WO2016006700A1 - 樹状細胞免疫受容体活性化剤、樹状細胞免疫受容体活性化方法、破骨細胞形成抑制剤、破骨細胞形成抑制方法、樹状細胞分化・増殖阻害剤、樹状細胞分化・増殖阻害方法、サイトカイン産生抑制剤、サイトカイン産生抑制方法、治療方法及びスクリーニング方法 - Google Patents
樹状細胞免疫受容体活性化剤、樹状細胞免疫受容体活性化方法、破骨細胞形成抑制剤、破骨細胞形成抑制方法、樹状細胞分化・増殖阻害剤、樹状細胞分化・増殖阻害方法、サイトカイン産生抑制剤、サイトカイン産生抑制方法、治療方法及びスクリーニング方法 Download PDFInfo
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- dendritic cell
- dcir
- inhibitor
- immune receptor
- osteoclast formation
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
Definitions
- the present invention relates to dendritic cell immune receptor activator, dendritic cell immune receptor activation method, osteoclast formation inhibitor, osteoclast formation inhibitor, dendritic cell differentiation / proliferation inhibitor, dendritic cell
- the present invention relates to a differentiation / proliferation inhibition method, a cytokine production inhibitor, a cytokine production inhibition method, a treatment method, and a screening method.
- Dendritic cell receptor immunoreceptor (Dendric Cell Immunoreceptor, hereinafter also referred to as DCIR) is a membrane protein expressed in cells such as dendritic cells that are major antigen-presenting cells, osteoclasts that are bone-resorbing cells, It has a domain (CRD) that recognizes sugar chains in the extracellular region, and an immunosuppressive signaling motif (ITIM) in the cytoplasmic region.
- the present inventors have succeeded in producing a mouse (Dcir ⁇ / ⁇ mouse) deficient in the DCIR gene first, and that the mouse spontaneously develops autoimmune diseases such as Sjögren's syndrome and attachment inflammation with age. Reported (for example, see Japanese Patent Application Laid-Open Nos.
- DCIR is thought to play a role in negatively controlling osteoclast formation, dendritic cell differentiation / proliferation and production of inflammatory cytokines. Therefore, if a ligand that specifically acts on DCIR is discovered, it will be an effective means for suppressing or alleviating the symptoms of bone metabolic diseases and autoimmune diseases. In addition, since dendritic cells play a central role in the immune system, therapeutic effects can be expected even in diseases such as allergies. Based on the above findings, the present inventors have found that keratan sulfate-II (KS-II) transmits signals into osteoclasts via SHP-1 as an endogenous ligand that specifically binds to DCIR (for example, , See International Publication No. 2011/105424).
- KS-II keratan sulfate-II
- An object of the present invention is to provide a growth inhibitor, a dendritic cell differentiation / proliferation inhibition method, a cytokine production inhibitor, a cytokine production inhibition method, a treatment method, and a screening method.
- a sugar chain having a structure represented by the following formula as a basic structure (however, sialic acid does not exist at two non-reducing ends of the following structure, and x and y each independently represents 3 or 4)
- a dendritic cell immune receptor activator comprising a compound having an active ingredient as an active ingredient.
- a method for activating a dendritic cell immune receptor comprising bringing the dendritic cell immune receptor activator according to ⁇ 1> above into contact with a dendritic cell immune receptor of a cell.
- An osteoclast formation inhibitor comprising a compound having a sugar chain structure represented by the above formula as an active ingredient.
- ⁇ 4> A method for inhibiting osteoclast formation, comprising bringing the osteoclast formation inhibitor according to ⁇ 3> above into contact with an osteoclast dendritic cell immune receptor.
- a dendritic cell differentiation / proliferation inhibitor comprising, as an active ingredient, a compound having a sugar chain structure represented by the above formula.
- a dendritic cell differentiation / proliferation inhibition method comprising contacting the dendritic cell differentiation / proliferation inhibitor according to ⁇ 5> above with a dendritic cell immunoreceptor of dendritic cells.
- a cytokine production inhibitor comprising, as an active ingredient, a compound having a sugar chain structure represented by the above formula.
- a method for suppressing cytokine production comprising bringing the cytokine production inhibitor according to ⁇ 7> above into contact with a dendritic cell immune receptor of dendritic cells.
- a treatment method comprising administering the dendritic cell immune receptor activator according to ⁇ 1> to a patient having a bone metabolic disease, an autoimmune disease or an allergic disease.
- Dendritic cell immunoreceptor by measuring the binding of a test substance to dendritic cell immune receptor in the presence of a compound having a sugar chain structure represented by the above formula or using said compound as a control
- a screening method comprising screening an activator or a dendritic cell immune receptor antagonist.
- a novel dendritic cell immunoreceptor activator, dendritic cell immunoreceptor activation method, osteoclast formation inhibitor, osteoclast formation inhibitor, dendritic cell differentiation / proliferation inhibitor Dendritic cell differentiation / proliferation inhibition method, cytokine production inhibitor, cytokine production inhibition method, treatment method and screening method are provided.
- FIG. 5 shows the results of flow cytometry analysis of DCIR mRNA quantitative data during osteoclast formation and DCIR expression in osteoclasts of wild-type mice and Dcir ⁇ / ⁇ mice.
- the solid line with the peak on the right represents the wild type
- the solid line with the peak on the right represents the Dcir-/-type
- the gray area represents the isotype control.
- Data are representative of 2 or 3 independent tests, error bars represent mean ⁇ standard deviation (triple measurement), * P ⁇ 0.05, ** P ⁇ 0.01. .
- the data is the result of performing real-time PCR for a predetermined number of days, calculated by the 2- ddCt method, and relative to macrophage data on the second day of culture as 1.
- Data are representative of 3 or more independent tests, error bars represent mean ⁇ standard deviation (triple measurement). It is a flow cytometry analysis result of DCIR expression on the cell surface of BMM on the 4th day of culture.
- the blue solid line represents the wild type
- the red solid line represents the Dcir ⁇ / ⁇ type
- the gray area represents the isotype control.
- Data are representative of 3 or more independent trials.
- FIG. 6 is a diagram (magnification 10 times) showing the degree of osteoclast formation of wild type and Dcir ⁇ / ⁇ . Data are representative of 3 or more independent tests, error bars are mean ⁇ standard deviation (triple measurement), ** P ⁇ 0.01. It is quantitative data of osteoclast mRNA. Data are representative of 2 or 3 independent tests, error bars are mean ⁇ standard deviation (triple measurement), * P ⁇ 0.05, ** P ⁇ 0.01.
- Tnfsf11 (RANKL) after stimulation of non-enriched osteoblasts cultured in osteogenic medium with VD3 (10 ⁇ 8 M) and PGE2 (10 ⁇ 6 M) or AA (50 ⁇ g / ml) for 24 hours.
- Tnfsflb (OPG) gene expression was quantified by real-time PCR.
- Data are representative of 3 or more independent tests, error bars represent mean ⁇ standard deviation (triple measurement). It is a flow cytometry analysis result of the number of osteoclast precursors in 8-week-old wild type mice and Dcir ⁇ / ⁇ mice. Bone marrow cells gated as negative for CD3, B220 and Ter119 are plotted against CD11b and Ly6C.
- FIG. 4 shows an increase in TRAP-positive mononuclear cells (MNC) in Dcir ⁇ / ⁇ mice immediately before osteoclast fusion (magnification 4 times).
- MNC TRAP-positive mononuclear cells
- Non-adherent BM collected from wild-type mice and Dcir-/-mice was cultured for 5 days in the presence of M-CSF with different concentrations and RANKL at 25 ng / ml, and images when TRAP staining was performed ( 4 times) and the results of counting the number of cells with 3 or more nuclei. Data are representative of two independent tests, error bars represent mean ⁇ standard deviation (triple measurement), ** P ⁇ 0.01.
- Non-adherent BM collected from wild-type mice and Dcir-/-mice was cultured for 5 days in the presence of RANKL at different concentrations and 5 ng / ml M-CSF, and images obtained when TRAP staining was performed ( 4 times) and the results of counting the number of cells with 3 or more nuclei.
- Data are representative of two independent tests, error bars represent mean ⁇ standard deviation (triple measurement), * P ⁇ 0.05, ** P ⁇ 0.01.
- FIG. 4 shows that BMM proliferates in response to M-CSF.
- Data are representative of 3 independent tests, error bars represent mean ⁇ standard deviation (triple measurement), * P ⁇ 0.05, ** P ⁇ 0.01.
- FIG. 3 is a diagram showing images of TRAP-positive cells (4 ⁇ ) and the number of apoptotic cells, in which osteoclasts were induced from the same number of wild-type BMM and Dcir ⁇ / ⁇ BMM. The part marked with an asterisk in the image represents apoptotic cells.
- Data are representative of two independent tests, error bars represent mean ⁇ standard deviation (triple measurement).
- FIG. 5 shows that the same number of wild-type BMM and Dcir ⁇ / ⁇ BMM were induced in osteoclasts in the presence of 5 ng / ml M-SCF and RANKL at different concentrations.
