor 647-labeled Phalloidin (Invitrogen), and the non-specific antibody used was FITC-labeled Rat IgG2a κ Isotype control (BioLegend).

[0031] (Purification of recombinant Ly6G) The Ly6G-pET28a(+) plasmid was introduced into protein expression E. coli (ClearColi BL21(DE3) Electrocompetent Cells) by electroporation to construct Ly6G-pET28a(+)BL21. Ly6G-pET28a(+)BL21 was grown at OD 300 in LB medium supplemented with 50 μg / mL kanamycin. 600= 0.5, and then cultured at 130 rpm for 5 hours in the presence of 0.1 mM IPTG (Protein Ark) to induce Ly6G expression. The cells were harvested by centrifugation at 4,000 × g for 10 minutes, and the supernatant was removed. The pellet was suspended in Cell Lysis buffer (100 mM phosphate buffer, 300 mM NaCl, protease inhibitor), and lysed by adding Fast Break Cell Lysis Reagent 10× (Promega). The solution was frozen and thawed three times at -80°C to confirm that the viscosity of the solution had increased, and then Benzonase Nuclease (EMD Millipore Corp) and 100 mM MgCl 2 After thawing, the mixture was centrifuged at 10,000 rpm at 4°C for 30 minutes, and the supernatant and storage solution were removed. TM The beads were mixed with Ni-Particles (Promega) and mixed upside down at 4°C for 3 hours to adsorb the target protein. After washing three times with 50 mM phosphate buffer using a magnetic separation stand (Promega), the beads were washed once each with 100 mM phosphate buffer and 10 mM imidazole elution buffer, and then eluted with 500 mM imidazole elution buffer. The resulting protein solution was placed in Biodesign Dialysis Tubing. TM Dialysis was performed in PBS at 4°C using a 3500 MWCO (BioDesign Inc. of New York). Buffer exchange was performed twice at 1-hour intervals, and then dialysis was performed overnight. After dialysis, recombinant Ly6G was quantified using a BCA Protein Assay Kit (Pierce).

[0032] (CBB staining of recombinant Ly6G) After separating recombinant Ly6G by SDS-PAGE, the gel was washed with distilled water for 1 hour, incubated with TaKaRa CBB Protein Safe Stain (TaKaRa) at room temperature overnight, washed with distilled water for 1 hour, and detected with Odyssey (LI-COR).

[0033] (Western blotting) Western blotting was carried out according to the procedure described below. Confirmation of recombinant Ly6G expression Recombinant Ly6G was separated by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% skim milk solution for 30 minutes, the membrane was incubated with the primary antibody overnight at 4°C. The membrane was then washed with 0.2% Tween solution and incubated with the secondary antibody for 1 hour at room temperature. The membrane was then washed with 0.2% Tween solution and detected with Odyssey (LI-COR). The primary antibodies used were Rat-Anti-Ly6G Antibody (clone: ​​1A8, BioXcell) and Mouse-Anti-Histag mAb (clone: ​​OGHis, MBL), and the secondary antibodies used were IRDye 800CW Goat-Anti-Rat IgG Antibody (LI-COR) and IRDye 800CW Donkey-Anti-Mouse IgG Antibody (LI-COR).

[0034] Detection of Ly6G protein in the supernatant from the interaction of mast cells and neutrophils 1.0×10 6 mast cells and 1.0 × 10 6A coculture system was prepared using neutrophils, and the cells were sensitized with anti-DNP-IgE (clone: ​​H1-DNP-ε-26.82, 1 μg / mL) at 37°C for 1 hour and washed with HEPES Tyrode Buffer. The cells were then stimulated with DNP-HSA (50 ng / mL) for 0, 0.5, 1, 3, and 6 hours to specifically activate mast cells. The cells were centrifuged at 3,000 rpm for 5 minutes, and the supernatant was used as a sample. The obtained samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% skim milk solution for 30 minutes, the primary antibody was reacted overnight at 4°C. The cells were then washed with 0.2% Tween solution and reacted with the secondary antibody at room temperature for 1 hour. The cells were then washed with 0.2% Tween solution and detected with Odyssey (LI-COR). The primary antibody used was Rat-Anti-Ly6G Antibody (clone: ​​1A8, BioXcell), and the secondary antibody was IRDye 800CW Goat-Anti-Rat IgG Antibody (LI-COR).

