WO2005028667A1 - Drug targeting g protein-coupled receptor expressed in human mast cells and method of screening the same - Google Patents

Abstract

A large number of GPCRs expressed in human mast cells are identified by extracting RNA from human mast cells and performing DNA chip analysis or RT-PCR analysis with the use of the RNA. Agonists and antagonists to these GPCRs, functionally modifications thereof, antibodies specifically recognizing these GPCRs and antisense DNAs and siRNAs inhibiting the expression of the GPCRs are usable as human mast cell activation inhibitors and remedies for allergic diseases. By using cells expressing these GPCRs or membrane fractions of these cells, it is possible to screen a human mast cell activation inhibitor or a remedy for allergic diseases.

Description

 Specification

Drug targeting G protein-coupled receptor expressed in human mast cells and screening method thereof

 The present invention relates to a drug targeting a G protein-coupled receptor (hereinafter abbreviated as GPCR) expressed in human mast cells and a method for screening the same. Background art

It is known that mast cells are activated by each stimulus, cause degranulation, and release and produce many inflammatory mediators [Kurosawa Motohiro, edited by “Mast cell clinic”, Advanced Medicine. , P142 and p559 (2001); Crit, Rev. Immunol., 22, 115-140 (2002)]. The main fe species are as diverse as amines, arachidonic acid metabolites, proteases, cytokins and chemokines. For example, when an antigen is recognized by mast cells, degranulation promptly releases histamine and tryptic enzyme, as well as prostaglandin D ₂ (PGD ₂ ), leukotriene (LT), and platelet activating factor (PAF). ) And various chemokines such as macrophage inflammatory protein (MIP) -1 and various cytokins such as granulocyte macrophage colony stimulating factor (GM-CSF). It is known to be newly synthesized and released. In addition, it has recently been clarified that a large amount of mast cells is produced in humans in the case of the major basic protein, a cytotoxic protein that was previously thought to be produced by eosinophils. [: Blood, 1127-1134 (2001)]. Thus, since mast cells are thought to play a major role in the pathogenesis of various allergic diseases, it is possible to treat allergic diseases by controlling mast cell activation. Conceivable.

Although various existing allergic drugs that have the effect of suppressing the release of inflammatory mediators from mast cells are known, ^ research is mainly conducted using rodent mast cells. Whether it has any effect on mast cells has not been sufficiently studied. However, recently, a method for preparing human mast cells has been established, and it has become possible to analyze the effects of existing drugs on human mast cells, and to compare them with the effects on rodent mast cells. As a result, it has been clarified that drug mast cells have different reactivity to human mast cells [Kurosawa Motohiro, edited by “Mast Cell Clinic”, Advanced Medicine, P559 (2001)]. . For example, sodium cromoglycate, which is used as an inhibitor of the release of inflammatory mediators, significantly suppresses IgE-dependent release of inflammatory mediators in rat peritoneal mast cells, but has an effect on human mast cells. Clin was not strong ones. Exp. Allergy, 28, 1 228-1236 (1998) ] o hydrochloride Azerasuchin histamine from cultured human mast cells at high concentrations, PGD _2, LT free, GM-CSF and Suppresses the production of MIP-1 strain, but none of the activities was strong. Also, sublatast tosilate, which is used as an anti-cytodynamic drug, showed an inhibitory effect on rat mast cells for the release of inflammatory mediators, but no effect on human mast cells. Exp. Allergy, 28, 1228-1236 (1998)].

On the other hand, it is known that a drug acting on GPCR expressed in mast cells significantly suppresses the production of inflammatory mediators from cultured human mast cells. For example, / ^ adrenaline At low concentrations of receptor stimulant I Seo Pro tele Nord 10 nmol / 1, evening hiss from cultured human mast cells Min, LT, PGD _2, GM- CSF and MIP-1 Hino liberates 80% or more to suppress. [J. Allergy Cl in. Immunol., 103, 421-426 (1999)]. Therefore, agonists, antagonists or functional modifiers of GPCRs expressed on human mast cells suppress the production of inflammatory mediators from human mast cells, and are useful in the treatment of diseases involving mast cells. It is considered effective. In addition, administration of hismin promotes apoptosis of human mast cells [Exp. Dermatol., 12, 53-60 (2003)] and induces apoptosis of mast cells by human melanocyte stimulating hormone. [FEBS Lett., 87, -87-93 (2003)], and the like, it is considered that a drug acting on GPCR expressed in mast cells induces apoptosis of human mast cells.

However, information on GPCI expressed in human mast cells has been limited. Specifically, the GPCR expressed in human mast cells, complement third component receptor 1,? ₂ adrenergic receptor, adenosine A2b receptors, prostaglandin E receptor 2, prostaglandin E receptor 3 subtype la, prostaglandin E receptor 3 alternative tip variant f (also known as prostaglandin E receptor 3 subtype lb), Epstein-Barr virus induced gene 1 Inva virus-inducible gene 2

(Epstein-Barr virus induced gene 2), G protein-coupled receptor RE2, G protein-coupled receptor 35, C-XC motif chemokine receptor 3, and thrombin receptor are known [Blood, 98, 1127]. -1134 (2001); Blood, 100, 3861-3868 (2002); BMC Immunol., 3, 5 (2002)]. Comparison of genes expressed in cultured mast cells derived from human umbilical cord blood and cultured mast cells derived from mouse bone marrow revealed that the genes expressed in humans and mice do not always match [Blood,, 3861-3868 ( 2002)]. As mentioned above, there are species differences in the reactivity of drugs. Therefore, there was no guarantee that the GPCR expressed in animal mast cells was expressed in human mast cells, and it was necessary to examine the expression in human mast cells. Invention

 The present invention has clarified the GPCRs expressed in human mast cells, and provided a target for these GPCRs. An agent for suppressing the activation of human mast cells, a therapeutic agent for allergic diseases, and a screening method thereof. It is intended to be.

 The present inventors cultured human mast cells such as cultured mast cells derived from human umbilical cord blood and cultured mast cells derived from human peripheral blood under various conditions, extracted RNA, and analyzed the DNA chip using the RNA. Alternatively, by performing RT-PCR analysis, we succeeded in identifying a GPCR that was not previously known to be expressed in human mast cells, and developed drugs and antibodies targeting the GPCR and screening for the same. The present invention has been completed by providing a method.

 The present invention relates to the following (1) to (34).

(1) Cysteinyl leukotriene receptor 1 (CysLTR1), G protein-coupled receptor 91 (GPR91), G protein-coupled receptor 34 (GPR34), which are G protein-coupled receptors expressed in human mast cells ), EGF-like module-containing mucin-like hormone receptor-like sequence 2 (E R2), platelet activator receptor (PAFR), G protein-coupled receptor 105 (GPR105), seven transmembrane superfamily members 1 (TM7SF1), G protein-coupled receptor 37 (GPR37), CD97 antigen (CD97), melanocortin 1 receptor (MCI), G protein-coupled receptor FIRE (FIRE), formyl Ptide receptor 1 (fMLPl), G protein-coupled receptor 65 (GPR65), prostaglandin E receptor 3 alternative variant h (EP3 variant h) ヽ purinergic receptor P2Y8 (P2Y8), dopamine receptor D2 (D2), endothelial differentiation lysophosphatidic acid G protein-coupled receptor 2 (EDG2), adenosine A3 receptor (A3), complement fifth component receptor 1 (C5R1), opsin 3 (0PN3), G protein-coupled Receptor 27 (GPR27), G protein-coupled receptor 44 (CRTH2), endothelial differentiation sphingolipid G protein-coupled receptor 1 (EDG1), formyl peptide receptor-like 2 (FPRL2), chemokine ( C-C motif) receptor-like 2 (CCRL2), metabotropic glutamate receptor 6 (mglu6) cannabinoid receptor 2 (CB2), G protein-coupled receptor 39 (GPR39), purin receptor P2Y5 (P2Y5), Adenosine A1 Receptor (A1), Pyrimidine Receptor P2Y4 (P2Y4), Pros-U-Glandin E Receptor 3 alternative variant j (EP3 variant «i), adenosine A2a receptor (A2a), type 1 angiotensin receptor (AT1B), type B endothelin receptor (ETB), gonadotropin releasing hormone receptor (GNRHR) , Cadherin EGF LAG 7-pass G-type receptor 2 (CELSR2), Chemokine (CC motif) receptor 7 (CC 7), Histamine receptor H4 (H4), G protein-coupled receptor 12 (GPR12), Histamine receptor Body HI (HI), G protein-coupled receptor 32 (GPR32), tumor endothelial cell marker 5 (TEM5), G protein-coupled receptor 58 (GPR58), force doherin EGF LAG 7-pass G-type receptor 1 (CELSR1), G protein-coupled receptor 85 (GPR85), motilin receptor (Motilin R), G protein-coupled receptor 64 (HE6), force-doherin EGF LAG 7-through G-type receptor 3 (CELSR3 ), Calcitonin receptor-like (CALCRL), retinal pigment epithelium-derived oral dopsin homolog (Rffl), G White matter-coupled receptor 18 (GP18), G protein-coupled receptor 35 (GPR35), Purine receptor P2Y10 (P2Y10), FLJ40279 (C-TESTI2027296), G protein-coupled receptor 141 (GPR141), testis Descending-related G protein-coupled receptor (hRUP16), inversion inversion-related receptor (ITR), G protein-coupled receptor MRGX2 (MRGX2), traceamine receptor 1 (SNORP33) 7-time transmembrane receptor CBRC7TM— 375 (C-THYMU2011548), Adrenomedullin receptor (GlOd), G protein-coupled receptor 107 (GPR107), G protein-coupled receptor 126 (GPR126), G protein-coupled receptor 41 (GPR41), G protein-coupled receptor 43 (GPR43), G protein-coupled receptor 68 (GPR68), gastrin-releasing peptide receptor (GRPR), G protein-coupled receptor 1019 (HE0AD54), putative G protein-coupled receptor hGPCRlO (hRUP9), membrane-type progestin receptor α: (mPRa), membrane-type progestin receptor α (mPRg), putative G protein-coupled type Receptor GPCR1 (SRL), Platelet activating receptor homologue H963 (H963), Prostaglandin E receptor 4 (EP4), Chemoin (C-C motif) receptor 1 (CCR1), Dopamine receptor A cell expressing a receptor selected from the group consisting of D5 (D5), leukotriene B4 receptor (LTB4R) and chemokine (C-X-C motif) receptor 5 (CXCR5), or a membrane fraction of the cell. Including a step of contacting with a test substance, acting on the receptor and inducing apotosis in human mast cells, (b) suppressing activation, (c) suppressing degranulation, (d) inflammatory A method for screening a substance that exhibits at least one of the following actions: mediae production inhibition, (e) inhibition of cytokine production, and (f) inhibition of chemokine production.

(2) G protein-coupled receptors expressed on human mast cells are GPR34, PAFR, GPR105, TM7SF1, GPR65, P2Y8, EDG2, A3, C5R1 ヽ EDG1 ヽ GPR39 _S EP3 variant A2a, ETB ヽ H4, CELSR2, TEM5 , C-TESTI2027296 _N GPR141, ITR, MRGX2, G10d, GPR43, GRPR, HEOAD54, H963, EP4 and 2. The screening method according to claim 1, which is a receptor selected from the group consisting of LTB4R, the expression of which is varied by stimulating activation of human mast cells via an IgE receptor.

 (3) G protein-coupled receptor expressed on human mast cells stimulates lipopolysaccharide on human mast cells selected from the group consisting of PAFR ヽ CD97, MCI ヽ C5R1, A2a, AT1B, CELS2, GCR7 and EP4 2. The screening method according to claim 1, which is a receptor whose expression fluctuates due to the change.

(4) human mast cells expressed in human mast cells are selected from the group consisting of PAFRs A2b, CD97, EDG2, 0PN3, CRTH2 _N EDG1, H4, GPR32 and CELSR1; 2. The screening method according to claim 1, which is a receptor whose expression varies when stimulated. -

(5) G protein-coupled receptor expressed in human mast cells is selected from the group consisting of PAFR, GPR105, CD97, MC1, A3, 0PN3, EDG1, CCRL2, P2Y5, A2a, CCR7, CELSR2, CELSR1 and EP4 2. The screening method according to claim 1, which is a receptor whose expression is fluctuated by co-stimulation of human mast cells with insulin and lipopolysaccharide.

(6) A G protein-coupled receptor expressed in human mast cells is a human selected from the group consisting of GPR34, GPR105, TM7SF, fMLPl, P2Y8, A3, CRTH2, CCRL2 _N P2Y5, A2a, H4, CALCRL and EP4. 2. The screening method according to claim 1, wherein the mast cell is a receptor whose expression fluctuates by glucocortide treatment. .

(7) expressed in human mast cells, and a G protein-coupled receptor expression level by stimulation of the glucocorticoid varies complement third component receptor 1 (C3aRl), GPR34, ₂ adrenergic receptor (? 2), GP painting, WSF, fMLPl, P2Y8, A3, CRTH2, CCRL2, P2Y5, A2a, Epsuba virus-inducible gene 2 (EBI2), thrombin receptor (PARI), H4, G protein-coupled type Contacting a test substance with a cell expressing a receptor selected from the group consisting of receptor RE2 (E2), CALCRL and EP4, or a membrane fraction of the cell, and acting on the receptor; A method for screening a substance capable of controlling a ruticoid action.

(8) G protein-coupled receptor expressed in human mast cells, CysLTR1, GPR91, GPR34, EMR2, PAFRs GPR105, TM7SF1, GPR37, CD97, MCI 、 FIRE ヽ fMLPl ヽ GPR65, EP3 variant h, P2Y8, D2 , EDG2, A3, C5R1, 0PN3, GPR27, CRTH2 EDG1, FPRL2, CCRL2, mglu6, CB2, GPR39, P2Y5, Al, P2Y4, EP3 variant j ヽ A2a, AT1B, ETB, GNRHR ヽ CELSR2, CCR7, H4, GPR12 HI ヽ GPR32, TEM5, GPR58, CELSR1, GPR85, Motilin R, HE6, CELSR3, CALCRL, RRH, GPR18, GPR35, P2Y10, C-TESTI2027296, GPR141, hRUP16 _s ITR, MRGX2, SNORF33, C-TH 丽 2011548, Gd , GPR107, GPR126, GPR41, GPR43, GPR68, GRPR ヽ HEO, hRUP9, mPRa, mPRg, SRL ヽ H963, EP4, CAG1, D5, LTB4R and CXCR5 Alternatively, using a function modifier, (a) induction of apoptosis, (b) suppression of activation on human mast cells (C) suppression of degranulation, (d) suppression of production of inflammatory mediators, (e) suppression of cytokine production, and (f) suppression of chemokine production.

(9) Using an agonist against A2a or HEOAD54, which is a G protein-coupled receptor expressed in human mast cells, induces (a) induction of apoptosis and (b) activation of human mast cells (C) inhibiting degranulation, (d) inhibiting the production of inflammatory mediators, (e) inhibiting the production of cytokines, and (f) inhibiting the production of chemokines.

 (10) 0 protein-coupled receptor expressed in human mast cells, 0? & 91, 0 卩 1134,? Against human mast cells using an antagonist to a receptor selected from the group consisting of humans 1 ^^^ 05, D2, D5, EDG2, EP3 variant h, EP3 variant j, EP4, ETB ヽ HI ヽ H4 and GPR43 , (A) induction of apoptosis, (b) suppression of activation, (c) suppression of degranulation, (d) suppression of production of inflammatory mediators, (e) suppression of production of cytodynamics (F) A method of performing at least one of chemokine production suppression.

 (11) CysLTR1, GPR91, GPR34, EMR2, PAFR, GPR105, 'TM7SF1, GPR37, CD97, MC1, FIRE, fMLPl, GPR65, EP3 variant h, which are G protein-coupled receptors expressed in human mast cells

P2Y8, D2, EDG2, A3, C5R1 0PN3, GPR27, CRTH2, EDG1 FPRL2, CCRL2, mglu6, CB2, GPR39, P2Y5, Al, P2Y4, EP3 variant j A2a AT1B, ETB, GNRHR CELSR2 _S CCR7, H4, G4 , GPR32, TM5, GPR58, CELSR1, GPR85, Motil in R, HE6, CELS 3 CALCRL RRH GPR18, GPR35, P2Y10, C-TESTI2027296, GPR141, hRUP16, ITR MRGX2, SNORF33, C-THYMU2011548, GlOd GPR107, GPR126 GPR107 Atopic skin using an agonist, angonist, or a function modifier for a receptor selected from the group consisting of GPR43, GPR68, GRPR HEOAD54, hRUP9, mPRa mPRg, SRL H963, EP4, C1, D5, LTB4R and CXCR5 How to treat inflammation, asthma, chronic obstructive pulmonary disease or allergic diseases.

 (12) A method for treating atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease using an agonist against A2a or HEOAD54 which is a G protein-coupled receptor expressed on human mast cells.

 (13) G protein-coupled receptor expressed in human mast cells, selected from the group consisting of GPR91, GPR34, PAFR, GPR105, EP3 variant h, D2, EDG2, EP3 variant _j, ETB ヽ HI and GPR43 A method for treating atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic diseases using angonist for the receptor.

 (14) C3aRl, GPR34, β, GPR105, TM7SF, fMLPl, P2Y8, A3, CRTH2, CCRL2, a G protein-coupled receptor that is expressed in human mast cells and whose expression level fluctuates due to glucocorticoid stimulation A method for controlling the action of a glucocorticoid on cells using an agonist, an engonist or a functional modifier for a receptor selected from the group consisting of P2Y5, A2a, EBI2, PARI ヽ H4, RE2, CALCRL and EP4.

(1 5) is a G protein-coupled receptors expressed in human mast cells, CysLTRl, GPR91, GPR34, EMR2 , PAFR GPR105, TM7SF1, GPR37 S CD97, MCI FIRE fMLPl, GPR65, EP3 variant h, P2Y8, D2, _{EDG2, A3, C5R1 S 0PN3,} GPR27, CRTH2, EDG1, FPRL2, CCRL2, mglu6, CB2, GPR39, P2Y5, Al, P2Y4, EP3 variant j s A2a AT1B, ETB, GNRHR, CELSR2, CCR7, H4, GPR1, HI GPR32 _S TEM5, GPR58, CELSR1, GPR85, Motil in R, HE 6, CELSR3, CALCRL, RRH, GPR18, GPR35, P2Y10, C-TESTI2027296 _N GPR141, hRUP16, ITR, MRGX2, SNORF33, C-THYMU2011548, G10d , GPR126, GPR41, GPR43 _N GPR68, GRPR, HEOAD54, hRUP9, mPRa, mPRg, SRL and agonists, gangsters or functional modifications to receptors selected from the group consisting of H963, EP4, CCR1, D5, LTB4R and CXCR5 The medicament according to any one of (a) to (: i), comprising a substance as an active ingredient. (a) An agent for inducing apoptosis of human mast cells.

 (B) human mast cell activity inhibitor

 (c) Inhibitor of degranulation of human mast cells

 (d) Inhibitor of mast cell production of human mast cells

 (e) Human mast cell cytokine production inhibitor

 (: E) Chemokine production inhibitor for human mast cells

 (g) Remedies for atopic dermatitis

 (h) remedies for asthma

 (i) Chronic obstructive pulmonary disease drug

 (j) A therapeutic agent for allergic diseases

(16) The medicament according to any one of ( _a ) to (j), which comprises, as an active ingredient, an agonist against A2a or HEOAD54, which is a G protein-coupled receptor expressed in human mast cells.