- FIG. 3 shows that Akt signals in response to M-CSF and RANKL in Bcir of Dcir ⁇ / ⁇ mice are enhanced. The lower number in each lane indicates the relative signal concentration of phosphorylated kinase. Data are representative of 3 or more independent trials. It is the bone structure analysis result of the tibia of an 8 week-old wild type mouse
- FIG. 8 shows micro CT images and bone parameter measurement results of femur trabeculae of 8-week-old wild type mice and Dcir ⁇ / ⁇ mice. Data are representative of two or more independent tests, error bars represent mean ⁇ standard error (4-6 replicates), * P ⁇ 0.05. It is a figure which shows the mode of bone formation of the wild type mouse
- Error bars represent mean value ⁇ standard error (4 to 6 measurements), and * P ⁇ 0.05 and ** P ⁇ 0.01. It is a flow cytometry analysis result of IFN- ⁇ positive T cells in peripheral blood. Data are representative of 3 independent trials. It is a flow cytometry analysis result of IFN- ⁇ positive T cells in lymph nodes. Data are representative of 3 independent trials. Error bars represent mean value ⁇ standard deviation (4-6 replicate measurements), * P ⁇ 0.05, ** P ⁇ 0.01. Serum concentrations of IFN- ⁇ in 12-week-old wild-type mice and Dcir ⁇ / ⁇ mice (each 10 mice) were analyzed by ELISA. The P value of wild type and Dcir-/-type by unpaired two-sided t-test was 0.10.
- Data are based on 3 independent trials.
- the black circles in the figure represent the data for each wild type individual, and the white circles represent the data for each Dcir-/-type individual.
- Data are representative of 3 independent trials.
- the effect of external addition of IFN- ⁇ on osteoblast formation is shown.
- the image of the alizarin red stained area was analyzed with ImageJ.
- Data are representative of 3 or more independent tests, error bars represent mean ⁇ standard deviation (triple measurement), * P ⁇ 0.05, ** P ⁇ 0.01. It shows a state of expression of osteogenesis-related genes after 14 days of culture in the presence of IFN- ⁇ .
- Data are representative of 3 or more independent tests, error bars represent mean ⁇ standard deviation (triple measurement), * P ⁇ 0.05, ** P ⁇ 0.01.
- FIG. 3 shows that binding of DCIR-Fc to asialotransferrin is CRD-dependent, but binding to heparin is CRD-independent.
- the results of DCIR-Fc and DCIR-Fc mutants (E197A-Fc, S199A-Fc, E197A / S199A-Fc) and the negative control Fc are shown. It is a result of a sugar chain microassay showing that DCIR-Fc binds to NA2.
- Data are representative of 3 or more independent tests, error bars represent mean ⁇ standard deviation (triple measurement), * P ⁇ 0.05, ** P ⁇ 0.01. It is a graph which shows that osteoclast formation is suppressed by NA2 addition.
- Data are representative of 3 or more independent tests, error bars represent mean ⁇ standard deviation (triple measurement), * P ⁇ 0.05, ** P ⁇ 0.01. It is a figure which shows the influence with respect to the signal through M-CSF and RANKL of NA2 when wild-type BMM is pre-treated with NA2 and stimulated with M-CSF or RANKL. It is a figure which shows that NA2 specifically controls the signal pathway by M-CSF and RANKL via DCIR.
- Dcir ⁇ / ⁇ BMM was treated with NA2 (1 ⁇ g / ml) for 6 hours at 37 ° C. in serum free medium before stimulation with M-CSF and RANKL. Thereafter, stimulation with M-CSF (20 ng / ml) or RANKL (100 ng / ml) was given for the time indicated in the figure. Data are representative of 3 independent trials. It is a figure which shows the production amount of the inflammatory cytokine when the stimulation by LPS is given to the bone marrow-derived dendritic cells in the presence or absence of NA2.
- a numerical range indicated by using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
- “Dendritic cell immunoreceptor activation” means that the dendritic cell immunoreceptor activator of the present invention is brought into contact with the dendritic cell immune receptor activator of the present invention. Means that the activity of the dendritic cell immunoreceptor is increased as compared with the case where the dendritic cell immunoreceptor is not contacted.
- “Inhibition of osteoclast formation” means that the osteoclast formation inhibitor of the present invention is brought into contact with the dendritic cell immune receptor, and the osteoclast formation inhibitor of the present invention is brought into contact with the dendritic cell immune receptor. It means that the formation of osteoclasts is suppressed as compared with the case where it is not allowed to occur. “Suppression of cytokine production” means that the cytokine production inhibitor of the present invention is brought into contact with the dendritic cell immune receptor, and the cytokine production inhibitor of the present invention is not brought into contact with the dendritic cell immune receptor. This means that production is suppressed.
- “Inhibition of dendritic cell differentiation / proliferation” means that the dendritic cell differentiation / proliferation inhibitor of the present invention is dendritic by contacting the dendritic cell differentiation / proliferation inhibitor of the present invention with a dendritic cell immune receptor. This means that dendritic cell formation is inhibited more than when not in contact with cellular immune receptors.
- treatment includes, in addition to eliminating symptoms of a disease to be treated, suppression of severity, reduction or alleviation of symptoms, and the like.
- the dendritic cell immune receptor activator of the present invention is a sugar chain having a structure represented by the following formula as a basic structure (however, sialic acid does not exist at the two non-reducing ends of the following structure, and x and y represents a compound having 3 or 4 (hereinafter also referred to as a specific sugar chain-containing compound) as an active ingredient.
- a compound having such a structure is a ligand that specifically acts on DCIR has not been reported so far.
- the specific sugar chain-containing compound has a sugar chain having a structure represented by the above formula as a basic structure
- the structure is not limited as long as the effect of the present invention is achieved. Therefore, it may have one branched chain at any site of the above basic structure or fucose may be added, and a compound having a sugar chain having such a structure is also included in the scope of the present invention. . Provided that no sialic acid is present at the two non-reducing ends of the structure. Examples of such sugar chain structures include the following.
- the method for producing the specific sugar chain-containing compound is not particularly limited, and examples thereof include collection from a living body, production by a genetic engineering method, and production by an organic synthetic chemical method.
- the structure or type is not limited as long as the effect of the present invention is achieved.
- a compound having a structure that is the same as or similar to the above, or a compound that is not derived from a living body is preferable.
- the biological compound include proteins (including peptides).
- the specific sugar chain-containing compound is a protein
- examples where the specific sugar chain-containing compound is a protein include those in which the reducing end of the sugar chain structure is bound to transferrin, acidic glycoprotein, fetuin, chicken-derived egg white protein, and the like.
- the specific sugar chain-containing compound is a compound not derived from a living body, the reducing end of the sugar chain structure is bonded to polyacrylamide (PAA), paranitrophenylphenol (pNP), aminopyridine (PA), etc. Can be mentioned.
- PAA polyacrylamide
- pNP paranitrophenylphenol
- PA aminopyridine
- the specific sugar chain-containing compound may be subjected to various modifications depending on the application.
- multimerization carriers such as micro beads and porcelain beads, proteins such as BSA and HSA, etc., including peptides
- amino group modification biotinylation, myristoylation, palmitoylation, acetylation, maleimidation, etc.
- Carboxy group modification (amidation, esterification, etc.)
- thiol group modification farnesylation, geranylation, methylation, palmitoylation, etc.
- hydroxyl group modification phosphorylation, sulfate, octanoylation, palmitoylation, palmitoleylation, Acetylation etc.
- fluorescent labeling PEGylation, unnatural amino acid, D-amino acid, introduction of various spacers, etc.
- the dendritic cell immune receptor activator of the present invention may contain components other than the specific sugar chain-containing compound depending on the use mode.
- components other than the specific sugar chain-containing compound include a medium used for preparing a dendritic cell immune receptor activator and a pharmaceutical additive.
- Additives for formulation include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, suspending agents, emulsifiers, buffers And solvents.
- the form of the dendritic cell immune receptor activator of the present invention is not particularly limited, and can be selected according to the use.
- forms suitable for oral administration such as tablets, granules, powders, capsules, suspensions, syrups, emulsions, limonades, ampoules for injection, freeze-dried powder for injection, dry powder for pulmonary administration Etc.
- the dendritic cell immune receptor activation method of the present invention includes contacting the dendritic cell immune receptor activator of the present invention with a dendritic cell immune receptor of a cell.
- the method for bringing the dendritic cell immunoreceptor activator into contact with cells is not particularly limited, and examples thereof include surgical treatment such as oral administration, intravenous administration, and indwelling.
- the amount of the dendritic cell immunoreceptor activator to be contacted with the dendritic cell immunoreceptor is not particularly limited, and includes the state of activity of the dendritic cell immune receptor, the intended degree of activation, and a specific sugar chain-containing compound. It can be selected according to the type and amount of other components used.
- DCIR dendritic cell immunoreceptor activation method of the present invention
- the biological mechanism negatively controlled by DCIR when the biological mechanism negatively controlled by DCIR is excessive for some reason, DCIR is produced.