[0035] Detection of phosphorylation of mast cell signaling molecules by Ly6G protein 1.0×10 6 Mast cells were sensitized with anti-DNP-IgE (clone: ​​H1-DNP-ε-26.82, 1 μg / mL) at 37°C for 1 hour and washed with HEPES Tyrode Buffer. After that, the mast cells were stimulated with DNP-HSA (50 ng / mL) (0, 2, 5, 15, 30 minutes) to specifically activate the mast cells, and lysis buffer (1% NP-40, 60 mM octylpyranoside, protease inhibitor) was added to lyse the cells. The cells were left on ice and centrifuged at 14,000 rpm for 10 minutes, and the supernatant was used as a sample. The obtained sample was separated by SDS-PAGE and transferred to a nitrocellulose membrane. 50% Intercept TMAfter blocking with Blocking Buffer (LI-COR) for 30 minutes, the primary antibody was reacted overnight at 4°C. After washing with 0.2% Tween solution, the secondary antibody was reacted at room temperature for 1 hour. After washing with 0.2% Tween solution, the cells were detected with Odyssey (LI-COR). Primary antibodies include Rabbit-Anti-phospho Lyn [Y396] Antibody (abcam), Rabbit-Anti-phospho Syk [Y525 / 526] Antibody (cell signaling), Rabbit-Anti-phospho LAT [Y191] Antibody (cell signaling), Mouse-Anti-phospho PLCγ1 [Y783] Antibody (BioLegend), Mouse-Anti-phosphoErk [T202 / Y204] Antibody(eBioscience), Rabbit-Anti-phospho p38 [T180 / Y182] Antibody(cell signaling), Rabbit-Anti-phospho JNK[T183 / Y135] Antibody(cell signaling), Rabbit-Anti-phospho Akt [T308] Antibody(cellsignaling), Mouse-Anti β-Actin Antibody(MBL) and Rabbit-Anti β-Actin Antibody (MBL) was used, and the secondary antibodies used were IRDye680CW Donkey-Anti-Mouse IgG Antibody (LI-COR), IRDye 800CW Donkey-Anti-Mouse IgG Antibody (LI-COR), IRDye 800CW Sheep-Anti-Rabbit IgG Antibody (LI-COR), and Alexa fluor 680 Goat-Anti-Rabbit IgG Antibody (Invitrogen).

[0036] (Detection of Ly6G-binding mast cells) 1.5×10 6Mast cells were sensitized with anti-DNP-IgE (clone: ​​H1-DNP-ε-26.82, 1 μg / mL) at 37°C for 1 hour, washed with HEPESTyrode Buffer, and incubated with recombinant Ly6G (10 μM) for 30 minutes. Mast cells were then stimulated with DNP-HSA (50 ng / mL) for 1 hour to specifically activate them. The cells were centrifuged at 1,500 rpm for 5 minutes, suspended in FACS Buffer, and Fc block (5 μg / mL) was added, followed by incubation with the antibody at 4°C for 1 hour. The cells were washed twice with FACS Buffer, passed through a nylon filter (41 μm), and detected with FACS Verse (BD). The specific antibody used was FITC-labeled Anti-Mouse Ly6G Antibody (Invitrogen), and the non-specific antibody used was FITC-labeled Rat IgG2a κ Isotype control (BioLegend).