 (A) human mast cell apoptosis inducer

 (b) human mast cell activation inhibitor

 (c) Inhibitor of degranulation of human mast cells

 (d) Inhibitor of mast cell production of human mast cells

 (e) Human mast cell cytokine production inhibitor

 (f) Chemokine production inhibitor for human mast cells

 (g) Remedies for atopic dermatitis

 (h) remedies for asthma

 (i) Chronic obstructive pulmonary disease drug

 (j) Remedies for allergic diseases

 (17) GPR91, GPR34, PAFR, GPR105, D2, D5, EDG2, EP3 variant h, EP3 variant j ヽ EP4, ETB, HI ヽ H4 and GPR43, which are G protein-coupled receptors expressed in human mast cells , Any one of (a) to (j), which comprises, as an active ingredient, an engonist against a receptor selected from the group.

 (A) human mast cell apoptosis inducer

 (b) Human mast cell activity inhibitor

 (c) Inhibitor of degranulation of human mast cells

 (d) Inhibitor of mast cell production of human mast cells

 (e) Human mast cell cytokine production inhibitor

 (f) a chemokine production inhibitor for human mast cells

 (g) Remedies for atopic dermatitis

 (h) remedies for asthma

 (i) Chronic obstructive pulmonary disease drug

 (j) Remedies for allergic diseases

(18) expressed in human mast cells, and a G protein-coupled receptor expression level by stimulation of Darco corticoid _{varies, C3aRl, β GPR34, GPR105 S} fMLPl, P2Y8, A3, CRTH2, CCRL2 S P2Y5, A2a , EBI2, CELSR2, PAR1, H4, RE2, CALCRL and EP4 An agent for controlling the action of dalcocorticoid, which comprises an agonist, an engonist or a function modifying substance for the body as an active ingredient.

(19) CysLTRU GPR9, a G protein-coupled receptor expressed on human mast cells, for the manufacture of a pharmaceutical composition for the treatment of atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease GPR3, EMR2, PAFR _S GPR105, TM7SF1, GPR37, CD97, MCI ヽ FIRE ヽ fMLPl, GP65, EP3 variant h, P2Y8, D2, EDG2, A3, C5R1, 0PN3, GPR27, CRTH2, EDG1, FPRL2, CCRL2, mglu6 _s CB2, GPR39, P2Y5, Al ヽ P2Y4, EP3 variant j _s A2a, AT1B, ETB ヽ GNRHR ヽ CELSR2 CCR7, H4, GPR12, Hl, GPR32 麵 ヽ GPR58, CELSR1, GPR85, Moti lin R, thigh, CELSR3 CALCRL, RRH, GPR18, GPR35, P2Y10, C-TESTI2027296, GPR141, hRUP16, ITR, MRGX2, SNORF33, C-THYMU2011548.G10d, GPR107, GPR126, GPR41, GPR43, GPR68, CM¾, HEOAD54, hidden 9, mPRa, mPRgァ An agonist or an antagonist of the receptor selected from the group consisting of H963, EP4, CCR1, D5, LTB4R and CXCR5. Or use of functional modifiers.

(20) CysLTR1, GPR91, GPR34, EMR2, PAFR _S GPR105, TM7SF1 _S GPR37, CD97, MC1, FIRE, fMLPl, GPR65, EP3 variant h, P2Y8, which are G protein-coupled receptors expressed on human mast cells _S D2, EDG2 _N A3, C5R1, 0PN3, GPR27, CRTH2, EDG1, FPRL2, CCRL2, mglu6, CB2, GPR39, P2Y5, Al, P2Y4, EP3 variant A2a ヽ AT1B, ETB ヽ GNRHR ヽ CELSR2, CCR7, HPR12 , HI ヽ GPR32, TEM5, GPR58, CELSRK GP 85 _S Motil in R HE6, CELS 3, CALCRL, RRH, GPR18, GP 35 _S P2Y10, C-TESTI2027296, GPR141, hRUP16, ITR _S MRGX2, SNORF33, C-THYMU2011548. Specifically reacts with a receptor selected from the group consisting of G10d, GPR107, GPR126, GPR41 ヽ GPR43, GPR68, GRPR ヽ HE0AD54, hRUP9 mPRa, mPRg ヽ SRLH963, EP4, CCR1, D5, LTB4R and CXCR5, and A geometrical body having at least one of the following actions (a) to (f) on human mast cells:

 (a) Induction of apoptosis

 (b) Suppression of activation

 (c) Suppression of degranulation

 (d) Inhibition of production of inflammatory mediator Yuichi

 (e) Suppression of cytokine production

 (f) Suppression of chemokine production

(2 1) was expressed in human mast cells, and a G protein-coupled receptor expression level by stimulation of Darco corticoid varies, C3aRl, GPR34, βヽ_{GPR105, TM7SF S fMLPl, P2Y8,} A3, CRTH2, CCRL2 An antibody that specifically reacts with a receptor selected from the group consisting of, P2Y5, A2a, EBI2, PARI, H4, RE2, CALCRL and EP4, and has an effect of controlling the action of dalcocorticoid.

A (2 2) G protein coupled receptors expressed in human mast cells, CysLTRl, GPR91, GPR34, EMR2 , PAFRヽGPR105, TM7SF1ヽGPR37, CD97, MCIヽFIREヽ_{fMLPl, GPR65 S EP3 variant h,} P2Y8 _{, D2 S EDG2, A3, C5R1} , 0PN3, GPR27, C Akira, EDG1ヽ _{FPRL2, CCRL2 S raglu6 s CB2,} GPR39, P2Y5, Al, P2Y4, EP3 variant j, A2aヽAT1B, ETBヽGNRHR, CELSR2, CCR7, H4, GPR12, HI ヽ GPR32, TEM5, GPR58, CELSR1, GPR85, Motil in R, HE 6, CELSR3, CALCRL ヽ RRH ヽ GPR18, GPR35, P2Y10, C-TESTI2027296, GPR14U hRUP16, ITR, MRGX2, SNORF33, C- THYMU2011548, G10d _s GPR107, GPR126, GPR41, GPR43, GPR68 _S GRPR ヽ HEOAD54, hRUP9, mPRa, mPRg, SRL, drawing, A method for detecting or isolating mast cells using an antibody that specifically reacts with a receptor selected from the group consisting of EP4, CCR1, D5, LTB4R and CXCR5.

 (23) using the antibody according to claim 20, for human mast cells, (a) induction of apoptosis, (b) suppression of activation, (c) W regulation of degranulation, (d) inflammation A method for suppressing at least one of the following: (1) suppression of cytokine production and (f) suppression of chemokine production.

 (24) A method for treating atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease using the antibody according to (20).

 (25) A method for controlling the action of glucocorticoid on cells using the antibody according to (21).

 (26) Any of (a) to (: i) containing the antibody according to (20) as an active ingredient.

(A) human mast cell apoptosis inducer

 (b) human mast cell activation inhibitor

 (c) Inhibitor of degranulation of human mast cells

 (d) Human mast cell inflammatory mediator production inhibitor

 (e) Human mast cell cytoin production inhibitor

 (f) Chemokine production inhibitor for human mast cells

 (g) Drug for treating atopic dermatitis ''

 (h) remedies for asthma

 (i) Drug for treating chronic obstructive pulmonary disease

 (j) A therapeutic agent for allergic diseases

 (27) An agent for controlling the action of dalcocorticoid, comprising the antibody according to claim 21 as an active ingredient.

 (28) Use of the antibody according to (20) for the manufacture of a pharmaceutical composition for treating atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease.

(29) CysLTR1, GPR91, GPR34, EMR2, PAFR GPR105, TM7SF1 GPR37, CD97, MC1, FIRE fMLPl GPR65, EP3 variant h, P2Y8, D2, EDG2, which are G protein-coupled receptors expressed in human mast cells A3, C5R1 0PN3, GPR27, CRTH2 , EDG1, FPRL2, CCRL2, mglu6 CB2, GPR39, P2Y5, Al, P2Y4, EP3 variant j, A2a, AT1B ETB GN thigh, CELSR2, CCR7, H4, GPR12 , HI GPR32 S TEM5, GPR58, CELSR1 _S GPR85, MotilinR, HE6, CELSR3, CALCRL, RRH, GP 18, GPR35, P2Y10, C-TESTI2027296, GPR141, hRUP16, ITR, MRGX2, SNORF33, C-THYMU2011548, G10d, GPR107, GPR126, GP41 _{S GPR43, GPR68 S GRPR, HEOAD54,} hRUP9, mPRa mPRg SRL, H963, EP4, CCR1, D5, LTB4R and CXCR5 suppress the expression of a ή Ru receptor selected from the group consisting of, siRNA against the gene encoding the receptor or Using antisense DNA, (a) induction of apoptosis, (b) suppression of activation The method of performing (c) inhibition of degranulation, (d) inflammatory mediator evening one production suppressing, one with least ingredients of (e) production of a cytokine-suppressing and (f) chemokine production inhibited.

(30) a G protein-coupled receptors expressed in human mast _{cells, CysLTRl, GPR91 N GP 34,} EM 2 N PAFRヽGP 105, TM7SF1, GPR37, CD97 , MCIヽFIRE, fMLPl, GPR65, EP3 variant h , P2Y8, D2, EDG2, A3, C5R1, 0PN3, GPR27, CRTH2, EDG1 ヽ FPRL2, CCRL2 _S mglu6, CB2, GP 39, P2Y5, Al, P2Y4, EP3 variant A2a ヽ AT1B, ETB ヽ GNRHR ヽ CELSR2, CCR7 _{, GPR12, Hl, GPR32 N TEM5} , GPR58, CELSR1, GPR85, Moti l in R, HE6, CELSR3, CALCRL, RRH, GPR18, GPR35, P2Y10, C-TESTI2027296s GPR141, hRUP16, ITR, MRGX2, SNORF33 C-THYMU2011548 S GlOd ヽ GPR107, GPR126, GPR41, GPR43, GP68, GRPR ヽ HEOAD54, hRUP9, mPRa, fflPRg ヽ SRL ヽ H963, EP4, CCR1, D5, LTB4R and suppress the expression of a receptor selected from the group consisting of CXCR5, A method for treating atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease, using siRNA or antisense DNA against a gene encoding the receptor.

 (31) C3aRl, GPR34, β, GPR105, T7SF, fMLPl, P2Y8, A3, CRTH2 CCRL2, a G protein-coupled receptor that is expressed in human mast cells and whose expression level fluctuates due to glucocorticoid stimulation , P2Y5, A2a, EBI2, PARI ヽ H4, RE2, CALCRLL and EP4, suppressing the expression of a receptor selected from the group consisting of: A method for controlling the action of glucocorticoid on mast cells.

(32) CysLTR1, GPR91, GPR34, EMR2, PAFR ヽ GPR105, TM7SF1, GPR37, CD97, MC1, FIRE ヽ fMLPl _s GPR65, EP3 variant h, P2Y8, which are G protein-coupled receptors expressed in human mast cells , D2, EDG2, A3, C5R1, 0PN3, GPR27, CRTH2, EDG1 ヽ Basket 2, CCRL2, mglu6, CB2, GPR39, .P2Y5, Al, P2Y4, EP3 variant j, A2a, AT1B ヽ ETB ヽ GNRHR ヽ CELSR2, CCR7 , H4, GPR12, HI ヽ GPR32, TEM5, GPR58, CELSRU GPR85, Motilin R, HE6, CELSR3, CALCRL, RRH, GPR18, GPR35, P2Y10 ヽ C-TESTI2027296, GPR1 1, hRUP16, IR, MRGX2, SNO F33 , C-THYMU2011548, G10d, 'Receptor selected from the group consisting of GPR107, GPR126, GPR41, GPR43, GPR68, GRPR ヽ HEO ヽ 9 mPRa, mPRg ヽ SRL ヽ H963, EP4, CCR1, D5, LTB4R and CXCR5 Any one of (a) to (: i) containing, as an active ingredient, siRNA or antisense DNA for a gene encoding the receptor, which suppresses body expression. Pharmaceutical.

 (a) Inducer of human mast cell apoptosis

 (b) human mast cell activation inhibitor

 (c) Inhibitor of degranulation of human mast cells

 (d) Human mast cell inflammatory mediator production inhibitor

 (e) Human mast cell cytokine production inhibitor

 (f) Chemokine production inhibitor for human mast cells

 (g) Remedies for atopic dermatitis

 (h) Asthma treatment

 (i) Drug for treating chronic obstructive pulmonary disease

 (j) Remedies for allergic diseases

(33) C3aRl, GPR34, (31, GPR105, WSF _S fMLPl, P2Y8, A3, CRTH2 _{CCRL2 S P2Y5 A2a, EBI2, PAR1} , H4, RE2, CALCRL and suppress the expression of a receptor selected from the group consisting of EP4, comprising as an active ingredient an siRNA or antisense DNA for the gene encoding the receptor, A regulator of the action of glucocorticoid.

(34) a G protein-coupled receptor expressed on human mast cells for the manufacture of a pharmaceutical composition for treating atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease, CysLTRls GPR91, GPR34, EMR2, PAFR ヽ GPR105, TM7SF1, GPR37, CD97, MC1, FIRE ヽ fMLPl, 5, EP3 variant h, P2Y8, D2, EDG2, A3, C5R1, 0PN3, GP27 CRTH2, 'EDG1, FPRL2 , CC L2 _N mglu6, CB2, GPR39, P2Y5, Al, P2Y4, EP3 variant j ヽ A2a, AT1B ヽ ETB ヽ GN thigh, CELSR2, CCR7, H4, GPR12, Hl, GPR32, TEM5, GPR58 _S CELSR1 ヽ GPR85 _N Motilin R ヽ HE 6, CELSR3, CALGRLs RRH, GPR18, GPR35, P2Y10, C-TESTI2027296 _S GPRU1, hRUP16, ITR, MRGX2, SNORF33, C-THYMU2011548 _N G10d, GPR107, GPR126, GPR41 GPR43, GPR68, GRPR, HEOAD54 Use of siRNA or antisense DNA against a gene encoding said receptor, which suppresses the expression of a receptor selected from the group consisting of listening, raPRa, mPRg, SRL, sight EP4, CCR1 ヽ D5, LTB4R and CXCR5.

 Hereinafter, the present invention will be described in detail.

 (1) Identification of GPCR expressed in human mast cells

 Human mast cells can be obtained from human lung, skin, fetal liver and the like by known methods [J. Immunol. Methods, 169, 153 (1994); J. Immunol., 138> 861 (1987); J. Allergy Clin. Immunol , 107, 322 (2001); J. Immunol. Methods., 40> 101 (2000)]. In addition, known methods [J. Immunol., 157> 343, (1996); Blood, 91> 187 (1998); J. Allergy Clin. Immunol., Dish, 141 (2000) ·; Blood, 97, 1016 ( Blood, 98, 1127 (2001); Blood, Sato, 3861 (2002); Blood, 97, 2045 (2001)], and mononuclear cells prepared from human umbilical cord blood, peripheral blood, bone marrow, lung or skin. It can be prepared by culturing spheres in the presence of stem cell factor (hereinafter abbreviated as SCF) and differentiating into mast cells. The mast cells prepared by differentiating the mononuclear cells by culture in this way are also called cultured mast cells.

 By extracting the culture odor and RNA from the various human mast cells described above, and using the RNA to analyze the expression levels of various GPCR mRNAs by DNA chip or RT-PCR, the expression in human mast cells has been achieved until now. GPCRs that were not known to do so can be identified.

 Furthermore, by culturing the prepared human mast cells under various conditions related to mast cell activation and identifying GPCRs whose expression levels fluctuate under those conditions, the mast cells have an inhibitory effect on mast cell activation. More suitable GPCRs can be selected to screen for different drugs.

Examples of culture under various conditions include, for example, culture in the presence of cytokines, growth factors, and chemokines, culture under conditions that activate mast cells, culture in the presence of various drugs, and components derived from microorganisms and viruses. And the like in the presence of E. coli. Examples of cytokines include inu-leukin 4 (IL-4), inu-Iu-Iu-Lukin 6 (IL-6), and inu-i-Iu-Fron-a (IFN-a). Conditions for activating mast cells include, for example, activation stimulation via IgE receptor, lipopolysaccharide (LPS) stimulation, calcium ionophore stimulation, compound 48/80 stimulation, peptidoglycan stimulation, mast cells Stimulation using a GPCR ligand expressed in the above. The drug has, for example, glucoamylase corticoid such as prednisolone or dexamethasone, immunosuppressants such as FK-506 or cyclosporin, the inflammatory mediator evening one release inhibiting the action of such 3 ₂ adrenergic receptor agonists and phosphodiesterase inhibitors Drugs, etc.

For the DNA chip analysis, for example, a commercially available Affymetrix GeneChip can be used. Known methods for RT-PCR analysis! : PCR Protocols, Academic Press (1990); Proc. Natl. Acad. Sci. USA, 87, 2725 (1990); 6) (1997)] can be used. As a quantitative RT-PCR method, Takara Shuzo's kit (For Real Time PCR TaKaRa Ex Taq-PC Version) for real-time RT-PCR can also be used.

 In the present invention, GPCRs that have been first revealed to be expressed in human mast cells include, for example, cystinyl leukotriene receptor 1 (CysLTRl), G protein-coupled receptor 91 (GPR91), and G protein-coupled receptor. 34 (GPR34), mucin-like hormone receptor-like sequence containing EGF-like module

2 (EMR2), Platelet activating factor receptor (PAFR), G protein-coupled receptor 105 (GPR105), 7-transmembrane superfamily member 1 (M7SF1), G protein-coupled receptor 37 (GPR37) , CD97 antigen (CD97), melanocortin 1 receptor (MCI), G protein-coupled receptor FIRE (FIRE), formyl peptide receptor 1 (fMLPl), G protein-coupled receptor 65 (GPR65), prostaglandin E-receptor 3 native variant h (EP3 variant h), purinergic receptor P2Y8 (P2Y8), dopamine receptor D2 (D2), endothelial differentiation lysophosphatidic acid G protein-coupled receptor 2 (EDG2), adenosine A3 receptor Body (A3), complement component 5 receptor 1 (G51), opsin

3 (0PN3), G protein-coupled receptor 27 (GPR27), G protein-coupled receptor 44 (CRTH2), endodermal differentiation sphingolipid G protein-coupled receptor 1 (EDG1), formyl peptide receptor-like Two

(FPRL2), Chemokine (C-C motif) receptor-like 2 (CCRL2), Methotropic bomiotropic receptor 6 (mglu6), Cannabinoid receptor 2 (CB2), G protein-coupled receptor 39 ( GPR39), purine receptor P2Y5 (P2Y5), adenosine A1 receptor (A1), pyrimidine receptor P2Y4 (P2Y4), prostaglandin G receptor E receptor 3 oryzae native variant j (EP3 variant j) _N adenosine A2a receptor (A2a;), type 1 angiotensin receptor (AT1B), type B endoselin receptor (ETB), gonadotropin releasing hormone receptor (GNRHR), cadherin EGF LAG 7-pass G-type receptor 2 (CELSR2), Chemokine (C-C motif) receptor 7 (CCR7), histamine receptor H4 (H4), G protein-coupled receptor 12 (GPR12), histamine receptor HI (HI), G protein-coupled receptor 32 (GPR32 ), Tumor endothelial cell marker 1 (TEM5), G protein-coupled receptor 58 (GPR58), Phosphorus EGF LAG 7-pass G-type receptor 1 (CELSR1), G protein cleaved receptor 85 (GPR85), motilin receptor (Motil in R), G protein-coupled receptor 64 (HE6), force-doherin EGF LAG G-type receptor 3 (CELSR3), calcitonin receptor-like (CALCRL), retinal pigment epithelium-derived oral dopsin homolog (RRH), G protein-coupled receptor 18 (GPR18), G protein-coupled receptor 35 (GPR35), Purine receptor P2Y10 (P2Y10), FLJ40279 (C-TESTI 027296), G protein-coupled receptor 141 (GPR141), Testicular descent-related G protein-coupled receptor (hRUP16), Intimal hyperplasia-related receptor (ITR), G protein-coupled receptor MR (S2 (MRGX2), traceamine receptor 1 (SNORF33), seven transmembrane receptor CBRC7 MJ75 (C-THYMU2011548), adrenomedullin receptor (GlOd), G protein-coupled receptor 107 (GPR107), G protein-coupled receptor 126 (GPR126), G protein-coupled receptor 41 (GPR41), G protein-coupled receptor 4 3 (GPR43), G protein-coupled receptor 68 (GPR68), gastrin-releasing peptide receptor (GRPR), G protein-coupled receptor 1019 (HEOAD54), putative G protein-coupled receptor hGPCR10 (hRUP9), membrane Progestin receptor (mPRa), M progestin receptor a (mPRg), putative G protein-coupled receptor GPCR1 (SRL), platelet activation receptor homolog H963 (H963), prostaglandin E receptor 4 ( EP4), chemokine (CC motif) receptor 1 (CCR1), dopamine receptor D5 (D5), leukotriene B4 receptor (LTB4R) and chemokine (C-X-C motif) receptor 5 (CXC5) I can give it. In the present invention, each GPCR is defined by the amino acid sequence and the base sequence of the database accession numbers shown in Tables 1, 3, 5, and 7. However, allyl variants are also included.