- the biological mechanism is suppressed by activating it.
- Biological mechanisms negatively controlled by DCIR include osteoclast formation, osteoclast proliferation response, cytokine production, dendritic cell proliferation response, dendritic cell function control, monocyte response, neutrophil function Control etc. are mentioned. Therefore, according to the dendritic cell immune receptor activation method of the present invention, osteoclast formation, dendritic cell differentiation / proliferation, cytokine production, and the like can be achieved.
- the type of cells having DCIR activated by the dendritic cell immunoreceptor activation method of the present invention is not particularly limited as long as the effects of the present invention are obtained. Examples thereof include dendritic cells, osteoclasts, macrophages, monocytes, neutrophils and the like.
- the osteoclast formation inhibitor of the present invention contains a specific sugar chain-containing compound as an active ingredient.
- the specific sugar chain-containing compound contained as an active ingredient acts as a ligand that activates DCIR.
- DCIR plays a role in negatively controlling osteoclast formation. Therefore, osteoclast formation can be suppressed by bringing the osteoclast formation inhibitor of the present invention into contact with DCIR to activate DCIR.
- osteoclast formation inhibitor of the present invention and the specific sugar chain-containing compound contained as an active ingredient include the dendritic cell immune receptor activator of the present invention and the specific sugar chain-containing compound contained as an active ingredient. This can be the same as the above-described specific embodiment.
- the osteoclast formation inhibitory method of the present invention includes contacting the osteoclast formation inhibitor of the present invention with an osteoclast dendritic cell immune receptor.
- the method for bringing the osteoclast formation inhibitor into contact with the dendritic cell immune receptor is not particularly limited, and examples thereof include oral administration, intravenous administration, and surgical treatment such as indwelling.
- the amount of osteoclast formation inhibitor to be brought into contact with the dendritic cell immune receptor is not particularly limited, and is used in combination with the state of osteoclast formation, the degree of intentional suppression of osteoclast formation, and a specific sugar chain-containing compound. Can be selected according to the type and amount of the components.
- the balance between bone formation by osteoblasts and bone resorption by osteoclasts is some reason. If the bone resorption by osteoclasts is relatively dominant, the osteoclast formation is intentionally suppressed to restore balance and maintain bone homeostasis. .
- the dendritic cell differentiation / proliferation inhibitor of the present invention contains a specific sugar chain-containing compound as an active ingredient.
- the specific sugar chain-containing compound contained as an active ingredient acts as a ligand that activates DCIR.
- DCIR plays a role in negatively controlling dendritic cell differentiation / proliferation. Therefore, the dendritic cell differentiation / proliferation can be inhibited by bringing the dendritic cell differentiation / proliferation inhibitor of the present invention into contact with DCIR to activate DCIR.
- dendritic cell differentiation / proliferation inhibitor of the present invention and the specific sugar chain-containing compound contained as an active ingredient include the dendritic cell immune receptor activator of the present invention and the specific sugar contained as an active ingredient.
- specific examples of the chain-containing compound can be the same as those described above.
- the dendritic cell differentiation / proliferation inhibition method of the present invention includes contacting the dendritic cell differentiation / proliferation inhibitor of the present invention with a dendritic cell immunoreceptor of the dendritic cell.
- the method for contacting the dendritic cell differentiation / proliferation inhibitor with the dendritic cell immune receptor is not particularly limited, and examples thereof include oral administration, intravenous administration, and surgical treatment such as indwelling.
- the amount of the dendritic cell differentiation / proliferation inhibitor to be contacted with the dendritic cell immunoreceptor is not particularly limited, and the state of dendritic cell differentiation / proliferation and the degree of inhibition of the intended dendritic cell differentiation / proliferation It can be selected according to.
- the cytokine production inhibitor of the present invention contains a specific sugar chain-containing compound as an active ingredient.
- the specific sugar chain-containing compound contained as an active ingredient acts as a ligand that activates DCIR.
- DCIR plays a role in negatively controlling the production of certain cytokines. Therefore, cytokine production can be suppressed by bringing the cytokine production inhibitor of the present invention into contact with DCIR to activate DCIR.
- Specific embodiments of the cytokine production inhibitor of the present invention and the specific sugar chain-containing compound contained as an active ingredient are specific examples of the dendritic cell immune receptor activator of the present invention and the specific sugar chain-containing compound contained as an active ingredient. It can be the same as that mentioned above as a specific aspect.
- the method for inhibiting cytokine production of the present invention includes contacting the cytokine production inhibitor of the present invention with a dendritic cell immune receptor of dendritic cells.
- the method for bringing the cytokine production inhibitor into contact with the dendritic cell immune receptor is not particularly limited, and examples thereof include surgical treatment such as oral administration, intravenous administration, and indwelling.
- the amount of cytokine production inhibitor to be brought into contact with the dendritic cell immune receptor is not particularly limited, and the state of cytokine production, the degree of intended suppression of cytokine production, and the type and amount of other components used with the specific sugar chain-containing compound It can be selected according to etc.
- the kind of cytokine whose production is suppressed by the method for suppressing cytokine production of the present invention is not particularly limited as long as the effect of the present invention is obtained.
- Examples include IFN- ⁇ , IL-6, IL-12, IL-23, IL-1, IL-17, and IL-17F.
- the treatment method of the present invention comprises administering the dendritic cell immune receptor activator of the present invention to a patient with bone metabolic disease, autoimmune disease or allergic disease.
- the dendritic cell immune receptor activator of the present invention when brought into contact with DCIR, the specific sugar chain-containing compound contained as an active ingredient acts as a ligand for activating DCIR.
- a biological mechanism that is negatively controlled by DCIR can be intentionally suppressed. Examples of such biological mechanisms include osteoclast formation and cytokine production. Therefore, the therapeutic method of the present invention is a method for treating various bone metabolic diseases associated with excessive bone resorption, and various autoimmune diseases or allergic diseases accompanied by excessive immune reaction or inflammation due to the production of inflammatory cytokines.
- the specific sugar chain-containing compound acts in a DCIR-specific manner to suppress the functions of dendritic cells, osteoclasts, etc., it is possible to provide a therapeutic method that can limit the action point of medicinal effect and has few side effects.
- Examples of the bone metabolic diseases include osteoarthritis, spondyloarthropathy, osteoporosis, Paget's disease, osteoarthritis, marble bone disease, and periodontal disease.
- Examples of autoimmune diseases include rheumatoid arthritis, multiple sclerosis, Guillain-Barre syndrome, Goodpasture syndrome, type I diabetes, thyroiditis, ulcerative colitis, Sjogren's syndrome, and the like.
- Examples of allergic diseases include bronchial asthma, atopic dermatitis, conjunctivitis, food allergy, anaphylaxis, contact dermatitis, allergic rhinitis, chronic glomerulonephritis and the like.
- the method of administering the dendritic cell immunoreceptor activator of the present invention to a patient is not particularly limited, and examples thereof include surgical treatment such as oral administration, intravenous administration, and indwelling.
- the amount of dendritic cell immunoreceptor activation administered to the patient is not particularly limited, and the type and condition of symptoms, the age and physique of the patient, the state of DCIR activity, the degree of intended DCIR activation, and the specific sugar chain content It can be selected according to the type and amount of other components used together with the compound.
- the specific mode of the dendritic cell immunoreceptor activator administered to a patient in the treatment method of the present invention and the specific sugar chain-containing compound contained as an active ingredient is the dendritic cell immunoreceptor activator of the present invention.
- the specific sugar chain-containing compound contained as an active ingredient can be the same as described above as a specific embodiment.
- the screening method of the present invention measures the binding of a test substance to a dendritic cell immune receptor in the presence of a specific sugar chain-containing compound or using a specific sugar chain-containing compound as a control, and dendritic cell immune receptor activity. Screening for an agent or dendritic cell immune receptor antagonist.
- the test substance is a DCIR activator or Whether it is a DCIR antagonist can be determined.
- Measurement of binding to DCIR can be performed in vitro or in vivo. When screening in vivo, the binding may be compared using as an index the amount of osteoclast formation, the degree of dendritic cell differentiation / proliferation, the amount of cytokine production, and the like.
- ⁇ Test Example 1 M-CSF-induced bone marrow formed at the mRNA and protein levels in the presence of macrophage colony-stimulating factor (M-CSF) and nuclear factor ⁇ B ligand receptor activator (RANKL), factors involved in osteoclast differentiation DCIR was expressed in macrophage cells (BMM) and osteoclasts (FIGS. 1-3). Gene expression increased with osteoclast maturation. DCIR was detected by CD11b positive cells (OsteoMacs) but not by enriched osteoblasts (FIG. 4).
- M-CSF macrophage colony-stimulating factor
- RNKL nuclear factor ⁇ B ligand receptor activator
- M-CSF is known to be involved in macrophage adhesion and osteoclast survival, but changes in Dcir-/-BMM adhesion activity and long-term cultured Dcir-/-osteoclast apoptosis was not seen (FIGS. 21-23). This means that DCIR-mediated signal negatively regulates proliferation induced by M-CSF.