[0037] (Measurement of calcium ion concentration in mast cells) 2.0×10 5 Mast cells were treated with anti-DNP-IgE (clone: ​​H1-DNP-ε-26.82, 1 μg / mL), recombinant Ly6G (2.5 μM), Fluo4-AM (DOJINDO, 3.5 μM), and Fura Red. TM The cells were treated with 5 μM β-AM (Invitrogen, 5 μM) and seeded on a glass bottom dish coated with 5 μg / mL Poly-D-Lysine hydrobromide (SIGMA) and sensitized for 35 minutes at 37°C. The cells were washed with HEPES Tyrode buffer and soaked in HEPES Tyrode buffer. Images were taken at 5 second intervals using a confocal laser microscope, and antigen stimulation (50 ng / mL) was performed 1 minute after the start of imaging. Images were then taken for 9 minutes (10 minutes in total). The fluorescence intensity of calcium fluorescent indicators Fluo 4 and Fura Red was measured at each time point. The graph shows the ratio of the fluorescence intensity of Fluo 4 to Fura red (Fluo 4 / Fura red) and the fluorescence intensity ratio (Ft) at each time point was calculated relative to the fluorescence intensity at the start of imaging (F 0 ) value.

[0038] (Ly6G fragment peptide) The peptides used were those with the following sequences (Cosmo Bio Co., Ltd.) obtained by fragmenting Ly6G (134 amino acids: SEQ ID NO: 10) into 20 amino acids each.

[0039] [Table 1]

[0040] (Measurement of degranulation amount) The amount of degranulation was measured using the β-hexosaminidase assay. 5 Mast cells were sensitized with anti-DNP-IgE (clone: ​​H1-DNP-ε-26.82, 1 μg / mL) for 1 hour at 37°C, washed with HEPES Tyrode buffer, and reacted with recombinant Ly6G (2.5 μM) or peptide (10 μM) for 30 minutes at 37°C. Mast cells were then stimulated with DNP-HSA (50 ng / mL) for 30 minutes at 37°C to specifically activate them. The cells were centrifuged at 3,000 rpm for 5 minutes, and the supernatant was used as a sample. In addition, samples treated with 0.5% Triton X-100 were prepared for measuring the total amount of β-hexosaminidase. The supernatant was mixed with 2 mM p-NAG / 0.1 M citrate buffer, and reacted at 37°C for 1 hour in the dark. Thereafter, 0.1 M carbonate buffer was added to stop the reaction, and the absorbance at 405 nm was measured using a multiplate reader (TECAN). The relative amount of degranulation was calculated using the following formula.

[0041]

number

[0042] (Quantitation of cytokine secretion) The amount of cytokine secreted was quantified using ELISA. 6Mast cells were sensitized with anti-DNP-IgE (clone: ​​H1-DNP-ε-26.82, 1 μg / mL) for 1 hour at 37°C, washed with HEPES Tyrode buffer, and reacted with recombinant Ly6G (2.5 μM) or Ly6G peptide (10 μM) for 30 minutes at 37°C. Then, the cells were stimulated with DNP-HSA (50 ng / mL) at 37°C (recombinant Ly6G: 8 hours, Ly6G peptide: 3 hours) to specifically activate the mast cells. The cells were centrifuged at 3,000 rpm for 5 minutes, and the supernatant was used as a sample. Measurements were performed using a Mouse TNF-α Duoset ELISA kit (R&D Systems) according to the attached protocol.

[0043] (statistics) Results were analyzed using GraphPad Prism (GraphPad Software Inc, California, USA). Statistical significance was determined by unpaired, two-tailed Student's t-test or One-way Analysis of Variance (ANOVA). Two-way ANOVA was used to examine the time course of intracellular calcium ion concentrations. Differences were considered significant when P values ​​were 0.05 or less with 95% confidence intervals. All results are presented as the mean ± SEM of at least three independent experiments. EXAMPLES

[0044] (Confirmation of differentiation and purification rate of mast cells and neutrophils) In this example, we performed an analysis of the interaction between mast cells and neutrophils and the molecular functions associated with the interaction. Mouse bone marrow-derived mast cells were used as mast cells, and mouse bone marrow-isolated neutrophils were used as neutrophils (Figure 1A). Bone marrow cells were collected from mouse bone marrow and cultured for one month in the presence of growth factors to obtain mast cells. In addition, neutrophils were purified using an isolation kit. Analysis was performed using a flow cytometer using marker proteins as indicators, and it was confirmed that the differentiation rate of mast cells was 95% or higher, and the purification rate of neutrophils was 90% or higher (Figure 1B). EXAMPLES