Of the above GPCR, as a GPCR that expressed by activated stimulation through IgE receptors varies GPR34, PAFRヽ _{GPR105, WSF1 S GP 65 N P2Y8} , EDG2, A3, C5R1ヽEDG1, GPR39, EP3 variant j, A2a, ETB, H4, CELSR2, TEM5, C-TESTI2027296 _S GPR141, ITR, MRGX2 _N GIOd ヽ GPR43, GRPR, HEOAD54, H963 _N EP4 and LTB4R. In addition, it has been reported that complement third component receptor 1 (C3aRl), adrenergic receptor (/ 32) and G protein-coupled receptor RE2 (RE2) are expressed in human mast cells. However, for the first time in the present invention, it was revealed that the expression fluctuates in IgE-treated cells.

 GPCRs whose expression fluctuates due to LPS stimulation include C3aRl, PAFR ヽ CD97, MC1, C5R1, A2a, AT1B, CELSR2, CCR7 and EP4, and GPCRs whose expression fluctuates due to IFN-a stimulation include C3aRl , PAFR ヽ A2b, CD97, EDG2, 0PN3, CRTH2, EDG1, prostaglandin E receptor 2 (EP2), H4, GPR32 and CELSR1, whose expression fluctuates with co-stimulation of IFN-α and LPS Examples of GPCRs include C3aRl, PAFRPRGPR105, CD97, MCI ヽ A3, 0PN3, EDG1, CCRL2, P2Y5, A2a, CCR7, RE2, CELSR2, CELSR1 and EP4. Of these, C3aRl, EP2 and RE were reported to be expressed in human mast cells, but for the first time in the present invention, it was clarified that the expression fluctuated due to IFN-α or LPS stimulation.

The GPCR expression in the glucocorticoid treatment varies, C3aRl, GPR34, β, GPR105 , TM7SF S fMLPl, P2Y8, A3, CRTH2, CCRL2, P2Y5, A2a, Epusu evening Inba one Virus induced gene 2 (EBI2), The thrombin receptor (PARI), H4, RE2, CALCRL and EP4 can each be mentioned. Of these, C3aRl, 32s EBI2, PARI and RE were reported to be expressed in human mast cells, but for the first time in the present invention it became clear that the expression was fluctuated by glucocorticoid treatment. .

 (2) Screening method for substances that suppress the activation of human mast cells targeting GPCR expressed in human mast cells

A GPCR to be screened is selected from the GPCRs expressed in human mast cells found in (1), and a known method using the cells expressing the GPCRs or the membrane fraction of the cells (see, eg, 2001). -245666) enables screening of agonists, antagonists or functionally modified substances of the selected GPCR. These substances cause human mast cells to undergo (i) induction of apoptosis, (ii) suppression of activation, (iii) suppression of degranulation, and (iv) inflammatory mediator via the GPCR. Has at least one of the following effects: (a) suppression of cytokine production, (V) suppression of cytokine production, and (vi) suppression of chemokine production. It can be used as a therapeutic drug for inflammatory lung diseases, allergic diseases and the like. Therefore, the screening method described below is based on the GPCiU, which acts on human mast cells by (i) induction of apoptosis, (ii) suppression of activation, (iii) suppression of degranulation, and (iv) inflammatory mediation. The present invention provides a method for screening a substance that suppresses the activation of human mast cells, which has at least one action of suppressing production overnight, (V) suppressing production of cytokin, and (vi) suppressing production of chemokine. Inflammatory media include amines such as histamine, Descriptor evening over peptidase such Protea Ichize, Arakidon acid metabolites, such as PGD ₂ and LT, the PAF, etc., as the site force in GM- the CSF and the like, as the chemokines MIP-1 flight and the like.

 In addition, the same method can be used to screen for GPCR agonists, angelic gonists, or functionally modified substances that are expressed in the human mast cells found in (1) and whose expression level fluctuates due to glucocorticoid stimulation. . Since these substances have an effect of controlling the action of glucocorticoid through the GPCR, they can be used as a regulator of the action of dalcocorticoid.

 The following briefly describes the screening method. The selected GPCR-expressing cells used for screening include mast cells derived from human peripheral blood, mast cells derived from human umbilical cord blood, mast cells prepared from human lung, skin, and tonsils, and cell lines established from human mast cells Transformed cells obtained by introducing an expression vector containing DNA encoding the GPCR into host cells such as animal cells and yeast can also be used. The transformed cells can be obtained by a known method (JP

 2001-245666). As a cell line established from human mast cells, LAD2 CLeuk. Res., 27, 671 (2003); which is known to well retain the properties of human mast cells; Leuk. Res., 27, 677 ( 2003)]. When a ligand, agonist or gonist specific for the selected GPCR is not used for screening, it is preferable to use a transformant containing DNA encoding the GPCR.

 (a) Ligand binding assay

 If the ligand, agonist, or gonist of the GPCR selected as a screening target is known, those labeled with a radioisotope, etc., and the cell expressing the GPCR or the membrane of the cell By using the fractions and performing ligand binding assay by a known method (Japanese Patent Application Laid-Open No. 2001-245666), it is possible to screen the GPCR agonist or angelic gonist. Hereinafter, a method using a labeled ligand will be described, but an agonist or an angelic gonist can also be used in the same manner as the ligand.

 Screening includes (i) contacting a labeled ligand with a cell expressing the GPCR or a membrane fraction of the cell, and (ii) labeling the labeled ligand and a test substance with the polypeptide of the present invention. Is measured by comparing the amount of the labeled ligand bound to the cell or the membrane fraction when the cell is contacted with the cell containing the partial peptide or the membrane fraction of the cell, and the results are compared. In the case where the test substance is present as compared to the case of (ii) (ii), if the amount of the labeled ligand bound decreases, the test substance is selected as an agonist or an agonist of the GP. Whether the selected test substance is an agonist or an antagonist of the GPCR can be confirmed by the following methods (b) to (d).

 The measurement of the binding amount of the labeled ligand can be performed as follows.

(I) a test substance and a radioactive isotope-labeled ligand of a given size, added to a buffer containing a certain amount of the cells expressing the GPCR or a membrane fraction of the cells, (ii) ) Prepared by adding only a certain amount of radiolabeled ligand labeled with a radioisotope, (iii) By adding a radiolabeled and fixed amount of radioactive ligand and a large excess of unlabeled ligand And react. The reaction is carried out at about 0-50 ° (preferably about 4-37 ° C.) for about 20 minutes-24 hours, preferably about 30 minutes-3 hours. (I)-(iii) After filtering with a glass fiber filter paper and washing with an appropriate amount of buffer, the amount of radioactivity remaining on the glass fiber filter paper is determined by liquid scintillation counting. Measure with a radiation measurement device such as Yuichi or Yukaichi. (I) and (ii) the values obtained by subtracting the amount of radioactivity (non-specific binding amount: NSB) of (i ii) from the amount of radioactivity (total binding amount: B) of (i) and (ii) It is calculated as the amount of each specific binding.

 (b) GTPa S-bonded assembly

 Using a labeled GTPaS and a cell expressing the GPCR selected as a screening target or a membrane fraction of the cell, a GTPaS binding assay is performed by a known method (Japanese Patent Application No. 2001-245666). The GPCR agonist, angel gonist, or function-decorating substance can be screened. As a cell containing a GPCR expressed in human mast cells, a transformant containing DNA encoding a GPCR expressed in mast cells can also be used. The transformant can be constructed by a known method (JP-A-2001-245666).

 For example, an agonist of the GPCR can be searched for by bringing a test substance into contact with a selected GPCR-expressing cell or a membrane fraction of the cell and performing a GTPas-binding assay.

 If the agonist of the GPCR is known, the agonist is brought into contact with the selected GPC-expressing cell or the membrane fraction of the cell in the presence of the test substance, and GTPaS-binding assay is carried out. You can search for an agonist, an angel, or a function modifier.

 When the GPCR is a constitutively active GPCR, the test substance is brought into contact with the selected GPCR-expressing cell or the membrane fraction of the cell, and GTPase binding is performed, whereby the GPCR agonist and enzyme You can search for gonists or functional modifiers.

According to the method described in the literature (Mol. Pharmacol., 7, 848-854 (1995), W098 / 46995), the amount of GTPaS bound to G can be determined by converting GTPyS labeled cells into GPCR-expressing cells or It can be added to the cell membrane fraction of the cell and measured as the amount of GTPas binding to the cell or cell membrane fraction. GTPaS binds to G in the same way as GTP, but does not undergo carohydrolysis, so the amount of binding can be measured accurately. As the labeled GTPa S, for example, GTPa S labeled with ³⁵ S can be used. Specifically, in a buffer containing a certain amount of a cell expressing the GPCR or a cell membrane fraction of the cell, (i) a test substance, a certain amount of agonist, GDP and a certain amount of GTP labeled with a radioactive element A) S, (ii) —quantitative agonist, GDP and a fixed amount of radioactively labeled GTPa S, ^) — quantitative anist, GDP, radioisotope Prepare a reaction mixture prepared by adding a certain amount of radioactive GTPaS labeled with and a large excess of unlabeled GTPaS, and react them. The reaction is carried out at O to 50 ° C, preferably 4 to 37 ° C, for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. (I) to (ii i) After reacting them, filter them with a glass fiber filter paper or the like, wash them with an appropriate amount of buffer, and measure the amount of radioactivity remaining on the glass fiber filter paper by liquid thinning force. Measure with a first-class radiation measurement device. (I) and (ii) the value obtained by subtracting the radioactivity (non-specific binding: NSB) of (i ii) from the radioactivity (total binding: B) of each (i) and (ii) It is calculated as the specific binding amount. '

By comparing the specific binding amount of GTPaS in the absence of the test substance in (ii) with the specific binding amount of GTPrS in the presence of the test substance in (i), the test substance in (i) Substances that reduce the amount of specific binding of GTPγS in the presence can be selected as antagonists for the selected GPCR. On the other hand, a substance that enhances the specific binding amount of GTPaS can be selected as an agonist for the selected GPCR. A similar measurement was performed by the above method without adding an agonist to the systems (i) to (iii), and the specific binding amount of GTPaS in the absence of the test substance of (ii) and the amount of (i) By comparing the specific binding amount of GTPγS in the presence of the test substance, selecting the substance that increases the specific binding amount of GTPaS in the presence of the test substance in (i) as the agonist to the selected GPCR can do.

 When the selected GPCR is a constitutively active GPCR, a similar measurement is performed by the above method without adding an agonist to the system of (i) to (iii), and the GTP in the absence of the test substance of (ii) is performed. The specific binding amount of GTPaS is reduced in the presence of the test substance (i) by comparing the specific binding amount of αTPS with the specific binding amount of GTPaS in the presence of the test substance (i). The substance to be selected can be selected as an antagonist for the selected GPCR. On the other hand, a substance that increases the amount of specific binding of GTPaS can be selected as an agonist for the selected GPCR. ― '

 In addition, for the selected substance, a similar assay using another GPCR-expressing cell or a cell membrane fraction of the cell instead of the selected GPCR-expressing cell or the cell membrane fraction of the cell is carried out. You can confirm whether the agonist is specific to the selected GPCR or an angist.

 (c) GTPase activity measurement method

 GTPase activity is measured using a GPCR-expressing cell selected as a screening target or a membrane fraction of the cell by a known method (Japanese Patent Laid-Open No. 2001-245666). Gonists, or functional agents, can be screened. For example, by contacting a test substance with a cell expressing the selected GPCR or a membrane fraction of the cell, and measuring the GTPase activity, an agonist of the GPCR can be searched.

 If the agonist of the GPCR is known, the agonist of the GPCR is contacted with the selected GPCR-expressing cell or the membrane fraction of the cell in the presence of the test substance, and the GTPase activity is measured. It is possible to search for an orangist or function modifier.

 When the GPCR is a constitutively active GPCR, the test substance is brought into contact with the selected GPCR-expressing cell or the membrane fraction of the cell, and the GTPase activity is measured, whereby the GPCR agonist, angelic gonist, or Function modifying substances can be searched for.

GTPase activity can be determined by the method described in the literature (J. Biol. Che., 271, 1857-1860 (1996), W098 / 46995). Can be measured by adding GTP labeled with a radioisotope as a substrate. The GTP labeling the § position phosphoric acid with a radioactive same position elements, labeled, for example, ³² P - can be used [§ ³² P] GTP. Specifically, a buffer containing a fixed amount of the cell expressing the selected GPCR or the membrane fraction of the cell,

(i) a test substance, a certain amount of agonist and a certain amount of radioactive GTP labeled with a radioactive isotope added to GTP, and (ii) a certain amount of agonist and a certain amount of phosphoric acid are radioisotope. Prepare and react with the addition of a certain amount of radioactive GTP labeled with. The reaction is carried out at 0 to 50 ° C; preferably at 4 to 37 ° C for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. (I) and

(ii) After reacting each, the supernatant of the reaction solution is collected by centrifugation, and the radioactivity of the inorganic phosphoric acid (Pi) released into the supernatant is measured with a radiation measuring device such as a liquid scintillation counter. Measure and use as GTPase activity. Alternatively, the reaction solution is filtered through a glass fiber filter paper and the like, washed with an appropriate amount of the same buffer, and the filtrate is collected. May be measured by the radiation measurement device described above. (Ii) in the absence of the test substance GTPase activity in the presence of the test substance in (i) can be compared with the GTPase activity of it can. On the other hand, a substance that increases GTPase activity can be selected as an agonist for the selected GPCR.

 A similar measurement was performed by the above method without adding an agonist to the systems (i) and (ii), and the GTPase activity in the absence of the test substance (ii) and the GTPase activity in the presence of the test substance (i) By comparing with the GTPase activity, a substance that increases the GTPase activity in the presence of the test substance (i) can be selected as an agonist for the selected GPCR.

 If the selected GPCR is a constitutively active GPCR, perform the same measurement as described above without adding agonist to the systems (i) and (ii), and perform the same measurement in the absence of the test substance in (ii). By comparing the GTPase activity with the GTPase activity in the presence of the test substance (i) .—, the substance that decreases the GTPase activity can be selected as an antagonist to the selected GPCR. On the other hand, a substance that increases GTPase activity can be selected as an agonist for the selected GPCR.

 In addition, for the selected substance, a similar assay using another GPCR-expressing cell or a cell membrane fraction of the cell instead of the selected GPCR-expressing cell or the cell membrane fraction of the cell is carried out. You can confirm whether the agonist is specific to the selected GPCR or an angist. Examples of human mast cells include mast cells derived from human peripheral blood, mast cells derived from human umbilical cord blood, mast cells prepared from human lung, skin, and tonsils, and human mast cell lines such as LAD2.

 (d) Function Atsushi

By using a GPCR-expressing cell selected as a screening target and conducting a functional analysis by a known method (Japanese Patent Application Laid-Open No. 2001-245666), the GPCR agonist, angelic gonist, or function g-modifying substance is screened. can do.

For example, a test substance is contacted with a cell expressing the selected GPCR, and the response of the cell via the GPCR (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP Production, inositol phosphate production, cell membrane potential fluctuations, phosphorylation of intracellular proteins, C-fos activation, pH reduction, cell growth activity, melanin pigment diffusion or diffusion, or reporter gene expression) By measuring, the agonist of the GPCR can be searched.

When a GPCR agonist is known, the agonist is brought into contact with a cell expressing the selected GPCR in the presence of a test substance, and the GPCR-mediated cell response (eg, arachidonic acid release, acetylcholine release, Intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, cell proliferation activity, melanin pigmentation, Or the spread, or the expression level of a reporter gene, etc.), it is possible to search for an agonist, an antagonist, or a function modifying substance of the GPCR.

When the GPCR is a constitutively active GPCR, a test substance is brought into contact with a cell expressing the selected GPCR, and the response of the cell via the GPCR (for example, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release) , Intracellular cMP production, Intracellular cGMP production, Inositol monophosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation, c-fos activation, pH decrease, Cell proliferation activity, Melanin color By measuring the aggregation or diffusion of the element, or the expression level of the repo overnight gene).

You can search for GPCR agonists, angel gonists, or functional modifiers. These GPCR-mediated cellular responses are described in J. Biol. Cem., 271, 1857 (1996), Science, 268> 98 (1995), J. Pharmacol. Exp. Ther., 275, 1274 (1995). ), J. Biol. Chem., 272, 1822 (1997), J. Recept. Signal Transduct. Res., 17, 57 (1997), Endocrinology 138, 1400 (1997) _N Endocrinology 138, 1471 (1997), Nat Biotec nol., 16, 1334 (1998), Biochem. Biophys. Res. Commun., 251, 71 (1998), Br. J. Pharmacol., 125, 1387 (1998), Trends Biotechnol., 15, 487 (1998). 1997), Anal.Biochem., 252, 115 (1997), Nature, 358, 325 (1992), Nature, 393> 272 (1998), Cell, 9> 573 (1998), J. Biol. Chera ,, 272. , 27497 (1997), rends Pharmacol. Sci., 18, 430 (1997) Trends Pharmacol. Sci., 20, 370 (1999), W098 / 46995.