- IFN- ⁇ production was remarkable in peripheral blood, brachial lymph nodes, and axillary lymph nodes. It can be seen that IFN- ⁇ production is not significant in the mesenteric lymph nodes, and that the serum concentration of IFN- ⁇ in Dcir ⁇ / ⁇ mice tends to be higher than in wild type mice, and the cytokine environment of Dcir ⁇ / ⁇ mice It was shown that it was inclined toward the IFN- ⁇ environment (FIGS. 30 to 32). These results led to the hypothesis that IFN- ⁇ is a powerful stimulator of osteoblast formation.
- IFN- ⁇ acts on osteoblasts was supported by the bone structure of Ifn- ⁇ and Dcir double-deficient mice and Ifn- ⁇ -deficient mice being equivalent to that of wild-type mice. . That is, it was suggested that IFN- ⁇ , which is considered to be an osteoclast inhibitor, does not act on osteoclasts in a steady state but may act on osteoblasts (FIG. 35). Overall, increased bone formation in Dcir-/-mice occurs due to systemic production of IFN- ⁇ , indicating that the net balance of IFN- ⁇ in vivo leans toward bone formation.
- Example 1 Ligand binding is required for the receptor to initiate the signal cascade and exert its biological function. Since the in vitro culture system of osteoclast formation exists only in BMM, it was speculated that the ligand of DCIR is expressed in BMM or osteoclasts. Some macrophages and osteoclasts were positive for DCIR-Fc staining that occurred when fusion between the extracellular domain of DCIR and the Fc region of human IgG2 occurred, but the binding was a change in the amino acid of CRD Decreased by DCIR E197A / S199A-Fc, which is a DCIR-Fc variant that gives rise to.
- DCIR-Fc binding was reduced by EDTA and trypsin. This indicates that DCIR interacts with glycoproteins in macrophages and osteoclasts in a CRD-dependent or Ca 2+ -dependent manner (FIG. 36).
- NA2 is a functional DCIR ligand that suppresses osteoclast formation by negatively controlling the M-CSF signal cascade and the RANKL signal cascade.
- NA2 is a ligand that specifically and functionally binds to DCIR.
- Dendritic cells were prepared by induction of Flt3L differentiation as follows.
- the bone marrow cells of the femur of wild type mice were removed, and the erythrocytes were placed in a hemolysis buffer (140 mM NH 4 Cl and 17 mM Tris-HCl, pH 7.2) and placed on ice for 10 minutes.
- Cells were seeded in a medium containing 10% FBS, antibiotic mixture, essential amino acid, 2-mercaptoethanol in a well of 100 ng / ml human Flt3L recombinant to a million per well in a 24-well plate. .
- the obtained dendritic cells were activated with an agonist reagent (LPS) in the presence or absence of 1 ⁇ g / ml NA2 for 24 hours.
- LPS agonist reagent
- the supernatant was collected and the amount of inflammatory cytokines (IL-6 and p40) was examined by ELISA.
- IL-6 and p40 inflammatory cytokines
- the two bars on the left represent the amount of inflammatory cytokine produced when LPS was not stimulated
- the two bars on the right represent LPS-stimulated production
- the black bar represents the production when stimulated in the presence of NA2. Indicates the amount.
- mice All mice were bred at the University of Tokyo or Tokyo University of Science in accordance with the guidelines of the Zoological Society of Japan, and all experiments were approved by the University of Tokyo Institute of Medical Science or the Animal Science Committee of Tokyo University of Science. .
- the base sequence of the mouse DCIR gene is Gene ID: Clec4a2, NCBI Accession No. This corresponds to the sequence of bases 273 to 1061 in NM001170332.
- ⁇ -MEM ⁇ -minimum essential medium
- penicillin 100 units / ml
- streptomycin 100 ⁇ g / ml
- 10% heat-inactivated fetal bovine serum were added to isolate BM cells.
- Red blood cells were disrupted with lysis buffer (140 mM NH 4 Cl and 17 mM Tris-HCl, pH 7.2) on ice for 10 minutes and pre-incubated for 1 hour in 100 mm diameter dishes.
- Non-adherent cells were collected and seeded with 50,000 cells per well in a 96-well plate in the presence of 20 ng / ml human M-CSF recombinant (R & D Systems) for 2 days Oita.
- Cells grown by M-CSF were used as bone marrow-derived macrophages (BMM).
- BMM was further cultured in the presence of 20 ng / ml M-CSF and 100 ng / ml soluble human RANKL recombinant (Oriental Yeast Co., Ltd.), and the medium was changed every 2 days. .
- BMM was cultured for 6 days in the presence of 20 ng / ml M-CSF, and the medium was changed every 2 days.
- BMM cultured in 100 mm dishes were dissociated with Cell Dissolution Solution Non-zymatic 1 ⁇ (Sigma-Aldrich) and the same number of BMM was reseeded to induce osteoclast formation.
- Adherent cells were fixed with 10% formalin for 3 minutes and further fixed with fixation buffer (50% ethanol and 50% acetone) for 1 minute. The fixed cells were stained with naphthol AS-MX phosphate (Nacalai Tesque) and Fast Red (Nacalai Tesque) for 10-15 minutes at room temperature and washed with water for 3 minutes. The number of TRAP-positive cells having 3 or 10 cell nuclei as multinucleated osteoclasts was counted.
- Mouse DCIR cDNA was amplified using sense: 5'-cgggaccccaccgggcttcagaaatacacttatg (SEQ ID NO: 1) and antisense: 5'-cggatattctataagtttttttttttca (SEQ ID NO: 2) as primers.
- the product obtained by PCR was cloned into pMXs-IRES-Puro (provided by Professor Toshio Kitamura of the University of Tokyo) at the BamHI and EcoRI sites.
- the pMXs vector was isolated from E. coli (DH5 ⁇ ).
- the DCIR Y / F mutant was produced using KOD-Plus-Mutageness Kit (Toyobo Co., Ltd.). That is, forward primer for substituting phenylalanine for tyrosine in ITIM of DCIR: 5′-cactTttgcagaagtgagattcaagaatgaatc (SEQ ID NO: 3, capital letter T is adenosine) and reverse primer: 5′-atttgagagcccatggtgggggattggtgcincctggtcin ) Designed. Inverse PCR was performed using the pMXs vector as a template, and The methylated DNA of the E. coli cell line was digested with digestible DpnI.
- the undigested PCR product was self-flyed at 16 degrees for 1 hour using T4 polynucleotide kinase and ligase.
- the plat-E packaging cell line (provided by Professor Toshio Kitamura of the University of Tokyo) was cultured in 10% FBS ⁇ -MEM and split at a ratio of 1: 5 every other day.
- Retroviral vectors were introduced into Plat-E cells using Lipofectamine 2000 (Invitrogen). After 8 hours of incubation, the medium was changed and incubated for an additional 24 hours. Retroviral supernatant was collected and frozen at ⁇ 80 ° C. until use.
- non-adherent bone marrow cells were cultured in 100 ⁇ l medium containing 20 ⁇ l retrovirus supernatant and the medium was changed every other day.
- BMM was recovered using Cell Dissolution Solution Non-ezymatic 1 ⁇ (Sigma-Aldrich) and placed in each well of a 96-well plate in the presence of M-CSF with varying concentrations. After 2-3 hours, 1.0 ⁇ Ci [3H] thymidine (PerkinElmer) was added and incubated for an additional 48 hours at 37 ° C. Cells were passed through a glass fiber filter using a Skatron microplate washer supernatant collection system (Molecular Device). The introduction of [ 3 H] thymidine into DNA was confirmed by a MicroBeta microplate counter system (PerkinElmer).
- FACS analysis Blood cell lysis of BM cells and peripheral blood mononuclear cells (PBMC) was performed on ice using hemolysis buffer (140 mM NH 4 Cl and 17 mM Tris-HCl, pH 7.2). BMM and PBS were separated and washed with FACS buffer (PBS containing 2% FBS) to remove serum components. The Fc receptor of these cells was blocked with 2.4G2, an Fc receptor blocker, at 4 degrees for 10 minutes. The cells were then washed twice with FACS buffer and incubated with fluorescently labeled antibody for 30 minutes on ice.
- hemolysis buffer 140 mM NH 4 Cl and 17 mM Tris-HCl, pH 7.2
- FACS buffer PBS containing 2% FBS
- APC- or PE-CD11b BioLegend
- FITC-Ly6c BioLegend
- APC-c-Fms BioLegend
- biotin-RANK eBioscience
- PE-Clec4a R & D Systems
- FITC-CD3, Pacific Blue-CD4, APC-CD8, BV510-CD11b, and PE-IFN- ⁇ were used.