[0045] (Transfer of Ly6G upon interaction between neutrophils and mast cells) The inventors have confirmed that when mast cells are specifically stimulated with an antigen, the morphology of the mast cells changes in association with a secretory response, and that neutrophils then change their morphology from round to spindle-shaped, migrate toward and adhere to the mast cells, and interact with them. Actin is a protein that is abundant in cells and forms the cytoskeleton. It is involved in interactions between various immune cells, and the present inventors have observed that actin accumulates at the adhesion surface where mast cells and neutrophils interact. To analyze the interaction between mast cells and neutrophils, we stained the neutrophil marker protein Ly6G and the cytoskeleton F-actin in both interacting cells and analyzed their localization changes. When mast cells and neutrophils were co-cultured, when mast cells and neutrophils were adhering to each other, Ly6G was observed to move from the neutrophils to the cell membrane surface of the mast cells near where the two cells were adhering, regardless of the presence or absence of antigenic stimulation (Fig. 2B: arrow). Furthermore, when antigenic stimulation was performed, the number of cells adhering to mast cells and neutrophils increased, and not only did the number of cells with Ly6G moving increase as previously observed, but it was also observed that Ly6G was taken up into the mast cells (Fig. 2B: arrowhead). Since Ly6G is a protein that is specifically expressed in neutrophils and not in mast cells, it was confirmed that Ly6G expressed on the cell membrane of neutrophils is cleaved, bound to the surface of mast cells, and taken up. In addition, in the results of this example, F-actin was not observed accumulating on the adhesion surface between mast cells and neutrophils. It is thought that the localization changes with a slight difference in timing, such as depolymerization occurring when Ly6G moves to the surface of the mast cell membrane, and repolymerization occurring after Ly6G is taken up into the cell, resulting in accumulation on the adhesion surface. EXAMPLES

[0046] (Cleavage of Ly6G from neutrophils and binding to mast cells) Based on the results of Example 3, the present inventors hypothesized that a factor controlling the movement of Ly6G at the adhesion surface between mast cells and neutrophils might be that mediators such as proteases secreted by mast cells cleave Ly6G on neutrophils, and the cleaved Ly6G migrates to the surface of mast cells and is taken up (see Figure 3). In this Example, it was confirmed that mediators such as proteases cleave Ly6G on neutrophils, and the cleaved Ly6G migrates to the surface of mast cells and is taken up.

[0047] Cleavage of Ly6G from neutrophils by mast cell activation We investigated whether inflammatory mediators secreted upon activation of mast cells have the effect of releasing Ly6G expressed in neutrophils. After co-culturing mast cells and neutrophils, the mast cells were specifically stimulated with an antigen, and the amount of Ly6G in the culture supernatant was detected by Western blotting. As a result, we confirmed that, compared to unstimulated cases, Ly6G in the culture supernatant was significantly increased 1 hour after antigen stimulation, and significantly decreased 3 and 6 hours after antigen stimulation (see Figure 4). This revealed that Ly6G on the cell membrane of neutrophils was cleaved and released into the culture supernatant following activation of mast cells by antigen stimulation. The decrease in the amount of Ly6G in the culture supernatant 3 and 6 hours after antigen stimulation may be due to the fact that Ly6G released into the culture supernatant bound to mast cells and was taken up by them, or that it was fragmented into smaller peptides by proteases contained in the secretory granules of mast cells.

[0048] (Binding of recombinant Ly6G to mast cells) We confirmed whether Ly6G cleaved from neutrophils binds to mast cells. In this example, recombinant Histag-fused Ly6G was purified with Ni magnetic beads using an E. coli expression system and used. After purification, Western blotting was performed, and bands derived from Ly6G were detected with anti-Ly6G antibody (clone: ​​1A8) and anti-Histag antibody (clone: ​​OGHis), confirming that the target protein had been purified (see Figure 5A). When this recombinant Ly6G was applied to IgE-sensitized mast cells and analyzed using a flow cytometer, Ly6G was observed to bind to mast cells. 4 When the percentage of mast cells with Ly6G bound to them was calculated, the percentage was 9.84% for mast cells alone (control), 19.1% for unstimulated mast cells treated with Ly6G, and 34.8% for mast cells stimulated with antigen (see Figure 5B). This shows that the percentage of mast cells with Ly6G bound to them increases when antigen stimulation is performed. Therefore, it is believed that there is a binding factor for Ly6G on the cell membrane of mast cells, and it was confirmed that this binding factor enhances the uptake of Ly6G into the cells following activation of mast cells by antigen. EXAMPLES