When monitoring the response of cells using the repo overnight system, for example, an appropriate method is to place the selected GPCR in the expressed cell downstream of the promoter sequence of the gene whose expression is induced by the activation of the selected GPCR. By introducing DNA linked to a suitable reporter gene, the activity of the selected GPCR can be measured by the expression of the repo overnight gene. As the promoter, for example, a promoter for the ICAM-1 gene, a promoter for the c-fos, a promoter for Krox-24 [Biochem. J., 320, 145 (1996)], etc. can be used. . In addition, the promoter may be an artificial promoter consisting of an appropriate transcription factor binding sequence and a basic promoter. Is the binding sequence of the transcription factor, the transcription involves transfer due to activation of the GPCR to couple to the active I spoon to SRE (serum responsive element) or GPCR activity I spoon to couple to G _s active I spoon in CRE (cAMP responsive element) can be used. Furthermore, by expressing the C-terminal 5 amino acids of Ga _s the expressing cells GPCR selected the chimeric Ga _s substituted at the C-terminus 5 amino acids of G «i, utilization of the original G _s and coupled to GPCR Seii匕The expression of the repo overnight gene can also be induced from the artificial promoter containing CRE that activates the transcription along with the activation of the GPCR coupled with the CRE.

 As the repo overnight gene, any repo overnight gene can be used. For example, the holu le luciferase gene, the 夕 mi 夕 luciferase gene, the lucifera gene, and the chlora mufenico The cetyltransferase gene, 3-glucuronidase gene, β-galactosidase gene, / 5-lactamase gene, equorin gene and green fluorescein protein gene can be used.

Specifically, cells expressing the selected GPCR are cultured in a multiwell plate or the like. If necessary, replace with a fresh medium or an appropriate buffer that is not toxic to cells. Prepare and incubate for a certain time. After incubation, the cell response (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, cell proliferation activity, activity to promote or suppress melanin pigment or diffusion, or expression of reporter gene, etc.). When the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult to be assayed by a degrading enzyme contained in a cell, an inhibitor for the degrading enzyme may be added for quantification. .

In addition, for activities such as suppression of cAMP production, the amount of cAMP production of cells It can be detected as a production inhibitory effect on the cells that have been increased.

 By comparing the degree of cell response in the absence of the test substance in (ii) with the degree of cell response in the presence of the test substance in (i), the response of the cell in the presence of the test substance in (i) is compared. The substance to be reduced can be selected as an antagonist for the selected GPCR. On the other hand, substances that increase cell response can be selected as agonists for the selected GPCR.

 A similar measurement is performed by the above method without adding an agonist to the systems (i) and (ii), and the degree of cell response in the absence of the test substance in (ii) and the test substance in (i) By comparing the degree of cellular response in the presence, the substance that increases cellular response in the presence of the test substance (i) can be selected as an agonist to the selected GPCR.

 In the case where the selected GPCR is a constitutively active GPCR, the same measurement is carried out without adding agonist to the system of (ii) and (ii) in the above-described manner, and the cells of the cell in the absence of the test substance of (ii) The degree of response is compared with the degree of cell response in the presence of the test substance (i), and a substance that reduces the response of the cell in the presence of the test substance (i) is selected as an antagonist to the selected GPCR. can do. On the other hand, a substance that increases the response of the cell can be selected as an agonist for the selected GPCR.

 In addition, for the selected substance, a similar process is performed using a cell expressing another GPCR or a cell membrane fraction of the cell instead of a cell expressing the selected GPCR or a cell membrane fraction of the cell. By doing so, it is possible to confirm whether the selected GPCR is an agonist or an angist.

The selected GPCR-specific agonist, antagonist, or functional modifier obtained as described above suppresses activation, granule suppression, and inflammatory mediation of human mast cells. Having at least one of cytokine production inhibition and chemokine production inhibition has the effect, for example, that the obtained agonist, angelic gonist or functional modification, the substance is cultured by adding human IgE After addition to human mast cells and incubating them, the human mast cells were activated by the addition of anti-human IgE antibody, etc., and the released (i) hissumin or hexosaminida, which is an indicator of degranulation, was released. _{Ichize, (ii) LTC 4, LTD} 4, LTE 4, PGD 2 such as inflammatory Medeie Isseki of -, (ii i) TNF- and GM- CSF, etc. site force-in, (iv) IL - 8ヽ1 -309, MIP-1 etc. in CBlood 100, 3 861 (2002)], and can be confirmed by comparison with the case where no agonist, angelic gonist or function modifying substance was added. In addition, human obesity cells are supplemented with an agonist, angonist, or a functional modifier to induce apoptosis, and the induction of apoptosis is detected by measuring chromatin DNA fragmentation or TUNEL method. It can be confirmed that it has an inducing action. As human mast cells, mast cells derived from human peripheral blood, mast cells derived from human umbilical cord blood, mast cells prepared from human lung, skin, and tonsils, and cell lines derived from human mast cells such as LAD2 can be used.

(3) Pharmaceutical composition containing a GPCR agonist, angonist, or a function modifying substance expressed in human mast cells

The agonist, angonist, and functional modifier for GPCR expressed in human mast cells obtained by the above screening method are useful as a safe, low-toxicity pharmaceutical composition that controls the function of human mast cells. is there. When the compound obtained by the above-mentioned screening method or a salt thereof is used as the above-mentioned pharmaceutical composition, it can be formulated according to a conventional method. For example, tablets, capsules, elixirs, microcapsules, and the like, coated with sugar as needed, orally, or aseptic solution with water or other pharmaceutically acceptable solutions, or suspensions It can be used parenterally in the form of injections, such as suspensions. For example, a unit required for generally accepted pharmaceutical practice together with a carrier, a flavor, an excipient, a vehicle, a preservative, a stabilizer, a binder and the like, which is a physiologically acceptable carrier or a salt thereof. The amount of the active ingredient in these preparations which can be produced by mixing them in a dosage form is such that an appropriate dose in the specified range can be obtained. Additives that can be incorporated into tablets, capsules, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, cocoa oil or cherry, and the like. When the preparation unit form is a capsule, the above type of material may further contain a liquid carrier such as oil and fat. Sterile compositions for injection should be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. Can be.

 As an aqueous solution for injection, for example, physiological saline, isotonic solution containing pudose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. Various solubilizing agents, for example, alcohols (for example, ethanol), polyalcohols (for example, propylene glycol, polyethylene glycol), nonionic surfactants (for example, polysorbate 80 (TM), HCO -50). As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as penzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, proforce hydrochloride, etc.), stabilizers (eg, human serum albumin polyethylene glycol, etc.), storage Agents (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection solution is usually filled into a suitable ampoule. Because the preparations obtained in this way are safe and low-toxic, they can be administered, for example, to humans and mammals (eg, rats, egrets, sheep, pussies, dogs, cats, dogs, monkeys, etc.). can do. The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method and the like. However, in the case of oral administration, in general, for an adult (as 60 kg), the dose is about 0 per day. 1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, etc. It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 6 O.kg. (4) Preparation and use of antibodies specifically recognizing GPCR expressed in human mast cells

(4-1) Preparation of antibodies specifically recognizing GPCR expressed in human mast cells According to a known method (Japanese Patent Application Laid-Open No. 2001-245666), polyclonal antibodies and monoclonal antibodies that specifically recognize GPCR expressed in human mast cells can be produced. By measuring the effects of the obtained antibodies on human mast cell apoptosis, activation, degranulation, production of inflammatory mediators, production of cytodynamics, production of chemokines, etc., (i) apoptosis Induction, (ii) suppression of activity, (iii) suppression of degranulation, (iv) suppression of production of inflammatory media-Yuichi, (suppression of production of cytodynamics, (vi) suppression of production of chemokines Similarly, an antibody having at least one action can be selected and obtained, and a polyclonal specifically recognizing a GPCR expressed in human mast cells and whose expression level fluctuates due to stimulation of dalcocorticoid. By measuring the effect of the obtained antibody on the action of glucocorticoid, glucococide can be produced through the GPCR. Antibodies having an effect of controlling the action of the ruticoid can be selected.

 (4-2) Use of antibodies that recognize GPCRs expressed in human mast cells

 (a) Immunological detection and quantification of GPCR expressed in human mast cells using an antibody that recognizes GPCR expressed in human mast cells

 Immunological methods for detecting GPCRs expressed in human mast cells include ELISA using a microtiter plate, fluorescent antibody method, western protocol, and immunohistochemical staining [Experimental Medicine, The Protocol Series, immunostaining and in situ hybridization, Yodosha (1997); J. Immunol. Methods, 150> 5, (1992)].

The immunological assay, Sandidzuchi ELISA method Epito one-flop of the antibodies were used two different monoclonal antibodies reactive with GPCR expressed in human mast cells in the liquid phase, with a radioisotope such as ^m I A radioimmunoassay method using a GPCR expressed in labeled human mast cells and an antibody recognizing the GPCR can be exemplified.

 The above detection or quantification method can be used for detection of human mast cells and diagnosis of diseases involving mast cells. In addition, cells or cells that express GPCR expressed in human mast cells can be identified using the above detection or quantification method. Cells and cell lines that express the GPCR expressed in human mast cells are useful for searching for the ligand, agonist or antagonist of the GPCR, and for analyzing the function of the GPCR.

 (b) Isolation of human mast cells using an antibody that recognizes GPCR expressed in human mast cells Known method [J. Immunol. Methods 169> 153 (1994); J. Allergy Clin. Immunol., 107, 322 ( 2001)], mast cells can be prepared with high purity from various human tissues using an antibody that recognizes GPCR expressed in human mast cells.

 (c) a drug containing an antibody that recognizes a GPCR expressed in human mast cells

Specifically recognizes the GPCR expressed in human mast cells obtained in (4-1), and (i) induces apoptosis, (ii) suppresses activation, (iii) suppresses degranulation, (iv ) Inflammatory media-suppression of production in the evening, (V) suppression of production of cytodynamics, (vi) antibodies having at least one action of suppressing production of chemokines are drugs, such as atopic dermatitis, asthma, It can be used as a therapeutic agent for chronic obstructive pulmonary disease or allergic disease. The antibody has an apoptosis-inducing effect on mast cells, an inhibitory effect on human mast cell activation, an inhibitory effect on human mast cell degranulation, an inhibitory effect on human mast cell inflammatory mediator production, and an inhibitory effect on human mast cells. Cytokine It can also be used as a drug having a production inhibitory action or a chemokine production inhibitory action on human mast cells. It has the effect of specifically recognizing GPCRs expressed in human mast cells and whose expression levels fluctuate due to glucocorticoid stimulation, and controlling the action of glucocorticoids, obtained in (4-1) The antibody can be used as a drug for controlling the action of dalcocorticoid.

 A drug containing an antibody that specifically recognizes a GPCR expressed in human mast cells can be administered alone as a therapeutic agent, but it is usually one or more pharmacologically acceptable. It may be admixed with more carriers to provide a pharmaceutical preparation prepared by any of the methods well-known in the art of pharmacy.

 It is desirable to use the most effective route for treatment, including oral administration, and parenteral administration such as buccal, respiratory, rectal, subcutaneous, intramuscular, and intravenous administration. . Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

 Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. For example, liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, soybean oil and the like. And preservatives such as p-hydroxybenzoic acid esters, and flavors such as peridotized flavor and peppermint as additives. Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl, etc. It can be manufactured using a binder such as alcohol, hydroxypropylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.

 Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. For example, an injection is prepared using a carrier comprising a salt solution, a pudose solution, or a mixture of both. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. A spray is prepared using the conjugate or the carrier which does not irritate the oral cavity and respiratory tract mucosa of the recipient and disperses the compound as fine particles to facilitate absorption. Specific examples of the carrier include lactose and glycerin. Preparations such as aerosols and dry powders are possible depending on the properties of the compound and the carrier used. Also, in these parenteral preparations, the components exemplified as additives in oral preparations can be added.

 The dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 mg / kg to 8 mg / kg per adult per day.

 (5) Method for suppressing the expression of GPCR gene expressed in human mast cells

Antisense RNA / DNA technology [Bioscience and India, 50, 322 (1992); Chemistry, 6> 681 (1991); Biotechnology, 9, 358 (1992); Trends Biotechnol., 10, 87 (1992); Trends Biotechnol., 10, 152 (1992); Cell engineering, 16, 1463 (1997)), Triple helix technology Trends Biotechnol., 10, 132 (1992)), Ribozyme technology (Curr. Opin. Chem. Biol., 3, 274 (1999); FEMS Microbiol. Rev ,, 23, 257 (1999); Front.Biosci., 4, D497 (1999); Chem, Biol., 6, R33 (1999); Nucleic Acids Res., 6, > 5237 (1998); Trends Biotechnol., 16, 438 (1998)], decoy DNA method [Japanese clinical, 56, 563 (1998); Circ. Res., 82, 1023 (1998); Exp. Nephrol., 5, 429 (1997); Japan Clinical, 54> 2583 (1996)], or the expression of any gene can be suppressed using siRNA (short interfering RNA).

 The siRNA is a short double-stranded RNA containing a partial sequence of the mRNA of a certain target gene, and can be expressed by RNA interference (RNAi) to suppress the expression of the gene. The sequence of siHNA can be appropriately designed based on the conditions of the literature [Genes Dev., 13, 3191 (1999)] from the base sequence of the mRNA of the target GPCR. Preferably, in the region 50 to 100 bases downstream from the start codon in the mRNA translation region, a sequence having 19 bases following M and a GC content of 30 to 70%, more preferably around 50%, is selected. The selected 19-base sequence or a complementary sequence, and two RNAs each having a sequence with TT or UU added to the 3 'end of the selected sequence are synthesized by DNA synthesis and annealed to produce an siRNA. Further, by inserting a DNA corresponding to the selected 19-base sequence into a vector for siRNA expression such as pSilencer 1.0-U6 (manufactured by Ambion), SUPER (OligoEngine), etc. It is possible to produce a vector expressing an siRNA capable of suppressing the expression.

 For example, it is possible to suppress the expression of a GPCR gene expressed in human mast cells using an antisense DNA or siRNA specific for a GPCR gene expressed in human mast cells. That is, by administering antisense DNA or RNAi specific to the GPCR gene, the production of the GPCR can be suppressed, and the function of human mast cells can be controlled.

 For example, if there is a patient whose receptor-mediated physiological action is enhanced due to an increased expression of a GPCR expressed in human mast cells or an increased expression of a ligand of the GPCR, an antisense specific for the GPCR gene By administering DNA or siRNA to a patient, the function of human mast cells can be controlled and a disease can be treated. That is, the antisense DNA or siRNA specific for the GPCR gene is useful as a therapeutic agent for diseases involving human mast cells.

 When an antisense DNA or siRNA specific to the GPCR gene is used as the therapeutic agent, the antisense DNA or siRNA may be used alone, or a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like. After insertion into the appropriate vector, the drug can be formulated, prescribed and administered according to the usual method described in (2) above.

 The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. BEST MODE FOR CARRYING OUT THE INVENTION

Example 1 Analysis of GP expressed when human cord blood-derived mast cells were cultured in each case

 (1) Preparation of human cord blood-derived mast cells

 Based on a known method [Blood, 111, 1016 (2001); Blood, 98. 1127 (2001); Blood, Michi, 3861 (2002)] from human cord blood obtained based on informed consent, Thus, human mast cells were prepared.

After obtaining CD34-positive mononuclear cells, which are precursor cells of mast cells, from human umbilical cord blood using a CD34 + isolation kit (Mi Uenyi Biotec), 100 ng / ml SCF, 50 Iscove's modified Dulbecco's medium (IMDM) containing ng / ml IL-6 and 5% fetal calf serum (hereinafter abbreviated as FCS) Cells were thread-swallowed for 18 weeks.

 (2) Preparation of mast cells treated with IgE receptor-mediated activation and glucocorticoid treatment

The cultured mast cells derived from human umbilical cord blood prepared in (1) above were cultured at 37 ° C. for 24 hours in the presence of l〃g / ml human myeloma IgE (Cosmo Bio). Next, after washing the cells, the cells were cultured at 37 ° C for 6 hours in the presence or absence of 1.5 µg / ml of rabbit ego anti-human IgE antibody (manufactured by Dako). In cells treated with human myeloma IgE and egret anti-human IgE antibody, an activation signal flows through the IgE receptor. These cells are called IgE-treated cells. Activation signals do not flow in cells treated with human myeloma IgE alone, so use these cells as controls. These φ cells are called control cells. Further, prior to treatment with Hitomiero Ichima IgE and Usagi anti-human IgE antibody, cells cultured for 48 hours in the presence of dexamethasone glucocorticoids Koi 10- ⁶ mol / 1 as de was also prepared at the same time. These cells are called IgE + Dex-treated cells. For culture, IMDM containing 100 ng / ml SCF, 50 ng / ml IL-6, and 5% FCS was used.

(3) Expression analysis using gene chip

 Analysis of GPCR expressed in mast cells cultured and prepared under the above conditions (2) using Affimetrics Genechip (human genome U133A probe array and human 'genome U133B probe' array). Was.

 The method of angular analysis is described in the protocol of Affymetrix Inc. (Expression Technical Technical Manual) and literature [Blood, 97, 1016 (2001), Blood, 98> 1127 (2001)] Blood, 100 , 3861 (2002)]. Hereinafter, the method is briefly described.

 Total RNA was obtained from the mast cells cultured and prepared under various conditions in the above (2) using an RNeggi 'mini' kit (RNeasy Mini Kits, Qiagen Iagen). Using a Superscript Choice system (Superscript Choice system manufactured by Invitrogen) and 7- (dT) 24 Primer (Amersham Biosciences), double-stranded cDNA was synthesized from the above RNA. Prepared. Using a BioArray High Yield RNA Transcript Labeling Kit [BioArray High-yield RNA Transcript Labeling Kit. Enzo Diagnostics, Inc.], biotinylated cRNA was prepared from the above cDNA. The biotinylated 'cRNA was hybridized with a human genomic U133A probe array and a human' genomic U133B pro 'array at 45 ° C for 16 hours. After washing the array, the hybridized cRNA was stained with streptavidin-phycoerythrin (streptavidin-phycoeirthrin, manufactured by Molecular Probes) to obtain a hybrid cell line. The measurement was carried out using an array scanner (Hewlett-Packard Gene Array Scanner).

The fluorescence intensity of each probe was quantified by using the software “GeneChip Analysis Suite” 5.0 manufactured by Affymetrix. Hereinafter, the expression levels of each gene were determined according to the algorithm of this software. The fluorescence intensity of each of the 10 probes corresponding to each gene and the fluorescence intensity of the corresponding single-base mismatch probe were measured, and the AD (average difference) value (fluorescence of each probe) was determined as the expression level of each gene. (The average value of the difference between the fluorescence intensity and the fluorescence intensity of the single base mismatch probe). The AD value of each gene was determined to be “Present” if it was determined to be expressed, and “Merginal” if it was determined to be unclear or not, based on the data analysis specifications of Affymetrix. Those that were determined not to have occurred had a flag of “Absent”. Further data analysis was performed using software GeneSpring 'version 5.1 (GeneSpring version 5.1, silicon' Silicon Genetics').

 When comparing the expression levels of each gene among control cells, IgE-treated cells, and IgE-Dex-treated cells, the AD values obtained above were calculated for all genes to correct the variance in signal intensity for each chip. Values were corrected for the median value of the chip. For each gene, calculate the ratio of the expression level in IgE-treated cells or IgE-Dex-treated cells to the control cells (relative expression level relative to control cells as 1), using the corrected AD value as the expression level did. Each gene was judged to be “expressed” when the value of the AD value was 100 or more and the flag was “Present”. Genes determined to be “expressed” in any of control cells, IgE-treated cells, and IgE-Dex-treated cells were defined as genes expressed in human mast cells. As a result of the above analysis, the names and abbreviations of GPCRs that were expressed in cultured mast cells derived from human umbilical cord blood, the registration number of the NCBI (National Center for Biotechnology Information) protein database of the amino acid sequence of the protein, and the nucleotide sequence of the cDNA Accession number based on NCBI nucleotide sequence data, Affymetrix's probe ID, which gene on the gene chip (A: human genome U133A probe 'array, B: human' genome U133B probe 'Array) is shown in Table 1. For the NCBI protein database and nucleotide sequence database, refer to the NCBI Inuichi Net site (http: 〃 丽 .ncbi.nlm.nih.gov /).