- DCIR-Fc-protein DCIR-Fc-protein variant, or Fc-protein to form a protein complex consisting of Fc-protein for detection of DCIR ligand and anti-IgG antibody Of FITC-labeled anti-human IgG (Jackson ImmunoResearch Laboratories) and a buffer containing calcium and magnesium (TBS containing 2 mM CaCl 2 , 2 mM MgCl 2 and 0.5% BSA) for 15 minutes at room temperature. Macrophages and osteoclasts were stained with the above protein complex for 1 hour at 4 ° C. and washed with Ca 2+ / Mg 2+ buffer.
- TBS buffer containing calcium and magnesium
- Flow cytometry was performed using FACSCanto II (Becton Dickinson) and analyzed using Flowjo software (Tree Star).
- FACSCanto II Becton Dickinson
- Flowjo software Te Star
- the cells were first stained with 2.4G2, and then the surface antigen before fixation was stained with FITC-CD3.
- immobilization / permeabilization buffer eBioscience
- the immobilization / permeabilization buffer was decanted and washed twice with FACS buffer containing 0.1% saponin. These cells were stained with primary antibodies for surface antigens and intracellular molecules and diluted with 0.1% saponin buffer.
- Quantification was performed using a CFX384 TM real-time system (Bio-Rad Laboratories, Inc.). The cycle number was 95 ° C for 1 minute in the first cycle, and the subsequent 44 cycles were 95 ° C for 3 seconds / 60 ° C for 30 seconds.
- DCIR sense 5'-cctggtgattacttatgctgtgtgt-3 '(SEQ ID NO: 5)
- antisense 5'-gtcagaagagccttgtttccttc-3' (SEQ ID NO: 6)
- GAPDH sense 5'-ttcaccaccgagga-3 (SEQ ID NO: 5)
- Antisense 5′-ggcatggactgtgtgtcatga-3 ′ (SEQ ID NO: 8) was used.
- the final primer concentration was 400 nM.
- GAPDH was used as an internal housekeeping gene.
- the target gene and housekeeping gene were simultaneously quantified in one plate using a water sample as a negative reference.
- RNA quality and initial quantity between samples were normalized with GAPDH.
- sense 5′-catttccccttactccccccgg-3 ′ (SEQ ID NO: 9)
- antisense 5′-gcatgagtgtccagagacccc-3 ′ (SEQ ID NO: 9) 10) was used to amplify the 686 pb product, and the PCR reaction was performed for 30 cycles. 10 seconds at 98 ° C. Le, 30 seconds at 55 ° C., was used for one minute went. GAPDH as an internal standard at 72 ° C..
- NP-40 solution 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , proteinase inhibitor cocktail (Thermo Fisher Scientific) and phosphatase inhibitor cocktail (Roche)).
- a lysate was obtained. Lysates were clarified by centrifuging on ice for 10 minutes and centrifuging at 15,000 rpm for 10 minutes. Equal volumes of lysate samples were separated on an SDS-polyacrylamide gel at 230 V for 30 minutes. The gel was electrically transferred onto the PVDF membrane at a constant current of 230 mA and a semi-dry system (Bio-Rad Laboratories, Inc.) in 25 minutes per membrane.
- the PVDF membrane was blocked at room temperature for 1 hour in 5% BSA (Sigma-Aldrich) dissolved in TBS containing 5% Tween 20, and incubated with the primary antibody at 4 ° C overnight, followed by HRP-labeled secondary antibody. Probed with.
- the PVDF membrane visualized the protein signal using ECL Prime Western Blotting Detection System (GE Healthcare).
- cDNA having amino acid sequences 70 to 238 covering the extracellular domain (EC) of mouse DCIR was subcloned into the pFUSE-hIgG2-Fc2 vector (Invitrogen) at the Bgl II site.
- the cDNA encoding EC was prepared using sense: 5′-ataagatctcaaaaagtactctcaactcttt-3 ′ (SEQ ID NO: 11) and antisense: 5′-ataagatcttaagttttttttttcatc-3 ′ (SEQ ID NO: 12) as primers.
- a site-specific mutant of the CRD domain in which glutamic acid and serine were substituted with alanine was prepared using the KOD-Plus-Mutageness kit (Toyobo Co., Ltd.). The following were used as primers.
- Plasmid vectors for obtaining DCIR-Fc (EC of mDCIR), DCIR-Fc mutant (inactive form of CRD domain), and Fc-protein (immunoglobulin constant region) were prepared. Subsequently, these plasmids were introduced into HEK293T cells using Lipofectamine LTX (Invitrogen), and the cells were incubated with Opti-MEM (Invitrogen) for 15 days. The medium was changed every 3 days. The Fc-chimeric protein was purified from the recovered supernatant by affinity chromatography using Protein A-Sepharose (GE Healthcare).
- BMM cultured for 6 days was collected with Cell Dissolution Solution Non-zymatic 1 ⁇ (Sigma-Aldrich) and resuspended in TSA buffer (TBS containing 2 mM CaCl 2 , 2 mM MgCl 2 and 0.5% BSA). It was. The BMM was then treated with 0.5 ⁇ l neuraminidase (Roche) per 100,000 cells for 30 minutes at 37 ° C. Thereafter, the cells were washed with TSA buffer and FACS analysis was performed.
- osteoclasts were induced from non-adherent BM cells in the presence of 1 ⁇ l neuraminidase (Roche). Neuraminidase was replenished when the medium was changed every other day.
- Non-decalcified bone tissue is placed in methyl benzoate for 15 minutes to facilitate the penetration of glycol methacrylate (GMA) into the tissue, then immersed in GMA (4 ° C.) containing 5% methyl benzoate for 2 hours. Changed three times. The bone tissue was then embedded in GMA. A 3 ⁇ m-thick tibia fragment was prepared using a microtome (Sakura Finetech Japan Co., Ltd.), and fluorescence in the calcification front was detected.
- GMA glycol methacrylate
- calcified surface calcified surface / bone surface [MS] / [BS];%), bone calcification rate ([MAR]; ⁇ m / day), bone formation rate ( [BFR] / [Bs]; ⁇ m 3 / ⁇ m 2 / day) was examined with an image analyzer (System supply) at a magnification of 400 times.
- Histomorphometric analysis of mouse tibia was performed at Kureha Analysis Center. Wild-type and Dcir ⁇ / ⁇ mouse bones were removed from 5-6 mice per group of 8 week old mice and fixed with 70% ethanol. In order to measure histomorphometric parameters of bone structure, femoral tissue fragments embedded in 3 ⁇ m thick GMA were stained with toluidine blue. The columnar bone parameter was defined as the area of secondary cancellous bone having a width of 1.05 mm in the distal direction from a point separated by 0.3 mm from the growth plate. The columnar bone at the metaphysis was observed with a HistometryRT CAMERA.
- osteoclasts [Oc.N] / 100 mm
- osteoclast surface area [Oc.S] / [BS];%) have one or more nuclei and form resorption pits on the columnar bone surface. Cells were counted as osteoclasts. Bone remodeling parameters including osteoblast count ([Ob.N] / 100 mm) and osteoblast surface area ([Ob.S] / [BS];%) were measured in cross sections stained with toluidine blue.
- Tb.Sp 1 / Tb.N ⁇ Tb.Th.
- Tb.Spac 1 / Tb.N
- PBMC Peripheral blood mononuclear cells
- phorbol myristate acetate final concentration: 500 ng / ml
- ionomycin final concentration: 50 ng / ml
- brefeldin A final concentration: 10 ⁇ g / ml
- Calvarial cells were prepared by continuous digestion and routine methods. That is, the calvaria was removed from a newborn mouse 1 to 2 days old and the adhesive mesenchymal tissue was removed. This calvaria was treated with a decomposition solution containing 0.1% collagenase (Wako Pure Chemical Industries, Ltd.) and 0.1% dispase (Roche Diagnostics Inc.) for 10 minutes at 37 ° C. with stirring. A first digestion was performed. The solution containing debris was discarded, and the remaining calvaria was further digested with the same decomposition solution at 37 ° C. for 1 hour.
- a decomposition solution containing 0.1% collagenase (Wako Pure Chemical Industries, Ltd.) and 0.1% dispase (Roche Diagnostics Inc.) for 10 minutes at 37 ° C. with stirring. A first digestion was performed. The solution containing debris was discarded, and the remaining calvaria was further digested with the same decomposition solution at 37 ° C. for 1 hour.
- the isolated cells were suspended in ⁇ -MEM (10% FCS, penicillin 100 units / ml, streptomycin 100 ⁇ g / ml) and seeded in a 12-well plate at a concentration of 200,000 per well. After culturing for 2 days, the cells were cultured in osteogenic medium (10% FBS, 50 ⁇ g / ml ascorbic acid (Sigma-Aldrich), 10 mM ⁇ -glycerophosphate (Calbiochem) and ⁇ -MEM supplemented with antibiotics) for 14 days or Incubated for 21 days. The medium was changed every 3 days.
- Mouse osteoblasts were cultured and differentiated as described above. Primary calvarial cells were cultured in the presence of mouse IFN- ⁇ recombinant (PeproTech). IFN- ⁇ was added every time the osteogenic medium was changed.