[0049] (Confirmation of the effect of Ly6G on the secretory response of mast cells) The results of Examples 2 to 4 revealed that Ly6G, which is expressed on neutrophils following activation of mast cells, is cleaved from neutrophils, migrates to and binds to the surface of mast cells, and is taken up into the mast cells (see Figures 2, 4 and 5). In order to elucidate the mechanism by which Ly6G regulates mast cell responses, we analyzed the effect of Ly6G on the secretion of inflammatory mediators. Inflammatory mediators secreted by mast cells include those (e.g., histamine) that are secreted immediately when mast cells are activated by antigens, and those (e.g., cytokines and chemokines) that are synthesized and secreted after mast cell activation. In this example, among the secretory reactions performed by activated mast cells, the degranulation reaction (histamine, etc.), which is an immediate reaction, and the amount of cytokine secretion, which requires time for secretion, were evaluated.

[0050] Effect of Ly6G on mast cell degranulation We analyzed the effect of Ly6G on the degranulation of mast cells. After IgE sensitization, mast cells were treated with recombinant Ly6G, and the amount of degranulation associated with antigen stimulation was measured. The amount of degranulation was evaluated by measuring the activity of an intragranular enzyme (β-hexosaminidase) that is secreted immediately upon activation of mast cells, similar to histamine. As a result, Ly6G treatment significantly reduced the amount of degranulation associated with antigen stimulation of mast cells (see Figure 6A). Furthermore, it was revealed that when the concentration of Ly6G used for treatment was changed, the amount of degranulation decreased in a Ly6G concentration-dependent manner (see Figure 6B). Furthermore, it was confirmed that Ly6G treatment did not affect the amount of degranulation in the absence of antigen stimulation. These results demonstrate that Ly6G can reduce the amount of degranulation secreted by mast cells in a concentration-dependent manner following antigen stimulation.

[0051] The effect of Ly6G on cytokine secretion from mast cells The effect of Ly6G on the amount of cytokine secreted by mast cells was analyzed. After IgE sensitization, mast cells were treated with recombinant Ly6G and stimulated with antigen. TNFα is a type of inflammatory cytokine secreted by mast cells, and it is known to induce neutrophil infiltration by regulating the expression of adhesion molecules on vascular endothelial cells. Here, the amount of TNFα secreted was evaluated using ELISA. It was revealed that Ly6G treatment significantly increased the amount of TNFα released following antigen stimulation (see Figure 7). Furthermore, it was confirmed that Ly6G treatment did not affect the amount of TNFα released in the absence of antigen stimulation. These results confirmed that Ly6G can regulate the amount of TNFα secreted by mast cells. EXAMPLES

[0052] (Effect of Ly6G on mast cell signaling) We analyzed the mechanism by which Ly6G regulates mast cell signaling.