Table 2 shows the results of these GPCR analyses. Each GPCR gene was abbreviated in Table 1, and the AD value before median value correction was shown as the gene expression level. The “F” column is a flag of the AD value, where P is “Present”, M is “Merginal”, and A is “Absent”. The column “Ratio” shows the ratio of the expression level to the control cells. Genes that were found to be expressed in human mast cells for the first time by the present invention are indicated by * in the column of “expression”. In addition, when the ratio of the expression level to the control cells in the IgE-treated cells is 1.8 or more or 0.5 or less, and it is determined that the expression is at least one of the control cells and the IgE-treated cells In addition, it was judged that the gene fluctuated due to the IgE treatment. Calculate the relative value (B / A) of the expression ratio (B) of the IgE-Dex treated cells assuming that the expression ratio (A) of the IgE-treated cells is 1; If it is determined that the expression is expressed in at least one of the IgE-treated cells and the IgE-Dex-treated cells, it is judged that the expression is fluctuated by glucocorticoid treatment, In the column of, if the value is 1.8 or more, it is indicated by “individual”. If it is 0.5 or less, it is indicated by “”. Table 1

 GPCR name Abbreviation Nucleotide sequence Amino acid sequence Probe ID H Complement third component receptor 1 C3aRl 丽 — 004054 Ρ-1 004045 209906— at A

G protein cleft type receptor 91 GPR91 —033050 ΝΡ 一 149039 223939— at B

G protein cryotype receptor 34 GPR34 NM— 005300 Ρ— 005291 223620—at B

Mucin-like hormone receptor containing EGF-like module

 ΤΓ Ρ9 images Π1 ^ Α Ί u l 〖Λ 〖Condition array 2

 ? 2 Adrenergic receptor β ΐ NMJ00024 P_000015 206170— at A Platelet activating factor receptor PAFR ΝΜ— 000952 P— 000943 211661_x_at A Adenosine A2b receptor A2b Μ— 000676 P— 000667 205891—at A

G protein ¾f-shape receptor 105 GPR105 丽 —014879 NP— 055694 206637-1 at A

7-times transmembrane super family 1 member 1 IM7SF1 thigh—003272 NP-1 003263 204137—at 'A

G protein toxin receptor 37 GPR37 U87460 AAC51281 209630 1 s-1 at A

CD97 antigen CD97 丽 — 001784 NP-1 001775 202910— s-1 at A Melanocortin 1 receptor MCI MJ02386 NP— 002377 205458-1 at A

G protein-coupled receptor FIRE FIRE Project 700 SEQ ID NO: 47 234675— x— at B Formyl peptide receptor 1 fMLPl NM— 002029 NP— 002020 205119—s—at A

G protein ^ 1 receptor type 65 GPR65 丽 — 003608 NP— 003599 214467— at A Pros evening glandin E receptor 3 EP3

 D38300 BAA07419 210374—x—at A. Alternative variant h varaint h

Purine receptor P2Y8 P2Y8 丽 — 178129 NP-1 835 230 229686—at. B Prostaglandin E receptor 3 EP3. A Oru Natipuno, 'Liant f variant f

 Dopamine receptor D2 D2 S69899 AAB20571 216938 1 x— at A Endothelial differentiation lysophosphatidic acid 丽 nm m MP ΠΠ1 Q9 ώυ¾ b d

G protein ephemeral receptor 2

 Dopamine receptor D2 D2 ■ —000795 NP— 000786 206590 1 x—at A adenosine A3 receptor A3 丽 —000677 NPJ00668 206171— at A complement fifth component receptor 1 C5R1 NM— 001736 NP— 001727 220088—at A Opsin 3 0PN3 丽 —014322 NP— 055137 219032— x—at A

G protein cinch receptor 27 · GPR27 MJ18971 NP-1 061844 221306—at A

G protein cleft receptor 44 CRTH2 ME04778 NP— 004769 206361— at A Endothelial differentiation sphingolipid

 , υ υ υ ichi

G protein type receptor 1

Phonoremil peptide receptor ft¾2 FPRL2 MJ02030 NP— 002021 214560—at A Pros evening glandin E receptor 2 EP2 MJ00956 NPJ00947 206631—at A

Mucin-like hormone receptor containing EGF-like module

 EMR2 Marauder— 013447 NP— 038475 232009—at B Volume 2

Chemokine (CC motif) receptor-like 2 CCRL2 NMJ03965 NP— 003956 211434—s-at A

6

Table 2 (continued)

Of the GPCRs listed in Tables 1 and 2, CysLTRl, GPR91, GPR34, EMR2, PAFR, GPR105, TM7SF1, GPR37, CD97, MC1, FIRE ヽ fMLPl, GPR65, EP3 variant h, P2Y8, D2, EDG2, A3, C5R1 ヽ 0PN3, GPR27, CRTH2, EDG1 ヽ FPRL2, CCRL2, mglu6, CB2, P2Y5, P2Y4, EP3 variant j, A2a, ETB, CELSR2, H4, Hl, CELSRl ヽ GPR85, HE 6, CELSR3, CALCRL ヽ RRH , EP4, CCR1 and D5 are the first GPCRs to be expressed in human mast cells. By comparing the expression levels in control cells and IgE-treated cells, GPCRs whose expression fluctuated when human mast cells were activated via the IgE receptor were identified. As a result, it has become clear for the first time that the expression of GPR34, GP fraction, and P2Y8 EDG1 EP3 variant j A2a ETB H4 RE2 and EP4 fluctuates due to activation via the IgE receptor. GPCRs whose expression fluctuates in IgE-treated cells are presumed to be involved in the control of mast cell activation, and those GPCRs are particularly useful as targets for therapeutic drugs for diseases involving mast cells. By comparing the expression levels in IgE-treated cells and IgE + Dex-treated cells, we identified GPCRs whose expression fluctuated when treated with dexamethasone. As a result, the expression of C3aRl, GPR34, ^ 2, GPR105, M7SF, fMLPl, P2Y8 _S A3, CRTH2, CCRL2, P2Y5, A2a, EBI2, PARI. Was first revealed this time. Glucocorticoid exerts anti-inflammatory effects, but also causes side effects. GPCRs whose expression fluctuates upon treatment with dexamethasone are likely to be involved in the efficacy or side effects of gusolecocorticoid, and those GPCRs are useful as targets for drugs that control the efficacy or side effects of glucocorticoid It is. Example 2 Analysis of GPCR expressed when human's peripheral blood-derived mast cells were cultured in various cases

(1) Preparation of mast cells derived from human peripheral blood

 Human peripheral blood-derived mast cells were prepared by a known method [Blood, 97, 1016 (2001)]. Human peripheral blood was obtained based on informative consent. The method is briefly described below.

 After fractionating malignant progenitor cell lineage negative human sphere cells from human peripheral blood, a serum-free, Iscov methylcellulose medium (manufactured by Stem Cell Technologies Inc.) The cells were cultured using IMDM containing 200 ng / ml SCF, 50 ng / ml IL-6, and 1 ng / ml IL-3. On day 42 of the culture, the cells were recovered by dissolving methylcellulose, and then subcultured into IMDM containing 100 ng / ml SCF, 50 ng / ml IL-6, and 5% FCS.

 (2) Preparation of mast cells activated by IgE receptor

 The cultured mast cells derived from human peripheral blood prepared in (1) above were cultured in the presence of human myeloma IgE in the same manner as in (2) of Example 1, and then in the presence or absence of a heron anti-human IgE antibody. And cultured. Cells treated with human myeloma IgE and Egret anti-human IgE antibody are called IgE-treated cells, and cells treated with only human IgE are called control cells.

 (3) Expression analysis using gene chip

Using a gene chip (human genome U133A probe array), GPCR expressed in the enriched cells prepared in (2) above was analyzed. The method described in Example 1 (3) was used. Each gene was judged to be “expressed” when the AD value was 100 or more and the flag was “Present”. Genes determined to be “expressed” in control cells or IgE-treated cells were defined as genes expressed in human mast cells. Names and abbreviations of GPCRs that were expressed in human peripheral blood-derived cultured enriched cells. Table 3 shows the probe IDs of TRIX. Table 4 shows the analysis results of these GPCRs. Each GPCR gene is indicated by the abbreviation in Table 3, and the AD value before correction by the median value is shown as the gene expression level. The “F” column is a flag of the AD value, P is “Present”, M is “Merginal”, and A is “Absent”. The "Ratio" column shows the ratio of the expression level to the control cells. Genes that were first revealed by the present invention to be expressed in human mast cells are indicated by * in the column of “expression”. In addition, the ratio of the expression amount of the IgE-treated cells to the control cells is 1.8 or more and 0.5 or less, and the control cells and the IgE-treated cells If the expression is judged to be "expressed" in at least one of the above, it is judged to be a gene whose expression fluctuates due to IgE treatment. Is indicated by "". O

GPCR name Abbreviation Nucleotide sequence Amino acid sequence Probe ID Complement 3rd component receptor 1 C3aRl Hire—004054 PJ04045 209906-1 at

EGF-like module containing mucin-like hormone receptor-like

 EMR2 丽 — 013447 PJ38475 207610 1 s—at Sequence 2

 ? 2 adrenergic receptor βΐ MM— 000024 MP— 000015 206170— at Platelet activating factor receptor PAFR M— 000952 NP— 000943 211661 1 x— at Adenosine A receptor A2b 丽 ichi 000676 PJ00667 205891— at

G protein cinch receptor 105 GPR105 丽 — 014879 NP— 055694 206637—at

7th super penetration family member 1 TM7SF1 M— 003272 NP— 003263 2041371 at

G protein C-type receptor 37 GPR37 U87460 AAC51281 209630—s—at

CD97 antigen CD97 丽 — 001784 NP— 001775 202910— s— at Melanoconoretin 1 receptor MCI MJ02386 NP— 002377 205458— at

G protein tongue ^ "concept 65 GPR65 NM_003608 NP 003599 214467— at Pros evening glandin Ε receptor 3 EP3

 D38300 BAA07419 210374— x— at Oru Natipari Pariant h varaint h

 Pros evening glandin E receptor 3 EP3

 D38298 AAC51281 2l0832_x_at Oriental Native Pariant f variant f

 Dopamine receptor D2 D2 S69899 AAB20571 216938-1 x— at endothelial differentiation lysophosphatidic acid G protein-coupled receptor

 EDG2 Kouichi 001401 NP— 001392 204038—s1 at condition 2

 Dopamine receptor D 2 D2 NM— 000795 NP— 000786 206590_x_at Fifth-component complement receptor 1 C5R1 M— 001736 NPJ01727 220088—at Adenosine A3 receptor A3 Thigh—000677 NP—000668 206171—at Psin 3 0PN3 NM — 014322 PJ55137 219032— x— at

G protein-coupled receptor 27 GPR27 MJ18971 NP— 061844 221306_at Endothelial differentiation sphingolipid G protein T-shaped receptor 1 EDG1 丽 —001400 NPJ01391 204642 at pros evening glandin E receptor 2 EP2 thigh—000956 PJ00947 206631 at chemokine (CC Motif) Receptor-like 2 CCRL2 MJ03965 PJ03956 211434-1 s- at male botropic glutamate receptor 6 mgl 6 P-000843 NP- 000834 208035 at

Table 4 (continued)

Of the GPCRs listed in Tables 3 and 4, EMR2, PAF _S GPR105, TM7SF1, GPR37, CD97, MCI ヽ GP 65 _N EP3 variant h, D2, EDG2, A3, C5R1, 0PN3, GPR27, EDG1 ヽ CCRL2 , mglu6, CB2, GP 39, Alヽ_{P2Y4, EP3 variant j s A2a,} ETBヽCELSR2, TEM5, GP 58, motilin R, EP4, CCR1, D5, LTB4R and CXCR5 are to be expressed in human mast cells This is the first GPCR that was revealed this time. As described in Example 1 (3), GPCR expressed in human mast cells is useful as a target for a therapeutic drug for diseases involving mast cells.

By comparing the expression levels in control cells and IgE-treated cells, GPCRs whose expression fluctuated when human mast cells were activated via the IgE receptor were identified. As a result, C3aRl, ^ 2, PAFR _S GPR105 _S TM7SF1, GPR65, EDG2, A3, C5R1 _S EDG1, GPR39, A2a ヽ ETB ヽ RE2 _N CELSR2 _S TEM5, EP4 and LTB4R are activated by IgE receptor-mediated activation. For the first time, it was revealed that the expression fluctuated. GPCRs whose expression fluctuates in IgE-treated cells are presumed to be involved in the control of mast cell activation. is there. () Expression analysis by quantitative RT-PCR

 Expression analysis using a gene chip may not be able to detect the expression of GPCRs with low expression levels. Therefore, expression analysis using quantitative RT-PCR was also performed. In addition, expression analysis using quantitative RT-PCR was performed on GPCRs without a probe on the gene chip. Table 5 shows the names and abbreviations of the GPCRs whose expression was analyzed by RT-PCR, the NCBI protein data of the amino acid sequence of the protein, the registration number of the NCBS, and the NCBI nucleotide sequence data of the cDNA base sequence. The accession number and the sequence number of the gene-specific primer are shown.

 Table 5

From the mast cells prepared in (2) above, DNasel treatment was performed using RNeasy Mini's kit (manufactured by Qiagen) to obtain total RNA, and then the first strand synthesis system for superscript RT-PCR ( (Invitrogen) was used to synthesize single-stranded cDNA. The procedure was performed according to the kit instructions. O'Rigo (dT) primer was used as the primer. All Alg Was diluted with water to a total volume of 2.1 ml. For the following quantitative RT-PCR analysis, a 10-fold diluted solution was used (10-1).

Quantitative RT-PCR was performed as follows. As a reagent, a kit manufactured by Takara Shuzo (For Real Time PCR TaKaRa Ex Taq R-PCR Version) was used. 10 l of the above single-stranded cDNA, 2.0 1 of 10 XR-PCR buffer (Mg free), 0.21 of 250 mM Mg ²⁺ solution ヽ 0.6 zl of dNTPs (10 t ol), 1.0〃1 of SYBR Green I After preparing a reaction mixture consisting of 1.5 〃1 of gene-specific primer (4 〃mol / l), 0.2 〃1 of ExTaq R-PCR Version, and 3 〃1 of water, PCR was performed using an ABI sequence detection system (ABI PRISM 7700, manufactured by Applied Biosystems). As the gene-specific primer, DNA consisting of the sequences of SEQ ID NOs: 1 to 46 shown in Table 4 was used. The reaction conditions are as follows: First, heat at 94 ° C for 5 minutes, repeat 45 cycles of 94 ° C for 30 seconds, 65 ° C for 1 minute, 72 ° C for 30 seconds, and finally repeat at 25 ° C for 2 seconds. The conditions for heating for a minute were used.

The expression level was represented by the number of cycles (Ct value) when the signal intensity after background correction reached 500. As a negative control, PCR using sterile water instead of single-stranded cDNA was also performed. After completion of the reaction, 5 μl was taken from the obtained PCR reaction solution, and 1.5% agarose gel [SeaKem GTG agarose (manufactured by CAMBREX) was used in Tris-acetate buffer ( Dissolved in 40 t ol / l tris-acetic acid, 1 liter of octaethyl ethylenediamine tetraacetic acid). The gel was stained for 30 minutes with a 1 / 10,000 dilution of SYBR Green, and the expected amplification of the DNA fragment was confirmed using FluorImager ™ (manufactured by Molecular Dynamics). When only the expected DNA fragment is amplified when using single-stranded cDNA, the fragment is not amplified when using sterilized water, and the Ct value when using single-stranded cDNA is 39 or less. Was determined to have gene expression. The results are shown in Table 6. For those with a Ct value of 39 or less when using sterile water, it was confirmed that the Ct value was lowered due to the amplification of the fragment resulting from primer-dimerization.

Table 6

As a result of analysis, GPR18 _S GPR35, P2Y10, C-TESTI2027296, 'GPR141, hRUP16, ITR, MRGX2, SNORF33, C-THYMU2011548,' G10d, GPR107, GPR126, GPR41, GPR43, GPR68, GRPR ヽ HEOAD54, hRUP9, mPRa It has now become clear for the first time that mPRg, SRL and H963 are expressed in cultured mast cells derived from human peripheral blood. Expression of GPR18, P2Y10, GPR16, GPR41, GPR43, GPR68, and H963 could not be detected by GeneChip, but expression was detected by RT-PCR analysis. GPCRs expressed in these human mast cells may be useful as targets for therapeutics for diseases involving mast cells.

 By comparing the expression level between control cells and IgE-treated cells, GPCH whose expression fluctuates when human mast cells are activated via the IgE receptor can be identified.

For the first time, it has been revealed that the expression of C-TESTI2027296, GPR14 ITR, MRGX2, G10d, GPR43, GRPR ヽ HEOAD54 and H963 fluctuates (Ct value fluctuates by 1 or more) due to IgE receptor-mediated activation. It has become. GPCRs whose expression fluctuates in IgE-treated cells are involved in the control of mast cell activation Thus, these GPCRs are particularly useful as targets for therapeutics for diseases involving mast cells. Example 3 Analysis of GPCRs Expressed When Human Peripheral Blood-Derived Mast Cells are Cultured in Each Case (2) (1) Preparation of mast cells using various culture conditions (activation of mast cells via LPS receptor) The cultured human mast cells derived from peripheral blood prepared by the method described in (1) of Example 2 were cultured at 37 ° C in the presence or absence of 30 ng / inl of IFN-α (R & D Systems). Cultured for 24 hours. Next, after washing the cells, the cells were cultured at 37 ° C for 2 hours or 6 hours in the presence or absence of 100 ng / ml LPS (derived from Salmonella typhi murium, manufactured by Sigma-Aldrich). For culture, IMDM containing 100 ng / ml SCF, 50 ng / ml IL-6, and 5% FCS was used.

 Cells treated only with IFN-α were treated with IFN-α, cells treated with IFN-α and LPS were treated with IFN-α + LPS, cells treated with LPS alone were treated with LPS, cells treated with IFN-α and LPS. Untreated cells are called control cells.

 (2) Expression analysis using jumpers

 GPCR expressed in the mast cells prepared in (1) above was analyzed using a gene chip (human 'genomic U133A protein' array). The method described in Example 1 (3) was used.