- Osteoblasts were prepared by culturing in 100 mm dishes for 5 days by the method described above. The osteoblasts were collected enzymatically and 20,000 cells per well were replated on Osteo Assay plates (Corning) in the presence of M-CSF and RANKL. After 3 days, cells were removed by ultrasonic disruption in 1M ammonia solution for 30 seconds. An image of the whole surface of the well coated with calcium phosphate was taken into a microscope (Keyence Corporation), and the area of the absorbed region was analyzed with ImageJ.
- a 96-well plate was coated with 10 mg / ml fibronectin (Sigma-Aldrich) and placed at 4 ° C. overnight. The wells were washed twice with PBS, and 50,000 BMMs cultured for 2 days were inoculated with M-CSF (20 ng / ml) or without M-CSF and allowed to pass for 2 minutes, 5 minutes, or 10 minutes. The cells were then washed twice with DMEM, fixed with methanol for 2 minutes, and stained with 0.5% distilled water of crystal violet. After washing 5 times with distilled water, 100 ml of 1% SDS was added to each well to solubilize the dye, and the absorbance at 595 nm was measured.
- Tnfsf11 (RANKL) Sense: 5′-cagcatcgctctgtttcctgta-3 ′ (SEQ ID NO: 33), Antisense: 5′-ctgcgtttcatggagtctca-3 ′ (SEQ ID NO: 34)
- Tnfsf11b (OPG) Sense: 5'-ggcgtgtacctgaggatcg-3 '(SEQ ID NO: 35), Antisense: 5'-gagaagaacccattctgacattt-3' (SEQ ID NO: 36) mRNA quality and quantity were normalized using GAPDH.
- the sugar chain-protein interaction was detected by an evanescent field fluorescence excitation detection system using a sugar chain microarray. That is, 3 spots of a sugar chain probe containing glycoprotein and glycoside-polyacrylamide (PAA) on a microarray grade epoxy activated glass slide (Schott) using a non-contact type microarray printing robot (MicroSys 4000, Genomic Solutions). Fixed one by one. The glass slide was incubated at 25 ° C.
- PAA glycoprotein and glycoside-polyacrylamide
- the prepared protein complex was applied to a glass slide at 100 ⁇ l per chamber and incubated at 20 ° C. for 3 hours.
- the binding was detected with an evanescent field fluorescence excitation scanner (SC-Profiler, GP Bioscience) without going through a washing step.
- SC-Profiler evanescent field fluorescence excitation scanner
- the data is Array Pro analyzer Ver. Analysis was performed with 4.5 (Media Cybernetics).
- the net intensity obtained by subtracting the raw intensity from the background intensity (net intensity) is shown as the average value ⁇ standard deviation of triplicate measurements.
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Abstract
Description
<1>下記式で表される構造を基本構造とする糖鎖(ただし、下記構造の2つの非還元末端にシアル酸が存在せず、x及びyはそれぞれ独立に3又は4を表す)を有する化合物を有効成分として含む、樹状細胞免疫受容体活性化剤。
<2>前記<1>に記載の樹状細胞免疫受容体活性化剤を細胞の樹状細胞免疫受容体に接触させることを含む、樹状細胞免疫受容体活性化方法。
<3>上記式で表される糖鎖構造を有する化合物を有効成分として含む、破骨細胞形成抑制剤。
<4>前記<3>に記載の破骨細胞形成抑制剤を破骨細胞の樹状細胞免疫受容体に接触させることを含む、破骨細胞形成抑制方法。
<5>上記式で表される糖鎖構造を有する化合物を有効成分として含む、樹状細胞分化・増殖阻害剤。
<6>前記<5>に記載の樹状細胞分化・増殖阻害剤を樹状細胞の樹状細胞免疫受容体に接触させることを含む、樹状細胞分化・増殖阻害方法。
<7>上記式で表される糖鎖構造を有する化合物を有効成分として含む、サイトカイン産生抑制剤。
<8>前記<7>に記載のサイトカイン産生抑制剤を樹状細胞の樹状細胞免疫受容体に接触させることを含む、サイトカイン産生抑制方法。
<9>前記<1>に記載の樹状細胞免疫受容体活性化剤を骨代謝疾患、自己免疫疾患又はアレルギー疾患の患者に投与することを含む、治療方法。
<10>上記式で表される糖鎖構造を有する化合物の存在下、又は前記化合物を対照として、被検物質の樹状細胞免疫受容体への結合性を測定し、樹状細胞免疫受容体活性化剤又は樹状細胞免疫受容体拮抗剤をスクリーニングすることを含む、スクリーニング方法。
本明細書において「~」を用いて示された数値範囲は、「~」の前後に記載される数値をそれぞれ最小値及び最大値として含む範囲を示す。
「樹状細胞免疫受容体活性化」とは、本発明の樹状細胞免疫受容体活性化剤を樹状細胞免疫受容体に接触させることで、本発明の樹状細胞免疫受容体活性化剤を樹状細胞免疫受容体に接触させない場合よりも樹状細胞免疫受容体の活性が高められた状態にすることを意味する。
「破骨細胞形成抑制」とは、本発明の破骨細胞形成抑制剤を樹状細胞免疫受容体に接触させることで、本発明の破骨細胞形成抑制剤を樹状細胞免疫受容体に接触させない場合よりも破骨細胞の形成が抑制された状態にすることを意味する。
「サイトカイン産生抑制」とは、本発明のサイトカイン産生抑制剤を樹状細胞免疫受容体に接触させることで、本発明のサイトカイン産生抑制剤を樹状細胞免疫受容体に接触させない場合よりもサイトカインの産生が抑制された状態にすることを意味する。
「樹状細胞分化・増殖阻害」とは、本発明の樹状細胞分化・増殖阻害剤を樹状細胞免疫受容体に接触させることで、本発明の樹状細胞分化・増殖阻害剤を樹状細胞免疫受容体に接触させない場合よりも樹状細胞の形成が阻害された状態にすることを意味する。
「治療」とは、治療対象である疾病の症状を消失させることのほか、重症化の抑制、症状の軽減又は緩和もこの用語に包摂される。
本発明の樹状細胞免疫受容体活性化剤は、下記式で表される構造を基本構造とする糖鎖(ただし、下記構造の2つの非還元末端にシアル酸が存在しせず、x及びyはそれぞれ独立に3又は4を表す)を有する化合物(以下、特定糖鎖含有化合物とも称する)を有効成分として含む。このような構造を有する化合物がDCIRに特異的に作用するリガンドであるという知見は、これまでに報告されていないものである。
特定糖鎖含有化合物が生体由来でない化合物である場合の例としては、上記糖鎖構造の還元末端がポリアクリルアミド(PAA)、パラニトロフェニルフェノール(pNP)、アミノピリジン(PA)等に結合したものを挙げることができる。
本発明の樹状細胞免疫受容体活性化方法は、本発明の樹状細胞免疫受容体活性化剤を細胞の樹状細胞免疫受容体に接触させることを含む。樹状細胞免疫受容体活性化剤を細胞に接触させる方法は特に制限されず、経口投与、静脈内投与、留置等の外科的処置などを挙げることができる。樹状細胞免疫受容体に接触させる樹状細胞免疫受容体活性化剤の量は特に制限されず、樹状細胞免疫受容体の活性の状態、意図する活性化の度合、特定糖鎖含有化合物とともに使用する他の成分の種類や量等に応じて選択できる。