[0053] Confirmation of sustained increase in calcium ion concentration in mast cells by Ly6G Changes in intracellular calcium ion concentration play an important role in mast cell activation and secretory responses. The increase in intracellular calcium ion concentration following antigen stimulation of mast cells is regulated by calcium ion efflux from the endoplasmic reticulum and calcium ion influx from the extracellular fluid. Therefore, we analyzed the effect of Ly6G on the increase in intracellular calcium ion concentration. Using the calcium fluorescent indicators Fluo 4 and Fura red, observations were performed using Fluo 4 and Fura red ratio imaging with a confocal laser scanning microscope (see Figure 8A). As a result, no change was observed in the increase in intracellular calcium ion concentration 1 minute after antigen stimulation, but a significant increase in intracellular calcium ion concentration was observed 9 minutes after antigen stimulation due to Ly6G treatment (see Figure 8B). Therefore, it was revealed that Ly6G treatment caused a sustained increase in intracellular calcium ion concentration in mast cells. It is believed that the increase in intracellular calcium ion concentration in mast cells is regulated mainly by two mechanisms: (1) calcium ion efflux from the endoplasmic reticulum, which occurs at an early stage, and (2) calcium ion influx from the extracellular fluid, which occurs after calcium ion release from the endoplasmic reticulum. Since Ly6G treatment did not significantly affect the increase in intracellular calcium ion concentration until 1 minute after antigen stimulation, it is believed that Ly6G regulates the influx of calcium ions from the extracellular fluid. From the above, it was confirmed that Ly6G regulates the intracellular calcium ion concentration in mast cells. This, together with the results of the above examples, confirmed that exogenous Ly6G administration can reduce the amount of degranulation secreted by mast cells.

[0054] Confirmation of sustained activation of mast cell signaling molecules by Ly6G It is known that activation of IgE receptors (FcεRI) on mast cells via antigens and antigen-specific IgE leads to activation of various signaling molecules, including Lyn, Syk, LAT, and PLCγ, which are important for the degranulation reaction of mast cells. It is also known that activation of transcriptional regulators such as MAPK (Erk, JNK, p38) promotes the transcription of cytokines and chemokines. Therefore, in this Example, the effect of Ly6G on the activation of signaling molecules in mast cells was analyzed. In this example, the phosphorylation state, which is an indicator of activation of Lyn, Syk, LAT, PLCγ, MAPK (Erk, JNK, p38), and Akt, which are signaling molecules important for mast cell signaling, was detected using Western blotting. As a result, sustained phosphorylation of Syk, LAT, PLCγ, Erk, p38, and Akt was observed in Ly6G-treated cells following antigen stimulation (see Figure 9). Thus, it was revealed that the activity of some of the important kinases and adaptor molecules in mast cell signaling via the IgE receptor (FcεRI) was sustained by Ly6G treatment compared to the untreated control. These results confirmed that Ly6G is involved in mast cell signaling. This, together with the results of the above examples, confirmed that exogenous Ly6G administration can reduce the amount of degranulation secreted by mast cells. In addition, in accordance with the results of the above examples, exogenous administration of Ly6G can suppress allergies by reducing the amount of degranulation secreted by mast cells. Furthermore, exogenous Ly6G administration does not simply reduce the amount of mast cell degranulation but also regulates the amount of inflammatory mediators secreted, i.e., exogenous Ly6G administration can suppress, treat, and even prevent allergies by regulating the amount of degranulation and the amount of inflammatory mediators secreted. EXAMPLES

[0055] (Confirmation of the active region of Ly6G) It is believed that the secretion-regulating effect of Ly6G on the amount of degranulation of mast cells is due to the fact that Ly6G is further fragmented after being cleaved from neutrophils, and that different actions are expressed specific to the fragmentation site. It has been reported that several peptides derived from eosinophil granule proteins cleaved by tryptase secreted by mast cells bind to mast cells and function. Therefore, we used peptides (SEQ ID NO: 1-9) obtained by fragmenting Ly6G (134 amino acids: SEQ ID NO: 10) into 20 amino acids with 5 amino acid overhangs, and analyzed the secretion of inflammatory mediators in the same way as recombinant Ly6G.

[0056] Confirmation of mast cell degranulation by Ly6G fragment peptide The effect of Ly6G peptide on the degranulation reaction of mast cells was analyzed. Mast cells after IgE sensitization were treated with Ly6G fragment peptides (#1 to #9 correspond to SEQ ID NOs: 1 to 9, respectively) and stimulated with antigen. The amount of degranulation was evaluated by measuring the activity of an intragranular enzyme (β-hexosaminidase) that is secreted in the same way as histamine. It was confirmed that administration of each Ly6G peptide reduced the amount of degranulation associated with antigen stimulation of mast cells, compared to the control (no peptide added) (see FIG. 10). In particular, it was confirmed that administration of Ly6G peptides #4 (SEQ ID NO: 4: CPFSDGFCVALEIEVIVDSH) and #9 (SEQ ID NO: 9: LLFSLVSVLLQTFL) significantly reduced the amount of degranulation associated with antigen stimulation of mast cells. As described above, each Ly6G peptide, like Ly6G, can reduce the amount of degranulation that accompanies antigenic stimulation of mast cells, and therefore, together with the results of the above examples, can not only suppress and treat allergies, but also prevent them.