Each gene was judged to be “expressed” when the AD value was 100 or more and the flag was “Present”. Genes determined to be “expressed” in any of control cells, IFN-a-treated cells, LPS-treated cells and IFN-a + LPS-treated cells were defined as genes expressed in human mast cells. Names and abbreviations of GPCRs that were expressed in cultured mast cells derived from human peripheral blood, the registration number of the NCBI protein database of the amino acid sequence of the protein, the registration number of the NCBI nucleotide sequence of the cDNA base sequence, and the affinity number Table 7 shows PIX's probe IDs. Table 8 shows the results obtained by culturing these GPCRs for 2 hours, and Table 9 shows the results obtained by culturing them for 6 hours. Each GPCR gene is indicated by the abbreviation in Table 7, and the AD value before correction with the median value is shown as the gene expression level. The column of “F” is a flag of AD value, P is “Present”, M is “Merginal”, and A is “Absent”. The column “Ratio” indicates the ratio of the expression level to the control cells. Genes that were first revealed by the present invention to be expressed in human mast cells are indicated by * in the “expression” column. Further, the ratio of the expression level to the control cell in each drug-treated cell is 1.8 or more and 0.5 or less, and "expressed" in at least one of the control cell and the drug-treated cell. When judged, the gene was judged to be a gene whose expression fluctuates due to the treatment with the drug. In the column of “fluctuation”, “1.8” or more was indicated by “individual” and 0.5 or less was indicated by “”. Table 7

 GPCR name Abbreviation Base sequence Amino acid sequence Probe ID Complement third component receptor 1 C3aRl 丽 — 004054 NPJ04045 209906_at

Mucin-like hormone receptor containing EGF-like module

 EMR2 page 013447 NPJ38475 207610—s—at-like sequence 2

 '? 2 Adrenergic receptor β— NM— 000024 NPJ00015 206170— at Platelet activating factor receptor PAF NMJ00952 NPJ00943 211661— x— at Adenosine A2b receptor A2b NMJ00676 NPJ00667 205891— at

G protein-coupled receptor 105 GPR105 NMJ14879 NPJ55694 206637—at

7 times super penetration family member 1 TM7SF1 M— 003272 NP— 003263 2041371 at

G protein-coupled receptor 37 GPR37 U87460 AAC51281 209630_s_at

CD97¾¾ '-CD97 NM— 001784 NPJ01775 202910 s— at melanocortin 1 receptor MCI M— 002386 NP— 002377 205458 at

Gl ^ white cylindrical receptor 65 GPR65 NM— 003608 NP— 003599 214467-1 at Pros evening glandin E receptor 3 EP3

 D38300 BAA07419 210374 ichi x— at Orevena Native Pariant h varaint

 Pros evening Gran E receptor 3 EP3

 D38298 'AAC51281 210832— x one at Oru Yunative Pariant f variant f

 Dopamine receptor D2 D2 S69899 AAB20571 216938— x— at lysophosphatidic acid

 EDG2 M— 001401 ML001392 204038— s—at G protein ^ = receptor type 2

 Dominamine receptor D2 D2 M— 000795 PJ00786 206590 1 x— at Adenosine A3 receptor A3 P 000677 NPJ00668 206171— at Complement component 5 receptor 1 C5R1 NM— 001736 NPJ01727 220088— at Opsin 30PN3 hiring— 014322 NPJ55137 219032— x— at

G protein cinch receptor 27 GPR27 KM— 018971 PJ61844 221306—at

G protein street receptor 44 CRTH2 丽 — 004778 NPJ04769 206361— at endothelial differentiation sphingolipid

 EDG1 NM— 001 willow NPJ01391 204641 at G protein

Pros evening glandin E receptor 2 EP2 M— 000956 PJ00947 206631—at Chemokine (C-C motif) receptor-like 2 CCRL2 NM— 003965 NPJ03956 211434— s—at NPJ00834 208035—at Table 7 (continued)

 GPCR name Abbreviation Base sequence Amino acid sequence Probe ID Cannabinoid receptor 2 CB2 MJ01841 PJ01832 206586—at

G protein receptor 39 GPR39 M— 001508 PJ01499 212909 at purine receptor P2Y5 P2Y5 NMJ05767 PJ05758 218589 at at adenosine A1 receptor A1 NMJ00674 NPJ00665 216220— s at pyrimidine receptor P2Y4 P2Y4 MJ02565 PJ02556 3 · EP3 L27489 AAC133731 210831— s—at Oriental Pariant 0 varinat j

Adenosine A2a receptor A2a NMJ00675 NP 000666 205013—s—at

Type 1 angiotensin Π receptor AT1B PJ00676 208016 s at

B-type endothelin receptor ETB MJ00115 PJ00106 204273— at gonadotropin-releasing hormone receptor GNRHR PJ00397 216341—s— at cadherin EGF LAG 7 transmembrane receptors 2 CELSR2 NM 001408 PJ01399 204029— at chemokine (CC motif) receptor 7 CCR7 NMJ01838 PJ01829 206337— at Thrombin receptor PARI μ NMJ01992 PJ01983 203989 x at Hisamine receptor Η4 H4 NMJ21624 PJ67637 221170— at

G protein ft receptor 12 GPR12 MJ05288 PJ05279 214558 at

G¾S protein ^ receptor RE2 RE2 MJ07369 PJ31395 214104— at Forced herin EGF LAG 7 transmembrane receptor 2 CELSR2 MJ01408 NP— 001399 36499 1 at

G protein microreceptor 32 GPR32 MJ01506 PJ01497 221469—at

G protein release receptor 58 GPR58 NMJ14626 PJ55441 221394— at cadherin EGF LAG 7 transmembrane receptor 1 CELSRl reaction — 014246 NPJ55061 41660— at motilin receptor Motilin R 丽 —001507 PJ01498 215910— s— at cadherin EGF LAG 7 Transmembrane receptor 3 CELSR3 NMJ01407 PJ01398 40020— at retinal pigment epithelium-derived oral dopsin homolog 丽 丽 J06583 NPJ06574 208314— at Pros evening glandin E receptor 4 EP4 NMJ00958 NPJ00949 204897 at Chemokine (CC motif) receptor 1 CCR1 NMJ295 NPJ01286 205098— at Dopamine receptor D5 D5 NMJ00798 PJ00789 208486 at Chemokine (C-X-C motif) receptor 5 CXCR5 NMJ01716 PJ01707 228065 at

Table 8

 3 Π IF-IFN-7 + L PS treatment

 I FN-treated cells LPS-treated cells

 GPCR-derived cells

 () Present

 AD AD Shire Hisashi Λ Π ノ: Hisashi

 Value

i ¹ ί

 Shirt r t

C3aRl 3277 Ρ 7493 P 1.9 pcs 2898 P 1, 1 5097 P 1.5 Thigh 2 2315 Ρ 3104 P 1, 1 3069 P 1.7 3694 P 1.5 β ΐ 9336 Ρ 10866 P 1.0 6817 P 0.9 9917 P 1.0

PAFR * 990 Ρ 2103 P 1.8 pieces 2939 P 3, 7 pieces 4009 P 3.8 pieces

A2b 753 Ρ 1810 .P '2.0 pieces 680 P 1.1 1332 P 1.7

GPR105 1352 Ρ 1032 P 0.6 764 P 0.7 693 P 0.5

TM7SF1% 1473 Ρ 1932 P 1.1 1251 P 1.1 1555 P 1.0

GPR37% 1446 Ρ 2326 P 1.4 1090 P 0.9 1054 P 0.7

CD97% 1180 Ρ 4301 P 3.1 pieces 1984 P 2.1 pieces 4852 P 3.9 pieces

MCI% 1485 Ρ 1271 P 0.7 586 P .0.5 357 P 0.2

GPR65% 1280 ρ1501 P 1.0 1315, P 1.3 1704 P, 1, 3

EP3

 氺 1035 ρ. 1111 P 0.9 837 P 1.0 938 P 0.9 variant h

 EP3

 138 η7 Ρ 1205 P 0.7 1186 P 1. 丄 1363 P 0.9 variant f

 D2% 463 Ρ 379 P 0.7 327 P 0.9 353 P 0.7

EDG2% 522 Ρ 3'87 P 0.6 '314 P 0.8 378 P 0.7

D2 441 Ρ 422 P 0.8 234 P 0.7 375 E 0.8

A3% 593 Ρ 726 P 1.0 430 P 0.9 270 P 0.4

C5R1% 278 Ρ 386 P 1.2 608 P 2.7 pieces 389 P 1.3 '

0PN3% 168 Ρ 109 A 0.5 144 A 1.1 92 A 0.5

GPR 7% 156 153 153 P 0.8 208 P 1, 7 200 P 1.2

CRTH2% 172 A 384 P 1.9 pieces 280 A 2.0 280 P 1.5

EDG1% 1213 Ρ 1229 P 0.9 963 P 1.0 420 P 0.3

EP2 198 Ρ 468 P 2.0 168 P 1.1 306 P 1.5

CCRL2% 2767 Ρ 3705 P 1.1 1744 P 0.8 3251 P 1.1 mglu6% 293 Ρ 347 P 1.0 152 P 0.6 360 P 1: 2 Table 8 (continued)

 3 toro PS processing

 I FN-treated cells LPS-treated cells

 GPC cell

 Hisashi Hisashi

AD value F AD value F ratio AD value F ratio AD value F ratio

CB2 137 A 233 P 1.4 211 A 1.9 236 M 1.6

GPR39 * 303 A 250 A 0.7 412 P 1.7 383 P 1 ..2

P2Y5 135 P 276 P 1.7 133 P 1.2 341 P 2.4 pieces

Al 252 A 250 P 0.8 184 P 0.9 235 P 0.9

Ρ2Υ4 167 A 87 A 0.4 108 A 0.8 170 M 1.0

ΕΡ3

 776 P 866 P 0.9 838 P 1.3 720 P 0.9 variant j

 A2a 656 P 428 P 0.6 1564 P 3.0 pieces 2517 P 3.6 pieces

AT1B 74 A 57 A 0.6 80 A 1.4 126 A 1.6

ETB 80 P 45 P 0.5 47 P 0.7 70 P 0.8

GNRHR 125 M 197 A 1.3 149 M 1.5 146 A 1.1

CELSR2 F 151 P 135 P 0.8 85 A 0.7 158 M 1.0

CCR7 * 103 A 128 A 1.0 188 A 2.3 711 P 6.5 pieces

PARI 556 P 796 P 1.2 285 P 0.6 824 P 1.4

H4 70 A 183 P 2.2 pieces 93 A 1.7 120 P 1.6

GPR12 227 P 238 P 0.9 151 M 0.8 167 1 P 0.7

RE2 96 A 81 A 0.7 26, k 0.3 43 A 0.4

CELSR2 82 A 103 A 1.1 75 A 1.1 123 A 1.4

GPR32 62 A 158 P 2.2 pieces 43 A 0.9 70 A 1.1

GPR58 F 117 A 74 A 0.5 93 A 1.0 111 P 0.9

CELSRl * 188 P 79 A 0.4 112 P 0.7 89 A 0.4

Motilin R 94 A 71 A 0.6 95 A 1.3 127 A 1.3

CELSR3 request 134 P 192 M 1.2 109 A 1.0 209 A 1.5

RRH 18 A 174 A 8.2 33 A 2.3 189 A 10.0

Table 9

 3 draw IFN-7 + LPS treatment

 IFN-treated cells LPS-treated cells

 GPCR-derived cells

 ) Present

 AD value F AD value F ratio AD value F ratio AD value F

C3aRl 3649 P 3261 P 1.0 1848 P 0.4 870 P 0.3

EMR2 1354 P 1295 P 1.1 1696 P 1.0 1244 P 1.1 j32 5116 P 4597 P 1.0 5837 P 0.9 3391 P 0.8

PAFR 2578 P 2093 P 0.9 3655 P 1.2 3434 P 1.6

A2b 647 P 630 P 1.1 653 P 0.8 586 P 1.1

GPR105 Thu 1183 P 867 P 0.8 986 P 0.7 450 P 0.4

TM7SF1 * 862 P 641 P 0.8 820 P 0.8 601 P 0.8

GPR37 Water 1043 P 711 P 0.8 1197 P 1.0 601 P 0.7

CD97 1164 P 858 P 0.8 1014 P 0.7 1185 P 1.2

MCI 743 P 529 P 0.8 463 P 0.5 372 P 0.6

GPR65 * 712 P 726 P 1.2 789 P 0.9 760 P 1.3

EP3

 415 415 P 424 P 1.2 560 P 1.1 331 P 0.9 variant h

 EP3

 654 P 570 P 1, 0 662 P 0.8 548 P 1.0 variant f

 D2 Thu 506 P 527 P 1.2 447 P 0.7.322 P 0.7

EDG2 Thu 215 P 335 P 1.8 pieces 399 P 1.5 211 P 1.2

D2 429P 436 P 1.1 452 P 0.9 581 P 1.6

A3 575 P 347 P 0.7 562 P 0.8 378 P 0.8

C5R1 151 A 84 P 0.6 83 A 0.5 77 A 0.-6

0PN3 * 145 P 123 A 1.0 131 P 0.7 97 M 0.8

GPR27 Thu 187 P 197 P 1.2 346 P 1.5 112 P 0.7

CRTH2 57 A 6 A 0.1 11 A 0.2 6 A 0.1

EDG1 N 602 P 237 P 0.4 420 P 0.6 144 P 0, 3

EP2 126 P 231 P 2.1 pieces 110 P 0.7 171 P 1, 6

CCRL2 Thu 995 P 668 P 0.8 761 P 0.6 439 P 0.5 mglu6 N 189 p 194 P 1.2 330 P 1.5 137 P 0.9 Table 9 (continued)

Table 7, among the GPCR listed in Table 8 and Table 9, EMR2, PAFR, GPR105, TWSF1 , GPR37, CD97, MC1, GPR65 N EP3 variant h N D2, EDG2, A3, C5R1 0PN3, GPR27, CRTH2 , EDG1, CCRL2, mglu6 CB2 GPR39, P2Y5, Al, P2Y4, EP3 variant j, A2a, AT1B ETB GNRHR CELSR2, CCR7, H4, GPR12, GPR32, GPR58, CELSR1, Moti lin R, CELSR3, RRH, DCR And And CXCR5 are the first GPCRs to be expressed in human mast cells. GPCRs expressed in these human mast cells are useful as targets for therapeutics for diseases involving mast cells. '

 By comparing the expression levels in control cells and IFN-a-treated cells, GPCRs whose expression fluctuated when human mast cells were activated via IFN-a receptor were identified. As a result, it was clear for the first time that the expression of C3aRl, PAFR, A2b, CD97, EDG2, 0PN3, CRTH2, EDG1 ヽ EP2, H4, GPR32, and CELSR1 fluctuated due to activation by IFN-y receptor. Became. GPCRs whose expression fluctuates in IM-treated cells are presumed to be involved in the control of mast cell activation, and those GPCRs are particularly useful as targets for therapeutic drugs for diseases involving mast cells. is there.

 By comparing the expression levels in control cells and LPS-treated cells, GPCRs whose expression fluctuated when human mast cells were activated via the LPS receptor were identified. As a result, it has become clear for the first time that the expression of C3aRl, PAFR, CD97, MC1, C5R1 ヽ A2a, AT1B ヽ CELSR2, CCR7 and EP4 fluctuates due to LPS receptor-mediated activation. GPCRs whose expression fluctuates in LPS-treated cells are presumed to be involved in the control of mast cell activation, and those GPCRs are particularly useful as targets for therapeutics for diseases involving mast cells.

 By comparing expression levels in control cells and IFN-α + LPS-treated cells, GPCRs whose expression fluctuates when human mast cells are activated through both IFN-α and LPS receptors are identified did. As a result, C3aRl, PAFR, GPR105, CD97, MC1, A3, 0PN3, EDG1, CCRL2, P2Y5, A2a, CCR7, RE2, CELSR2, CELSR1 and EP4 mediated both IFN-α and LPS receptors. For the first time, it has been clarified that the expression fluctuates due to the activity. GPCRs whose expression fluctuates when treated with IFN-α + LPS are presumed to be involved in the control of mast cell activation, and those GPCRs are particularly useful as targets for therapeutics for diseases involving mast cells. is there.

 By comparing the expression levels between LPS-treated cells and IFN-α + LPS-treated cells, the effect of IFN-α on LPS receptor-mediated activation of human mast cells can be examined. For the first time, CD97, A2a and CCR7 were found to increase in expression when stimulated with LPS alone, but further increased when pretreated with IFN-α. On the other hand, the expression of MC1 was also reduced by LPS alone stimulation, but it was found for the first time that the expression was further reduced by pretreatment with IFN-γ. These GPCRs are presumed to be deeply involved in the control of mast cell activation, and are particularly useful as targets for therapeutic agents for diseases involving mast cells. Example 4 Action of HEOAD54 agonist on IgE receptor-mediated activation of human mast cells (1) Action of HEOAD54 agonist on IgE receptor-mediated activation of human peripheral blood-derived mast cells

 5-Oxo-6E, 8Z, 11Z, 14Z-eicosatetraenoic acid (hereinafter abbreviated as 5-oxo-ETE) is a ligand for HEOAD54 and has recently been shown to act as an agonist [J. Biol Chem., 277, 31459 (2002); Mol. Pharmacol., 63> 471 (2003)]. In Example 2 (4), since it was found that HEOAD54 was expressed in human peripheral blood-derived mast cells, the effect of 5-oxo-ΕΤΕ on activation of human peripheral blood-derived mast cells was examined. Preparation and culture of mast cells derived from human peripheral blood were performed by the method described in Example 2.

l〃g / ml human myeloma IgE (Cosmo Bio), 100 ng / ml SCF, 50 ng / ml IL-6, Prepare 2.5 x 10 ⁵ cells / ml of human peripheral blood-derived mast cells in IMDM containing 5% FCS and 5%, and dispense 100 d per 1-well into a 96-well plate at 37 ° C. Cultured for 24 hours. The medium was removed by centrifugation, and Tyrode buffer (126.1 t ol / l NaCl, 4.0 t ol KC1 ヽ 1, 0 t ol / l CaCl ₂ , 0.6 corrupt ol / l MgCl ₂ , 0.6 vigorous ol / l K¾P0 _4, 10 Keio HEPES, 5.6 mmol / 1 D - glucose, 0.1% © shea serum albumin, after washing with pH 7.4), incubated with evening Gray one de buffer 100〃1 37 ° C for 30 minutes did. Next, a persian anti-human IgE antibody (manufactured by Dako) having a final concentration of 1.5 zg / ml was added, and the mixture was further incubated at 37 ° C for 30 minutes to induce degranulation. The supernatant was collected by centrifugation, and the amount of histamine in the supernatant was measured using a histamine immunoassay kit (manufactured by Immunotech) to measure the degree of the particles. (4) A similar experiment was performed without the addition of a heron anti-human IgE antibody to measure the amount of spontaneous release of Hismin in the absence of stimulation via the IgE receptor. In addition, a similar experiment was carried out with the addition of Triton X-100 (manufactured by Nacalai Tesque) at a final concentration of 1% in place of the heron anti-human HgE antibody. The amount of histamine was measured. '

 In the above case, when a Thai buffer containing various concentrations of 5-0X0-ETE (manufactured by Cayman) was used instead of the Thai buffer added before the addition of anti-human IgE antibody The effect of 5-0X0-ETE on degranulation was determined by measuring the amount of histamine released into the supernatant of the test. Table 10 shows the concentration of 5-0X0-ETE used and the degree of degranulation. The degree of degranulation was indicated by the ratio (%) of the amount of hissamine released into the supernatant to the total amount of hissamine. As shown in Table 10, 5-0X0-ETE was found to inhibit IgE receptor-mediated mast cell degranulation.

 Table 10.