本発明の破骨細胞形成抑制剤は、特定糖鎖含有化合物を有効成分として含む。有効成分として含まれる特定糖鎖含有化合物は、DCIRを活性化させるリガンドとして作用する。DCIRは破骨細胞の形成を負に制御する役割を果たしている。従って、本発明の破骨細胞形成抑制剤をDCIRに接触させてDCIRを活性化させることで、破骨細胞の形成を抑制することができる。
本発明の破骨細胞形成抑制方法は、本発明の破骨細胞形成抑制剤を破骨細胞の樹状細胞免疫受容体に接触させることを含む。破骨細胞形成抑制剤を樹状細胞免疫受容体に接触させる方法は特に制限されず、経口投与、静脈内投与、留置等の外科的処置などを挙げることができる。樹状細胞免疫受容体に接触させる破骨細胞形成抑制剤の量は特に制限されず、破骨細胞形成の状態、意図する破骨細胞形成の抑制の度合、特定糖鎖含有化合物とともに使用する他の成分の種類や量等に応じて選択できる。
本発明の樹状細胞分化・増殖阻害剤は、特定糖鎖含有化合物を有効成分として含む。有効成分として含まれる特定糖鎖含有化合物は、DCIRを活性化させるリガンドとして作用する。DCIRは樹状細胞分化・増殖を負に制御する役割を果たしている。従って、本発明の樹状細胞分化・増殖阻害剤をDCIRに接触させてDCIRを活性化させることで、樹状細胞の分化・増殖を阻害することができる。
本発明の樹状細胞の分化・増殖阻害方法は、本発明の樹状細胞の分化・増殖阻害剤を樹状細胞の樹状細胞免疫受容体に接触させることを含む。樹状細胞の分化・増殖阻害剤を樹状細胞免疫受容体に接触させる方法は特に制限されず、経口投与、静脈内投与、留置等の外科的処置などを挙げることができる。樹状細胞免疫受容体に接触させる樹状細胞の分化・増殖阻害剤の量は特に制限されず、樹状細胞の分化・増殖の状態や、意図する樹状細胞の分化・増殖の阻害の程度に応じて選択できる。
本発明のサイトカイン産生抑制剤は、特定糖鎖含有化合物を有効成分として含む。有効成分として含まれる特定糖鎖含有化合物は、DCIRを活性化させるリガンドとして作用する。DCIRはある種のサイトカインの産生を負に制御する役割を果たしている。従って、本発明のサイトカイン産生抑制剤をDCIRに接触させてDCIRを活性化させることで、サイトカインの産生を抑制することができる。
本発明のサイトカイン産生抑制方法は、本発明のサイトカイン産生抑制剤を樹状細胞の樹状細胞免疫受容体に接触させることを含む。サイトカイン産生抑制剤を樹状細胞免疫受容体に接触させる方法は特に制限されず、経口投与、静脈内投与、留置等の外科的処置などを挙げることができる。樹状細胞免疫受容体に接触させるサイトカイン産生抑制剤の量は特に制限されず、サイトカイン産生の状態、意図するサイトカイン産生の抑制の度合、特定糖鎖含有化合物とともに使用する他の成分の種類や量等に応じて選択できる。
本発明の治療方法は、本発明の樹状細胞免疫受容体活性化剤を骨代謝疾患、自己免疫疾患又はアレルギー疾患の患者に投与することを含む。
本発明のスクリーニング方法は、特定糖鎖含有化合物の存在下又は特定糖鎖含有化合物を対照として、被検物質の樹状細胞免疫受容体への結合性を測定し、樹状細胞免疫受容体活性化剤又は樹状細胞免疫受容体拮抗剤をスクリーニングすることを含む。
破骨細胞の分化に関係する因子であるマクロファージコロニー刺激因子(M-CSF)と核因子κBリガンドの受容体活性化因子(RANKL)の存在下でmRNA及びタンパク質レベルで形成したM-CSF誘発骨髄マクロファージ細胞(BMM)と破骨細胞にDCIRを発現させた(図1~3)。遺伝子の発現は破骨細胞の成熟に伴って増加した。DCIRはCD11b陽性細胞(OsteoMacs)によって検出されたが、エンリッチされた骨芽細胞には検出されなかった(図4)。
骨髄中の破骨細胞前駆体数の頻度を確認するため、骨髄中の造血細胞を調べた。フローサイトメトリー解析の結果、Dcir-/-マウスの破骨細胞前駆体数の頻度(CD11bloLy6Chi)は野生型のそれと同程度であった(図12、13)。このことから、DCIRが破骨細胞の分化に関係していることがわかった。
Dcir-/-マウスの骨の構造について、脛骨遠位骨幹端部の組織形態計測的な評価を行った。その結果、破骨細胞及び骨芽細胞の双方のパラメータが増加していた(図26)。マイクロCT撮影によると、8週齢のDcir-/-マウスの大腿骨に軽度の大理石骨病が発症しており(図27)、骨体積及び骨梁数が増加していた(図27)。さらに、動的組織形態計測による解析結果は骨梁表面における単位あたりの骨石灰化速度及び骨形成速度の上昇を示していた(図28)。これらの結果は、DCIRの欠損が、骨芽細胞による骨の形成が破骨細胞による骨の破壊よりも優勢であり、破骨細胞と骨芽細胞のバランスにより制御される骨代謝が骨形成に傾いていることを示唆している。
受容体がシグナルカスケードを開始してその生物学的機能を発揮するためには、リガンドの結合が必要である。破骨細胞形成のインビトロ培養系はBMMのみ存在するため、DCIRのリガンドはBMM又は破骨細胞に発現すると推測した。DCIRの細胞外ドメインとヒトIgG2のFc領域との融合が生じたときに生じるDCIR-Fc染色に対してマクロファージと破骨細胞の一部は陽性であったが、その結合はCRDのアミノ酸の変化を生じさせるDCIR-Fc変異体であるDCIR E197A/S199A-Fcにより減少した。
以下のようにして、Flt3Lの分化誘導により樹状細胞を作製した。野生型マウスの大腿骨の骨髄細胞を摘出し、赤血球を溶血バッファー(140mM NH4Cl及び17mM Tris-HCl、pH7.2)に入れ、氷上で10分間おいた。細胞を10%FBS、抗生物質混合物、必須アミノ酸、2-メルカプトエタノールを含む培地にて、100ng/mlのヒトFlt3L組み変え体の存在下、24ウェルプレートにウェルあたり100万個となるよう播種した。4日後、100ng/mlのヒトFlt3L組変え体(Milteny社)を各ウェルに添加してさらに4日間培養した。その後、細胞を回収し、96ウェルプレートにウェルあたり50万個の細胞を播種した。
(マウスの作製及び入手先)
Dcir-/-マウス及びIfnγ-/-マウスは既報に従い作製し、C57BL/6と12世代戻し交配した。Rag2-/-マウスは公益財団法人実験動物中央研究所より提供を受けた。Ifn-γ-/-Dcir-/-マウスの作製のため、当研究室でDcir-/-マウスをIfnγ-/-マウスと交配した。実験で使用したマウスはすべて8~12週齢のオスである。対照として用いた週齢及び性別が同じであるC57BL/6Jマウスは日本エスエルシー株式会社より購入した。すべてのマウスは公益財団法人日本動物学会のガイドラインに従って東京大学又は東京理科大学内で飼育し、すべての実験は東京大学医科学研究所又は東京理科大学の動物実験委員会の承認を得て行った。なお、マウスDCIRの遺伝子の塩基配列はGene ID:Clec4a2、NCBIのアクセッションNo.NM001170332のうち273番目~1061番目の塩基の配列に相当する。
各群の統計的差異はtwo-tailed unpaired students’ t-test (*P<0.05、**P<0.01、***P<0.001、NSは非有意)により判断し、P<0.05で統計的に有意な差があると判断した。
M-CSF依存性骨髄由来マクロファージを作製するため、α-minimal essential medium(α-MEM)(商品名Gibco、Life Technologies)で大腿骨の骨髄腔を洗い流し、ペニシリン(100単位/ml)、ストレプトマイシン(100μg/ml)及び10%熱非動化ウシ胎児血清を補充してBM細胞を単離した。赤血球を溶血バッファー(140mM NH4Cl及び17mM Tris-HCl、pH7.2)を用いて氷上で10分間かけて破壊し、100mm径ディッシュで1時間プレインキュベートした。非接着性細胞(造血細胞)を回収し、20ng/mlのヒトM-CSF組み換え体(R&D Systems社)の存在下で96ウェルプレートに1ウェルあたり50,000個の細胞を播種し、2日間おいた。M-CSFにより増殖した細胞を骨髄由来マクロファージ(BMM)とした。破骨細胞を作製するため、BMMをさらに20ng/mlのM-CSFと100ng/mlの可溶性ヒトRANKL組み換え体(オリエンタル酵母工業株式会社)の存在下で培養し、培地を2日おきに交換した。成熟したマクロファージを得るため、BMMを20ng/mlのM-CSFの存在下で6日間培養し、培地を2日おきに交換した。一部の実験では、100mmディッシュで培養したBMMをCell Dissociation Solution Non-ezymatic 1×(Sigma-Aldrich社)で解離し、同数のBMMを再播種して破骨細胞の形成を誘発した。
接着細胞を10%ホルマリンで3分間固定し、固定バッファー(50%エタノール及び50%アセトン)で1分間さらに固定した。固定した細胞をナフトールAS-MXリン酸塩(ナカライテスク株式会社)及びファストレッド(ナカライテスク株式会社)で10~15分間室温で染色し、3分間水洗した。細胞核が3以上又は10以上存在するTRAP陽性細胞を多核化した破骨細胞とし、その数を数えた。