[0057] Confirmation of cytokine response of mast cells using Ly6G fragment peptides The effect of Ly6G peptide on the secretion of cytokines (TNFα) from mast cells was analyzed. After IgE sensitization, mast cells were treated with Ly6G peptides #1 to #9 and stimulated with antigen. Administration of Ly6G peptide #4 increased the amount of TNFα secreted by mast cells following antigen stimulation, as compared to the control (no peptide added) (see FIG. 11). From the above, Ly6G peptide #4, like Ly6G, can regulate the amount of TNFα secreted by mast cells following antigen stimulation.

[0058] (General remarks) The results of the examples revealed that secretions from mast cells cleave Ly6G from the cell membrane of neutrophils (see FIG. 4). It was also revealed that Ly6G cleaved from neutrophils bound to mast cells, and that the percentage of mast cells that internalized Ly6G increased after antigen stimulation. This suggests that there is a binding factor for Ly6G on the cell membrane of mast cells, and that antigen-induced activation of mast cells enhances the internalization of Ly6G into the cells. Furthermore, the results of this study demonstrated that Ly6G has the ability to regulate the secretory response of mast cells following antigen stimulation, and Ly6G treatment significantly reduced the amount of degranulation. Ly6G treatment induced a sustained increase in intracellular calcium ion concentration, as well as sustained activation of Syk, LAT, PLCγ, Erk, p38, and Akt, which are important signaling molecules in mast cells. Since PLCγ is a calcium-binding protein, it is believed that sustained activation of PLCγ caused a sustained increase in intracellular calcium concentration. Furthermore, since it was expected that Ly6G would be further fragmented after cleavage from neutrophils and exert different regulatory effects specific to the fragmentation site, we analyzed the secretion response of inflammatory mediators using fragmented peptides of Ly6G. As a result, the amount of degranulation of mast cells following antigen stimulation was significantly reduced by Ly6G peptide treatment. Thus, it was revealed that Ly6G can control the secretion response of mast cells even when fragmented into peptides of about 20 amino acids. The sequence of peptide #4 is CPFSDGFCVALEIEVIVDSH, which is a negatively charged hydrophobic peptide. The sequence of peptide #9 is LLFSLVSVLLQTFL, which is an uncharged hydrophobic peptide. In this example, analysis using a co-culture system of mast cells and neutrophils revealed that Ly6G expressed in neutrophils following activation of mast cells is cleaved from neutrophils, migrates to and binds to the surface of mast cells, and is taken up into mast cells. It was revealed that Ly6G, a neutrophil-derived protein, functions as a mast cell response regulator.

[0059] From the above, this Example clarified that Ly6G expressed in neutrophils following activation of mast cells is cleaved from neutrophils, migrates to and binds to the surface of mast cells, and is taken up into mast cells. In addition, Ly6G, a neutrophil-derived protein, functions as a mast cell response regulator, and can suppress allergies by controlling the amount of inflammatory mediators secreted (see Figure 12). [Industrial Applicability]

[0060] It is possible to provide an allergy suppressant and a degranulation suppressant.

Claims

1. An allergy suppressant containing Ly6G.

2. An allergy suppressant comprising any one of the following polypeptides: A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4 A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:10 A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:9

3. A degranulation inhibitor containing Ly6G.

4. A degranulation inhibitor comprising any one of the following polypeptides: A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4 A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:10 A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:9

5. An agent for increasing TNFα release, comprising Ly6G.

6. An agent for increasing TNFα release, comprising any one of the following polypeptides: A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4 A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:10

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