(2) Effect of agonist of HEOAD54 on IgE receptor-mediated activation of human mast cell line LAD2

 LAD2 is a recently established human mast cell line that is known to well retain the properties of human mast cells. CLeuk. Res., 27, 671 (2003); Leuk. Res. 27, 677 (2003). )]. Therefore, the effect of 5-oxo-ETE on the activation of human mast cell line LAD2 was examined. LAD2 was cultured in a Stem Pro-34 medium (manufactured by Invitrogen) containing 100 ng / ml SCF. LAD2 was obtained from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD 20892-1881, USA).

l ^ g / ml (manufactured by Cosmo Bio) of Piochin binding Hitomiero Ichima IgE and prepared LAD2 in Stem Pro-34 medium containing 100 ng / ml of SCF 2. to be 5 X 10 ⁵ cells / ml And 96 ゥ el play The mixture was dispensed at a rate of 100 liters per well and cultured at 37 ° C for 24 hours. After removing the medium by centrifugation and washing with Tyrode buffer, 100% Tyrode buffer was added and incubated at 37 ° C for 30 minutes. Then, a final concentration of 10 μg / ml streptavidin-phycoerythrin (BD PharMingen, hereinafter abbreviated as streptavidin-PE) was added, and the mixture was further incubated at 37 ° C. for 30 minutes to allow degranulation. Induced. The supernatant was collected by centrifugation, and the degree of degranulation was determined by measuring the amount of histamine in the supernatant using a His-min-immunoassay kit. A similar experiment was performed without streptavidin-PE, to measure the spontaneous release of hismin in the absence of stimulation via the IgE receptor. In addition, the same experiment was carried out by adding Triton X-100 at a final concentration of 1% instead of streptavidin-PE, and the same experiment was performed to measure the total amount of hismin in LAD2.

 In the above, released into the supernatant when using a Thai buffer containing various concentrations of 5-OXO-ETE instead of the buffer to be added prior to the addition of streptavidin-PE The effect of 5-oxo-ETE on degranulation was examined by measuring the amount of hissamine produced. Table 11 shows the concentrations of 5-0X0-ETE and the degree of JW grains used. The degree of S½S grain was shown by the ratio (%) of the amount of histamine released into the supernatant to the total amount of histamine.

 Table 11

From the results of (1) and (2) above, it was revealed that 5-oxo-ETE treatment suppresses degranulation of human peripheral blood-derived mast cells and LAD2 induced by IgE receptor stimulation. Therefore, agonists of 5-oxo-ETE and HEOAD54 are considered to be drugs that suppress the activation of human mast cells by IgE receptor stimulation.

Similarly, by measuring the action of agonists of other GPCRs, it can be determined whether or not the agonist or gonist of each GPCR suppresses the activation of human mast cells stimulated by IgE receptors. Activation of human mast cells, instead of degranulation, TNF-chilled GM-CSF cytokine production, such as, IL-8, 1-309, chemokine production, such as MIP-1 Fly, LTC _4, LTD _4, LTE It can also be examined by measuring the production of inflammatory media such as PGD ₂ overnight [Blood 100. 3861 (2002)]. Instead of mast cells derived from human peripheral blood and LAD2, mast cells derived from human umbilical cord blood, mast cells prepared from human lung, skin, and tonsils can also be used. Example 5 Effect of agonists of various GPCRs on activation of human mast cells

In Examples 1 to 3, the effects of GPCRs, which were found to be expressed in human mast cells, on LAD2 were examined. (1) Action of various GPCR agonists on IgE receptor-mediated activation of human mast cells LAD2 was cultured by the method described in Example 4. LAD2 was prepared at a concentration of 2.5 × 10 ⁵ cells / ml in a Stem Pro-34 medium containing 100 mg / ml of human myeloma IgE and 100 ng / ml of SCF, and cultured at 37 ° C. for 24 hours. After removing the medium by centrifugation and washing with Tyrode buffer, add Tyrode buffer to make up to 4.0 x 10 ⁵ cells / ml, and add 50 μl / well to a 96-well plate. Dispensed. Next, 50〃1 of an evening buffer was added and incubated at 37 ° C for 30 minutes, and a final concentration of 10 µg / ml of a heron anti-human IgE antibody (manufactured by Dako) was added. Incubation was performed at 37 ° C for 20 minutes to induce degranulation. The supernatant was collected by centrifugation, and the degree of degranulation was measured by measuring the activity of /?-Hexosaminidase in the supernatant. ^ -Hexosaminidase activity was determined by adding the collected supernatant to a 4 l / l P-nitrophenyl N-acetyl-glucosaminide (Sigma) dissolved in 40 l / l citrate buffer (pH 4, 5). Was added to 50〃1 and incubated at 37 ° C for 1 hour.The absorbance at 405 nra of a sample containing 100 d of 0.2 mol / 1 glycine (pH 10.7) was added to the plate reader 1420 ARVOsx (PerkinElmer Co., Ltd.). Was used for the measurement.ゥ Similar experiments were performed without the addition of a heron anti-human IgE antibody to measure the amount of spontaneous ^ -hexosaminidase release in the absence of IgE receptor-mediated stimulation. In addition, the same experiment was performed by adding Triton X-100 at a final concentration of 1% in place of the Egret anti-human IgE antibody, and the total -hexosaminidase activity in LAD2 was measured.

In the above, instead of the tie-buffer buffer added before the addition of anti-human IgE antibody, released into the supernatant when Tyrode buffer containing various concentrations of GPCR agonists shown in Table 12 was used. The effect of each agonist on degranulation was determined by measuring the activity of // へ -hexosaminidase. However, in experiments using lysophosphatidylserine as an agonist for GPR34, the incubation after adding the Thai reagent containing the agonist was 10 minutes, and the final concentration of the heron anti-human IgE antibody was 15 μg. I went with / ffll. Table 12 shows the GPCR agonists used and their concentrations, and the degree of β-granules when they were used. The degree of degranulation was indicated by the ratio (%) of /?-Hexosaminidase activity in the supernatant to the total /?-Hexosaminidase activity.

Table 12

These results revealed that agonist treatment of A2a suppressed LAD2 degranulation induced by IgE receptor stimulation. Therefore, agonists of A2a are stimulated by IgE receptors. It is considered to be a drug that suppresses the activation of human mast cells.

On the other hand, by agonist treatment of D2, D5 ヽ GPR91, GPR105, GPR43, Hl, H4, GPR34, EP3 varaint h, EP3 variant f EP3 varaint_j, EP2 or EP4, LAD2 degranulation due to IgE receptor <f It was found to be enhanced. Therefore, the agonists of D2, D5, GPR91, GPR105, GPR43, HI ヽ H4, GPR34, EP3 varaint h, EP3 variant f, EP3 varaint j, EP2 or EP4, are responsible for IgE receptor-stimulated human mast cell activity. It is considered to be a drug that suppresses dani. Similarly, by measuring the action of agonists of other GPCRs, it can be examined whether or not the agonist or gonist of each GPCR suppresses the activation of human mast cells stimulated by IgE receptors. Activation of human mast cells, instead of degranulation, TNF-chilled GM- side Cain production such as CSF, IL-8, 1-309, chemokine production, such as _{MIP- 1, LTC 4, LTD 4} , LTE _4. It can also be examined by measuring the production of inflammatory media such as PGD2 overnight (Blood 100> 3861 (2002)). Instead of LAD2, mast cells derived from small umbilical cord blood, mast cells derived from human peripheral blood, and mast cells prepared from human lung, skin, and tonsils can also be used.

 (2) Effects of agonists of various GPCRs on the activity of human mast cells

Prepare LAD2 in Stem Pro-34 medium containing 1 zg / ml human myeloma IgE and 100 ng / ml SCF to 2.5 x 10 ^δ / ml and culture at 37 ° C for 24 hours did. After removing the medium by centrifugation and washing with buffer overnight, adjust the volume to 2.0 x 10 ⁵ cells / ml by adding tie-mouth buffer, and add 1 well to a 96-well plate. Per 100〃1 was dispensed. Next, Tyrode buffer containing various concentrations of GPCR agonists shown in Table 13 was added, and the mixture was incubated at 37 ° C for 20 minutes. The supernatant was collected by centrifugation, and the degree of degranulation of the agonist of each GPCR by single stimulation was measured by measuring the /?-Hexosaminidase activity in the supernatant. ^ -Hexosaminidase activity was measured in the same manner as in Example 5 (1). The same experiment was performed without adding the GPCR agonist to measure the amount of spontaneous? -Hexosaminidase release. In addition, the same experiment was carried out using Triton X-100 at a final concentration of 1% in place of the GPCR agonist, and the same experiment was carried out to measure the total /?-Hexosaminidase activity in LAD2.

Table 13 shows the GPCR agonists used and their final concentrations, and the degree of degranulation when they were used. The degree of degranulation was expressed as the ratio (/%) of /?-Hexosaminidase activity in the supernatant to the total ^ -hexosaminidase activity.

Table 13

These results revealed that A2a agonists did not induce LAD2 degranulation. On the other hand, EP2, the EP3 variant h, EP3 variant f, EP3 variant j s EP4, PAFR ETB EDG2 or Agonisuto processing GPR34, revealed that induces degranulation LAD2. Therefore, EP2, EP3 variant h, EP3 variant f EP3 variant j EP4, PAFR, ETB, EDG2 or GPR34 antagonists may be agents that suppress the activation of human mast cells.o

Similarly, by measuring the action of agonists of other GPCRs, it can be determined whether or not the agonist or gonist of each GPCR suppresses the activation of human mast cells stimulated by IgE receptors. Activation of human mast cells, instead of degranulation, TNF-chilled GM- cytokine production, such as CSF, IL- 8, 1-309, chemokine production, such as _{MIP-Ι α, LTC 4,} LTD 4, LTE ₄ , PGD2 It can also be examined by measuring the production and the like of inflammatory mediators such as [Blood 100, 3861 (2002)]. Instead of LAD2, mast cells derived from human umbilical cord blood, mast cells derived from human peripheral blood, and mast cells prepared from human lung, skin, and tonsils can also be used.

 Based on the results of (1), (2) and Example 4, the compounds (agonist, angonist, functional modifier) acting on GPCR expressed in human mast cells, antibodies, and the expression of GPCR are controlled. The compound, antibody, siRNA, or antisense DNA that regulates the signal transduction of the GPCR, or the compound, antibody, siRNA, or antisense DNA that is useful as the pharmaceutical of any of the following (b) to (j): it is conceivable that.

 (b) human mast cell activation inhibitor

 (c) Inhibitor of degranulation of human mast cells

 (d) Human mast cell inflammatory mediator production inhibitor

 (e) Human mast cell cytokine production inhibitor

 (f) Chemokine production inhibitor for human mast cells

 (g) Remedies for atopic dermatitis

 (h) Asthma treatment

 (i) Drug for treating chronic obstructive pulmonary disease

 (j) Drugs for allergic diseases Industrial applicability

 According to the present invention, GPCRs expressed in human mast cells have been identified, and these GPCRs have been targeted, and inhibitors of human mast cell activation and allergy to atopic dermatitis, asthma, chronic obstructive pulmonary disease, etc. A therapeutic agent for sexual diseases and a method for screening the same are provided.

"Sequence List Free Text"

SEQ ID NO: 1—Inventor: Hirohisa Saito Yoshimichi Okayama

 Inventor: Jun Kashiwakura-Kazumi Miura; Masaya Obayashi

 Inventor: Tetsuro Yoshida Katsutoshi Sasaki

Claims

The scope of the claims

1. Cysteinyl leukotriene receptor 1 (CysLTRl), G protein-coupled receptor 91 (GPR91, G protein-coupled receptor 34 (GPR34), which is a G protein-coupled receptor expressed in human mast cells EGF-like module-containing mucin-like hormone receptor-like sequence 2 (EMR2), platelet activating factor receptor (PAFR), G protein-coupled receptor 105 (GPR105), 7-transmembrane superfamily member 1 TM7SF1), G protein-coupled receptor 37 (GPR37), CD97 antigen (CD97), melanocortin 1 receptor (MCI), G protein-coupled receptor FIRE (FIRE), formyl peptide receptor 1 (fMLPl), G protein-coupled receptor 65 (GPR65), prostaglandin E receptor 3 alternative variant h (EP3 variant h), purine receptor P2Y8 (P2Y8), dopamine receptor D2 (D2), endothelial differentiation Lysophosphatidic acid G protein-coupled receptor 2 (EDG2), adenosine A3 receptor (A3), 5th complement component receptor 1 (C5R1), opsin 3 (0PN3), G protein-coupled receptor 27 (GPR27), G protein-coupled receptor 44 (CRTH2), endothelial differentiation sphingolipid G Protein light-coupled receptor 1 (EDG1), formyl peptide receptor-like 2 (FPRL2), chemokine (C-C motif) receptor 2 (CCRL2), mesobotropiglucamic acid receptor 6 (mglu6 ) _s Cannabinoid receptor 2 (CB2), G protein-coupled receptor 39 (GPR39), Purine receptor P2Y5 (P2Y5), Adenosine M receptor (A1), Pyrimidine receptor P2Y4 (P2Y4), Prostaglandin E receptor (3) Ornithine native variant 1, j (EP3 variant j), adenosine A2a receptor (A2a), type 1 angiotensin receptor (AT1B), type B endothelin receptor (ETB), gonadotropin releasing hormone receptor (GNRHR) ), Cadherin EGF LAG 7-pass G-type receptor 2 (CELSR2), Chemokine (C-C motif) ) Receptor 7 (CCR7), histamine receptor H4 (H4), G protein-coupled receptor 12 (GPR12), histamine receptor HI (HI), G protein-coupled receptor 32 (GPR32), tumor endothelial cells One strength one 5 (TEM5), G protein-coupled receptor 58 (GPR58), force doherin EGF LAG 7 penetration G-type receptor 1 (CELSR1), G protein-coupled receptor 85 (GPR85), Motilin receptor ( Motilin R), G protein-coupled receptor 64 (HE6), cadherin EGF LAG 7-pass G-type receptor 3 (CELSR3), calcitonin receptor-like (CALCRL), retinal pigment epithelium-derived oral dopsin homolog (RRH), G protein-coupled receptor i-book 18 (GPR18), G protein-coupled receptor 35 (GPR35), purine receptor P2Y10 (P2Y10), FLJ40279 (C-TESTI2027296), G protein-coupled receptor 141 (GPR141), testis Descending-related G protein-coupled receptor (hRUP16), Inversion inversion thickness-related receptor (ITR), G protein-coupled receptor MRGX2 (MRGX2), Tresamine receptor 1 (SNORF33), 7-transmembrane receptor CBRC7TMJ75 (C-THYMU2011548), Adrenomedullin receptor (GlOd), G protein-coupled receptor 107 (GP 107), G protein-coupled receptor 126 (GPR126) , G protein-coupled receptor 41 (GPR41), G protein-coupled receptor 43 (GPR43), G protein-coupled receptor 68 (GPE68), gastrin-releasing peptide receptor (GRPR), G protein-coupled receptor 1019 (HEOAD54), putative G protein-coupled receptor hGPCR10 (hRUP9), JI progestin receptor (fflPRa), membrane-type progestin receptor A (mPRg), putative G protein-coupled receptor GPCRi. (SRL), platelet activity Hidden receptor homolog H963 (H963), prostaglandin E receptor 4 (EP4), chemoforce in (CC motif) receptor 1 (CCR1), dopamine receptor D5 (D5), leukotriene B4 receptor ( LTB4R) and chemokine (CX-C motif) receptor 5 (CXCR5) Including expressing cells or bringing into contact with a test substance and the membrane fraction of the cell, it acts on the receptor, and to human mast cells (a) apoptosis induction, (b) activation suppressing, A screening method for a substance exhibiting at least one of (c) inhibition of degranulation, (d) inhibition of production of inflammatory mediator, (e) inhibition of cytokine production, and (f) inhibition of chemokine production.

 2. G protein-coupled receptors expressed in human mast cells are GPR34, PAFR, GPR105, TM7SF1, GPR65, P2Y8, EDG2, A3, C5R1, EDG1 ヽ GPR39, EP3 variant j ヽ A2a ヽ ETB, H4, CELSR2, TEMff ヽ

C-TESTI2027296, GPR141, ITR, MRGX2 _S G10d, GPR43, GRPR _S HEOAD54 _s H963, EP4 and LTB4R Expression is changed by stimulating activation of human mast cells via IgE receptor 2. The screening method according to claim 1, which is a receptor that performs the screening.

 3. The G protein-coupled receptor expressed in human mast cells stimulates human mast cells to lipopolysaccharide selected from the group consisting of PAFR, CD97, MC1, C5R1, A2a, AT1B, CELSR2, CCR7 'and EP4. 2. The screening method according to claim 1, which is a receptor whose expression fluctuates due to the following.

 4. G protein-coupled receptor expressed in human mast cells is selected from the group consisting of PAFR, A2b, CD97, EDG2, 0PN3, CRTH2, EDG1, H4, GPR32 and CELSRl. 2. The screening method according to claim 1, which is a receptor whose expression changes when stimulated.

 5. G protein-coupled receptor expressed in human mast cells is selected from the group consisting of PAFR, GPR105, CD97, MC1, A3, 0PN3, EDG1 ヽ CCRL2, P2Y5, A2a, CCR7, CELS2, CELSRl and EP4 2. The screening method according to claim 1, wherein the receptor is a receptor whose expression is fluctuated by co-stimulation of human mast cells with co-stimulatory inulin and lipopolysaccharide.

 6. The G protein-coupled receptor expressed in human mast cells is a human selected from the group consisting of GPR34, GPR105 TM7SF, fMLPl, P2Y8, A3, C thigh 2, GCRL2, P2Y5, A2a, Η4, CALCRL and ΕΡ4 2. The screening method according to claim 1, which is a receptor whose expression varies when mast cells are treated with glucocorticoid.

7. Complement third component receptor 1 (C3aRl), GP34, adrenergic receptor, a G protein-coupled receptor that is expressed in human mast cells and whose expression level fluctuates upon stimulation of glucocorticoid ? 2), GPR105, TM7SF, fMLPl, P2Y8, A3, CRTH2, CCRL2, P2Y5, A2a, Epusutai emissions - bar one virus induced gene 2 (EBI2), Suronpin receptor (PARI), H4 _S G protein-coupled receptor Contacting a test substance with a cell expressing a receptor selected from the group consisting of RE2 (RE2), CALCRL and EP4, or a membrane fraction of the cell; A method for screening for a substance whose action can be controlled.

8. CysLTR1, GPR91, GPR34, EMR2, PAFRs GP, M7SFU GPR37, CD97, MCI, FIRE ヽ fMLPl, GPR65, EP3 variant h, P2Y8, D2, which are G protein-coupled receptors expressed in human mast cells EDG2, A3, C5R1 ヽ 0PN3, GPR27, CRTH2, EDG1 ヽ FPRL2, CC L2, mglu6, CB2, GPR39 _S P2Y5, Al, P2Y4, EP3 variant j _N A2a ヽ AT1B ヽ ETB, GN thigh, CELSR2, CCR7, H4, GPR12, HI ヽ GPR32 ヽ TEM5 _S GPR58, CELSRl ヽ GPR85, Motilin R ヽ HE6, CELSR3, CALCRL, RRH ヽ GPR18, GPR35, P2Y10, C-TESTI2027296, GPR141, hRUP16, ITR, MRGX2, SNORF33, C-THYMU2011 GPR107, GPR126, GPR41, GPR43, GPR68, GRPR GRHEOAD54, hRUP9, mPRa ヽ raPRg, SRL ヽ H963 EP4, CCR1 ヽ D5s LTB4R and CXCR5 (A) induction of apoptosis, (b) At least one of the following: suppression of activation, (c) suppression of degranulation, (d) suppression of production of inflammatory mediators, (e) suppression of production of site power-ins, and (f) suppression of production of chemokines. How to do.

 9. Using agonists against A2a or HEOAD54, which are G protein-coupled receptors expressed on human mast cells, induce (a) induction of apoptosis, (b) suppression of activation, (c) A) inhibiting degranulation, (d) inhibiting production of inflammatory mediators, (e) inhibiting production of cytokines, and (f) inhibiting production of chemokines.

10. From G protein-coupled receptors expressed on human mast cells, GPR91, GPR34, PAFR, GPR105, D2, D5, EDG2, EP3 variant h, EP3 variant j, EP4, ETB ヽ Hl, H4 and GPR43 (A) induction of apoptosis, (b) suppression of activation, (c) suppression of degranulation, (d) A method for suppressing at least one of the production of an inflammatory mediator, the production of (e) a cytokine, and the production of a chemokine (f).

1 1. CysLTR1, GPR91, GPR34, EMR2, PAFR ヽ GPR105, TM7SF1, GPR37 _S CD97, MCI ヽ FIRE ヽ fMLP1, GPR65, EP3 variant h, which are G protein-coupled receptors expressed in human mast cells

P2Y8, D2, EDG2, A3, C5R1, 0PN3, GPR27, CRTH2, EDG1, FPRL2, CCRL2, mglu6, CB2, GPR39, P2Y5, Al, P2Y4 _N EP3 variant j, A2a, AT1B, ETB ヽ GNRHR ヽ CELSR2,,. CCR7, H4, GPR12, HI ヽ GPR32 TEM5, GPR58, CELSR1, GPR85, Motilin R, HE6, CELSR3, CALCRL, RRH, GPR18, GPR35, P2Y10, C-TESTI2027296 _S GPR1 1, hRUP16 _s ITR, M GX2 _S SNORF33 C-THYMU2011548, G10d, GPR107 _N GPR126, GPR41, GPR43, GPR68, GRPR ヽ HEOAD54, hRUP9, mPRa ヽ mPRg ヽ SRL, H963, EP4, CCR1, D5, LTB4R and CXCR5, an agonist for a receptor selected from the group consisting of: A method for treating atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease using an engonist or a functional modifier.

 12. A method for treating atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease using an agonist against A2a or HEOAD54, which is a G protein-coupled receptor expressed on human mast cells. '

 1 3. G protein-coupled receptors expressed on human mast cells, GPR91, GPR34, PAFR, GPR105,, D2, D5, EDG2, EP3 variant h, EP3 variant j, EP4, ETB ヽ HI ヽ H4 and GPR43 A method for treating atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease, using an gonist for a receptor selected from the group consisting of: · ，

14. G protein-coupled receptor expressed in human mast cells and whose expression level fluctuates upon stimulation of glucocorticoid, C3aRl, GPR34, β, GPR105, TM7SF, fMLPl, P2Y8, A3, CRTH2, CCRL2 _N P2Y5 _S A2a, EBI2 ヽ. A method for controlling glucocorticoid action on cells using an agonist, anthrone gonist, or a mechanical modifier for a receptor selected from the group consisting of PAR1, H4, RE2, CALCRL, and EP4 .

15 5. CysLTRl, GPR91, GPR34, EMR2, PAFR, GPR105, TM7SF1 ヽ GPR37, CD97, MC1, FIRE ヽ fMLPl, GP65, EP3 variant h, P2Y8, which are G protein-coupled receptors expressed in human mast cells , D2, EDG2, A3, C5R1 ヽ 0PN3 GPR27 _S C rinse, EDG1 ヽ FPR, CCRL2, mglu6, CB2, GPR39 _S P2Y5, Al, P2Y4, EP3 variant A2a _s AT1B ヽ ETB ヽ GNRHR ヽ CELSR2, CCR7, H4, GPR12ヽ HI ヽ GPR32, TEM5, GPR58, CELSR1, GPR85, Motilin R, HE6, CELSR3, CALCRL ヽ RRH ヽ GPR18, GPR35, P2Y10, C-TESTI2027296s GPR141, hRUP16, ITR, MRGX2, SNORF33, C-THYMU2011548, G10d, GPR107, GPR126, GPR41, GPR43, GPR68, GRPR The medicament according to any one of (a) to (j), which comprises, as an active ingredient, an agonist, an engonist or a function modifier for a receptor selected from the group consisting of D5, LTB4R and CXCR5.

 (a) Apoptosis inducer for human mast cells

 (b) Activation of human mast cells i 卬

 (c) Inhibitor of degranulation of human mast cells

 (d) Human mast cell inflammatory mediator production inhibitor

 (e) Human mast cell cytokine production inhibitor

 (f) Chemokine production inhibitor for human mast cells

 (g) Remedies for atopic dermatitis

 (h) remedies for asthma

 (i) Drug for treating chronic obstructive pulmonary disease '' (j) Drug for treating allergic disease

 16. The medicament according to any one of (a) to (j), which comprises, as an active ingredient, an agonist against A2a or HEOAD54, which is a G protein-coupled receptor expressed in human mast cells.

 (a) Apoptosis inducer for human mast cells

 (b) Human mast cell activity inhibitor

 (c) + degranulation inhibitor for human mast cells

 (d) Human mast cell inflammatory mediator production inhibitor

 (e) Human mast cell cytokine production inhibitor

 (: F) Chemokine production inhibitor for human mast cells

 (g) Remedies for atopic dermatitis

 (h) remedies for asthma

 (i) Chronic obstructive pulmonary disease drug

 (j) Remedies for allergic diseases

 17. Group consisting of GPR91, GPR34, PAFR, GPR105, D2, D5, EDG2, EP3 variant h, EP3 variant j ヽ EP4, ETB ヽ HI ヽ H4 and GPR43, which are G protein-coupled receptors expressed on human mast cells The pharmaceutical according to any one of (a) to (j), which comprises, as an active ingredient, an engonist against a receptor selected from the group consisting of:

 (a) Apoptosis inducer for human mast cells

 (b) human mast cell activation inhibitor

 (c) Inhibitor of degranulation of human mast cells

 (d) Inhibitor of mast cell production of human mast cells

 (e) Human mast cell cytokine production inhibitor

 (f) Chemokine production inhibitor for human mast cells

 (g) Remedies for atopic dermatitis

 (h) remedies for asthma

(i) Chronic obstructive pulmonary disease drug (j) Remedies for allergic diseases

 18. G protein-coupled receptor that is expressed in human mast cells and whose expression level fluctuates upon stimulation of darcocorticoid, C3aRl, βGPR34, GPR105, fMLPl, P.2Y8, A3, CRTH2, CCRL2 Control of dalcocorticoid action, which contains an agonist, angonist or a functional modifier as an active ingredient for a receptor selected from the group consisting of P2Y5, A2a, EBI2, CELSR2, PARI, H4, RE2, CALCRL and EP4 Agent.

1 9. CysLTRl _s GPR91, a G protein-coupled receptor expressed on human mast cells, for the manufacture of a pharmaceutical composition for the treatment of atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease , GPR34, EMR2, PAFR ヽ GPR105, TM7SF1, GPR37, CD97, MC1, FIRE ヽ fMLPl ヽ GPR65, EP3 variant h, P2Y8, D2, EDG2, A3, C5R1 ヽ 0PN3, GPR27, CRTH2, EDGU FPRL2, CCRL2, mglu6 CB2, GPR39, P2Y5, Al, P2Y4, EP3 variant j s A2a, AT1BヽETB, G _{image, CELSR2 S CCR7 H4, GPR12,} Hl, GPR32 TEM5, GPH58, CELSR1, GPR85, motilin R, HE6, CELSR3, CALCRLヽRRH ヽ GPR18, GPR35 P2Y10 ヽ G-TESTI2027296 _S GPR141, hRUP16, ITR, MRGX2, SNORF33, C-THYMU2011548, GlOd ヽ GPR107, GPR126, GPR41, GPR43 _S GPR68, GRPR ヽ HEOAD54, hRUP9, mPRa, mPRa, mPRg Agonists for receptors selected from the group consisting of EP4, CCR1, D5, LTB4R and CXCR5 The use of strike or function modifier substance.

2 0. A G protein-coupled receptors expressed in human mast _{cells, CysLTRl, GPR91, GPR34 N EMR2} , PAFRヽGPR105, TM7SF1, GPR37, CD 97 , MCIヽ _{FIRE S fMLPl s GPR65, EP3 variant} h, P2Y8 , D2, EDG2, A3, C5R1, 0PN3, GPR27, CRTH2, EDG1, FPRL2, CCRL2, mglu6, CB2, GPR39, P2Y5, Al, P2Y4, EP3 variant j, A2a, AT1B ヽ ETB ヽ GNRHR ヽ CELSR2, CCR7, H4 , GPR12 ヽ Hl, GPR32, TEM5, GPR58, CELSR1, GPR85, Motilin R, HE6, CELSR3, CALCRL, RRH, GPR18, GPR35, P2Y10, C-TESTI2027296, GPR1 1, hRUP16, ITR ヽ MRGX2, SNORF2011, C-THYMU , G10d, GPR107, GPR126, GPR41, GPR43, GPR68, GRPR, HEOAD54, hRUP9, mPRa, mPRg, SRLH963, EP4, CCR1, D5, LTB4R and CXCR5 And a human having at least one of the following actions (a) to (f) on human mast cells:

 (a) Induction of apoptosis

 (b) Suppression of activation

 (c) Suppression of degranulation

 (d) Suppression of production of inflammatory media

 (e) Suppression of cytokine production

 (f) Suppression of chemokine production

2 1. Expressed in human mast cells, and a G protein-coupled receptor expression level by stimulation of glucocorticoids Koi de _{fluctuates, C3aRl, GPR34 S? 2,} GPR105, TM7SF, fMLPl, P2Y8, A3, CRTH2 An antibody which specifically reacts with a receptor selected from the group consisting of CCRL2s P2Y5, A2a, EBI2, PARI ヽ H4, RE2, CALCRL and EP4, and has an effect of controlling the action of glucocorticoid.

2 2. CysLTR1, GPR91, GPR34, EMR2, PAFR, GPR105, TM7SF1, GPR37, CD97, MCI ヽ FIRE, fMLPl, GPR65 _S EP3 variant h, P2Y8 _S , which are G protein-coupled receptors expressed in human mast cells D2 EDG2, A3, C5R1 _S 0PN3, GPR27, par 2, EDG1, FPRL2 _N CCRL2, mglu6 _s CB2, GPR39 _S P2Y5, Al, P2Y4, EP3 variant j, A2a, AT1B ヽ ETB ヽ GNRHR ヽ CELSR2, CCR7, H4, GPR12, HI ヽ GPR32, TEM5, GPR58. CELSRl, GPR85, Motilin R, HE6, CELSR3 _S GALCRL, RH, GPR18 , GPR35, P2Y10, C-TESTI2027296s GPR141, hRUP16, ITR, MRGX2, SNO F33 _N C- THYMU2011548, GlOd ヽ GPR107, GPR126, GPR41, GPR43, GPR68, GRPR ヽ HEOAD54, hRUP9, mPRa, fflPRg ヽ RL A method for detecting or isolating mast cells using an antibody that specifically reacts with a receptor selected from the group consisting of CCR1, D5, LTB4R and CXCR5.

 23. Use of the antibody according to claim 20 to (a) induce apoptosis, (b) suppress activation, (c) suppress degranulation, and (d) inflammation of human mast cells A method of suppressing at least one of the following: (i) suppression of production of cytokines and (:) suppression of production of chemokines.

 24. A method for treating atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease using the antibody according to claim 20.

 25. A method for controlling the action of glucocorticoid on cells using the antibody according to claim 21.

26. The medicament according to any one of (a) to (: i), comprising the antibody according to claim 20 as an active ingredient. '

(a) Inducer of human mast cell apoptosis

 (b) Human mast cell activity inhibitor

 (c) Inhibitor of degranulation of human mast cells

 (d) Human mast cell inflammatory mediator production inhibitor

 (e) Human mast cell cytokine production inhibitor

 (: F) Chemokine production inhibitor for human mast cells

 (g) Remedies for atopic dermatitis.

 (h) Asthma treatment

 (i) Chronic obstructive pulmonary disease drug

 (j) Remedies for allergic diseases

 27. An agent for controlling the action of glucocorticoid, comprising the antibody according to claim 21 as an active ingredient.

 28. Use of the antibody according to claim 20 for the manufacture of a pharmaceutical composition for the treatment of atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease.

2 9. CysLTR1, GPR91, GPR34, EMR2, PAFR, GPR105, TM7SF1 ヽ GPR37, CD97, MC1, FIRE ヽ fMLPU GPR65, EP3 variant h, P2Y8, D2, which are G protein-coupled receptors expressed in human mast cells , EDG2, A3, C5R1 ヽ 0PN3, GPR27, CRTH2, EDG1, FPRL2, CCRL2, mglu6, CB2, GPR39, P2Y5, Al, P2Y4, EP3 variant j, A2a, AT1B ヽ ETB ヽ GNRHR ヽ CELSR2, CCR7, HPR12 , HI ヽ GPR32, TEM5 _S GPR58, CELSR1, GPR85, Motilin R, HE6, CELSR3, CALCRL, RRH, GPR18, GPR35, P2Y10, C-TESTI2027296, GPR141 _S hRUP16, ITR, MRGX2, SNORF33, C-THYMU2011548 GPR107, GPR126, GPR4U GPR43, GPR68, GRPR, HEOAD54, hRUP9, mPRa ヽ mPRg, SRL, art, EP4, CCRls D5, suppresses the expression of a receptor selected from the group consisting of LTB4R and CXCR5, encoding the receptor (A) apoptosis of human obesity cells using siRNA or antisense DNA Guide, (b) inhibition of the activity I spoon, inhibition of (c) degranulation, (d) A method for suppressing at least one of production of inflammatory mediators, (e) suppression of production of cytokines, and (f) suppression of production of chemokine.

30. CysLTRl, GPR91 GPR34, EMR2, PAFR ヽ GPR105, TM7SF1 ヽ GPR37, CD97, MCI, FIRE, fMLPl, GPR65, EP3 variant h ヽ P2Y8, a G protein-coupled receptor expressed in human mast cells D2, EDG2, A3, C5R1, 0PN3, GPR27, CRTH2, EDG1, FPRL2, CCRL2, mglu6, CB2, GPR39, P2Y5, Al, P2Y4, EP3 variant j s A2aヽAT1BヽETBヽGNRHRヽCELSR2, CCR7, H4, GPR12, HI ヽ GPR32 _N TEM5, GPR58, CELSR1, GPR85, Motilin R, HE6 CELSR3, CALCRL, RRH, GPR18, GPR35, P2Y10, C-TESTI2027296s GPR141, hRUP16, ITR, MRGX2, SNORF33, C-THdMU2011 Suppressing the expression of a receptor selected from the group consisting of GPR107, GPR126, GPR41 _S GPR43, GPR68, GRPR ヽ HEOAD54, hRUP9, mPRa EP mPRg, SRL, H963, EP4, CCR1 D5, LTB4R and CXCR5. Atopic dermatitis, asthma, chronic obstructive pulmonary disease using siRNA or antisense DNA against the encoding gene Or a method of treating allergic diseases.

31. G protein-coupled receptor expressed in human mast cells and whose expression level fluctuates upon stimulation of glucocorticoid, C3aRl, GPR34, β1, GPR105, TM7SF, fMLPl, P2Y8, A3, CRTH2, Using siRNA or antisense DNA against a gene encoding the receptor, which suppresses the expression of a receptor selected from the group consisting of CCRL2, P2Y5, A2a, EBI'2, PARI, H4, RE2, CALCRLL and EP4. A method for controlling the action of dalcocorticoid on human mast cells.

3 2. CysLTR1, GPR91, GPR34, EMR2, PAFR ヽ GPR105, TM7SF1, GPR37, CD97, MC1, FIRE ヽ fMLP1, GPR65, EP3 variant h, P2Y8 _N , which are G protein-coupled receptors expressed in human mast cells D2, EDG2, A3, C5R1, 0PN3, GPR27, CRTH2, EDG1 ヽ FPRL2, CCRL2, mgl'u6, CB2, GPR39 ヽ P2Y5, Als P2Y4, EP3 variant j, A2a ヽ AT1B, ETB, GNRHR ヽ CELSR2, CCR7 , GPR12, Hl, GPR3, TEM5, GP58 CELSR1, GPR85, Motilin R, HE6, CELSR3, CALCRL, RRH _N GPR18, GPR35, P2Y10 ヽ C-TESTI2027296 GPR141, hRUP16, ITR ヽ MRGX2, SNORF33, C-THYMU54811抑制 す る suppresses the expression of a receptor selected from the group consisting of GPR107, GPR126, GPR41, GPR43, GPR68, GRPR, HEOAD54, hRUP9, mPRa, mPRg ヽ SRL, H963, EP4, CCR1, D5, LTB4R and CXCR5. Any one of (a) to (j) containing as an active ingredient siRNA or antisense DNA for a body-encoding gene Medicine.

 (a) Inducer of human mast cell apoptosis

 (b) human mast cell activation inhibitor

 (c) Inhibitor of degranulation of human mast cells

 (d) Human mast cell inflammatory mediator production inhibitor

 (e) Human mast cell cytoin production inhibitor

 (f) Chemokine production inhibitor for human mast cells

 (g) Remedies for atopic dermatitis

 (h) Asthma treatment

 (i) Drug for treating chronic obstructive pulmonary disease

 (j) Remedies for allergic diseases

3 3. Expressed in human mast cells, and a G protein-coupled receptor expression level by stimulation of glucocorticoids Koi de fluctuates, C3aRl, GPR34, βヽGPR105, TM7SF, fMLPl, P2Y8, A3, CRTH2 S A receiver selected from the group consisting of CCRL2, P2Y5, A2a, EBI2, PARI, H4, RE2, CALCRL 'and EP4 An agent for controlling the action of dalcocorticoid, which comprises as an active ingredient siRNA or antisense DNA against a gene encoding the receptor, which suppresses the expression of a receptor.

34. CysLTRl ヽ GPR91, a G protein-coupled receptor expressed on human mast cells, for the manufacture of a pharmaceutical composition for the treatment of atopic dermatitis, asthma, chronic obstructive pulmonary disease or allergic disease , GPR34, EMR2, PAFR ヽ GPR105, TM7SF1, GPR37 _S CD97, MCI ヽ FIRE ヽ fMLPl, GPR65, EP3 variant h, P2Y8, D2, EDG2, A3, C5R1 ヽ 0PN3, GPR27, C 、, 廳, FPRL2 CCRL2 ヽ mglu6 , CB2, GPR39, P2Y5, Al , P2Y4, EP3 variant j s A2aヽAT1BヽETB, GnRHRヽCELSR2, CCR7, H4, GPR12, HIヽGPRs 32 _S orchid, GPR58, CELSR1ヽGPR85, motilin R, HE 6, CELSR3s CALCRL, RRH, GPR18, GPR35, Ρ2Π0, C-TESTI2027296, GPR141, hRUP16, ITR, MRGX2, SNORF33, C-THYMU2011548, G10d, GPR107, GPR126, — GPR41, GPR43, GPR68, GRPR ヽ HEOAD54, hRUPg , SRL ヽ, EP4, CCR1 ヽ D5, LTB4R and CXCR5, which suppress the expression of a receptor selected from the group consisting of The use of siRNA or antisense DNA for the gene encoding.