マウスDCIRのcDNAを、プライマーとしてセンス:5’-cgggatcccaccatggcttcagaaatcacttatg(配列番号1)及びアンチセンス:5’-cggaattctcataagtttattttcttca(配列番号2)を用いて増幅した。PCRで得られた産物を、BamHI及びEcoRIサイトでpMXs-IRES-Puro(東京大学の北村俊雄教授より提供を受けた)にクローニングした。pMXsベクターを大腸菌(DH5α)から単離した。DCIR Y/F変異体を、KOD-Plus-Mutagenesis Kit(東洋紡株式会社)を用いて作製した。すなわち、DCIRのITIMにおけるチロシンをフェニルアラニンに置換するためのフォワードプライマー:5’-cactTttgcagaagtgaagttcaagaatgaatc(配列番号3、大文字のTはアデノシン)及びリバースプライマー:5’-atttctgaagccatggtgggatccttggttaac(配列番号4、大文字のTはアデノシン)を設計した。pMXsベクターをテンプレートとした逆PCRを行い、典型的なE.coliセルラインのメチル化したDNAを消化可能なDpnIで消化した。消化していないPCR産物を、T4ポリヌクレオチドキナーゼ及びリガーゼを用いて16度で1時間セルフライゲートした。plat-Eパッケージングセルライン(東京大学の北村俊雄教授より提供を受けた)を10%FBS α-MEMで培養し、1日おきに1対5の割合で分割した。レトロウイルスベクターをPlat-E細胞にLipofectamine 2000(Invitrogen社)を用いて導入した。8時間インキュベートした後、培地を交換し、さらに24時間インキュベートした。レトロウイルス上清を回収し、使用するまで-80℃で冷凍した。DCIRのBMとDCIR変異体のBMをレトロウイルスによって形質導入するため、非接着性骨髄細胞をレトロウイルス上清20μlを含む培地100μlで培養し、培地を1日おきに交換した。
BMMをCell Dissociation Solution Non-ezymatic 1×(Sigma-Aldrich社)を用いて回収し、濃度を変化させたM-CSFの存在下で96ウェルプレートの各ウェルに300,000個ずつ入れた。2~3時間後、1.0μCiの[3H]チミジン(PerkinElmer社)を添加し、さらに48時間37℃でインキュベートした。細胞はSkatronマイクロプレートウォッッシャー上清回収システム(Molecular Device社)を用いてガラスファイバーフィルターを通した。[3H]チミジンのDNAへの導入は、MicroBetaマイクロプレートカウンターシステム(PerkinElmer社)によって確認した。
BM細胞と末梢血単核細胞(PBMC)の血液細胞溶解を溶血バッファー(140mM NH4Cl及び17mM Tris-HCl、pH7.2)を用いて氷上で行った。BMMとPBSを分離し、FACSバッファー(2%FBSを含むPBS)で洗浄し、血清成分を除去した。これらの細胞のFc受容体はFc受容体ブロッカーである2.4G2で、4度で10分間ブロックした。その後、細胞をFACSバッファーで2度洗浄し、蛍光標識した抗体とともに30分間氷上でインキュベートした。抗体としては、APC-又はPE-CD11b(BioLegend社)、FITC-Ly6c(BioLegend社)、APC-c-Fms(BioLegend社)、biotin-RANK(eBioscience社)及びPE-Clec4a(R&D Systems社)を使用した。細胞内分子の染色にはFITC-CD3、Pacific Blue-CD4、APC-CD8、BV510-CD11b、PE-IFN-γ(BioLegend社)を使用した。DCIRリガンド検出用のFc-タンパク質と抗IgG抗体とからなるタンパク質複合体を形成するため、10μg/mlのDCIR-Fc-タンパク質、DCIR-Fc-タンパク質変異体、又はFc-タンパク質を、5μg/mlのFITC標識抗ヒトIgG(Jackson ImmunoResearch Laboratories社)とともにカルシウム及びマグネシウムを含有するバッファー(2mM CaCl2、2mM MgCl2及び0.5%BSAを含むTBS)で、15分間室温でインキュベートした。マクロファージと破骨細胞を上記のタンパク質複合体で1時間、4℃で染色し、Ca2+/Mg2+バッファーで洗浄した。フローサイトメトリーをFACSCanto II(Becton Dickinson社)を用いて実施し、Flowjoソフトウェア(Tree Star社)を用いて分析した。細胞内染色のため、細胞をまず2.4G2で染色し、次いで固定前の表面抗原をFITC-CD3で染色した。FACSバッファーで洗浄後、固定/透過化バッファー(eBioscience社)を添加し、4℃で60分おいた。遠心分離後、固定/透過化バッファーをデカンテーションし、0.1%サポニン含有FACSバッファーで2回洗浄した。これらの細胞は表面抗原及び細胞内分子を一次抗体で染色し、0.1%サポニンバッファーで希釈した。
全RNAはBMM及び破骨細胞からGenEluteTM Mammalian Total RNA Miniprepキット(Sigma-Aldrich社)を用いて抽出した。全RNAの定量はNanoDropを用いて行った。1μgの全RNAをSuperscript II Reverse Transcriptase(Invitrogen社)を用いて逆転写し、1本鎖cDNAを合成した。定量PCRを全RNAに相当する5ng、SYBR Green I(タカラバイオ株式会社)、フォワード及びリバースプライマー(Operon社)を含めた全反応容量を10μlとし、2又は3サンプルとして行った。定量はCFX384TMリアルタイムシステム(バイオ・ラッド ラボラトリーズ株式会社)を用いて行った。サイクル数は1サイクル目を95℃で1分、続く44サイクルを95℃で3秒/60度で30秒とした。プライマーとしてはDCIRのセンス:5’-cctggtgattctatgctgtggt-3’(配列番号5)、アンチセンス:5’-gtcagaagagagccttgttccttc-3’(配列番号6)、GAPDHのセンス:5’-ttcaccaccatggagaaggc-3’(配列番号7)、アンチセンス:5’-ggcatggactgtggtcatga-3’(配列番号8)を用いた。プライマーの最終濃度は400nMであった。GAPDHはインターナルハウスキーピング遺伝子として用いた。目的の遺伝子とハウスキーピング遺伝子を、水サンプルをネガティブリファレンスとして1プレートで同時に定量した。サンプル間のRNAの品質と初期量の違いはGAPDHで正規化した。目的の遺伝子の相対的発現は、2-dCt(dCt=目的遺伝子の平均Ct値-ハウスキーピング遺伝子の平均Ct値)として計算した。目的の遺伝子の2日目に対する3日目及び4日目のfold changeは2-ddCt(ddCt=3日目及び4日目の各サンプルにおける(目的遺伝子の平均Ct値-ハウスキーピング遺伝子の平均Ct値)-2日目の(目的遺伝子の平均Ct値-ハウスキーピング遺伝子の平均Ct値)として計算した。標準偏差は既報に従い、数学的手法で計算した。RT-PCRをEx-taq(タカラバイオ株式会社)を用いて反応容量20μlとして行った。DCIRを増幅するためのプライマーとして、センス:5’-catttcccttatctcgccctgg-3’(配列番号9)、アンチセンス:5’-gcatgagtgtccaagatcc-3’(配列番号10)を使用し、686pbの産物を増幅した。PCR反応は30サイクルを98℃で10秒、55℃で30秒、72℃で1分間行った。内部標準としてGAPDHを使用した。
M-CSF及びRANKLを介したシグナルのウェスタンブロッティングのため、BMMを5ng/mlのM-CSFとともに12ウェルプレートに各ウェル250,000個ずつ再播種した。24時間培養後、刺激前にBMMをM-CSFを添加しない血清フリー培地にて6時間おいた。次いで、20ng/mlのM-CSF又は100ng/mlのRANKLを添加して所定の時間、細胞を活性化した。細胞は1%NP-40溶液(10mM Tris-HCl、pH7.5、100mM NaCl、5mM MgCl2、プロテナーゼ阻害剤カクテル(Thermo Fischer Scientific社)及びホスファターゼ阻害剤カクテル(Roche社))に溶解して細胞溶解物を得た。溶解物を氷上に10分おき、15,000rpmで10分間遠心分離して清澄化した。等量の溶解物サンプルをSDS-ポリアクリルアミドゲルで230Vにて30分間分離した。ゲルはPVDF膜上に定電流230mAとし、膜あたり25分でセミドライシステム(バイオ・ラッド ラボラトリーズ株式会社)により電気的に転写した。PVDF膜は5%のTween 20を含むTBSに5%BSA(Sigma-Aldrich社)を溶かした中で1時間室温でブロックし、一次抗体とともに4℃で一晩インキュベートした後、HRP標識二次抗体でプローブした。PVDF膜はECL Prime Western Blotting Detection System(GE Healthcare社)を用いてタンパク質シグナルを可視化した。
マウスDCIRの細胞外ドメイン(EC)をカバーするアミノ酸配列70~238番目のcDNAを、pFUSE-hIgG2-Fc2ベクター(Invitrogen社)にBgl IIサイトにてサブクローニングした。ECをコードするcDNAは、プライマーとしてセンス:5’-ataagatctcaaaagtactctcaacttctt-3’(配列番号11)、アンチセンス:5’-ataagatcttaagtttattttcttcatc-3’(配列番号12)を用いて作製した。グルタミン酸とセリンがアラニンに置換されたCRDドメインの部位特異的変異体を、KOD-Plus-Mutagenesisキット(東洋紡株式会社)を用いて作製した。プライマーとしては、以下のものを使用した。
・E197A センス:5’-AATGGTGCTCCCAGCAGTGGCAATGAA-3’(配列番号13)、アンチセンス:5’-TGTAAGACCGTGTTACCACGAGGGTCA-3’(配列番号14)
・S199A センス:5’-GAGCCCGCTAGTGGCAATGAAAAATGT-3’(配列番号15) 、アンチセンス:5’-ACCGTGTTACCACTCGGGCGATCACCG-3’(配列番号16)
・E197A/S199A センス:5’-GGTGCTCCCGCTAGTGGCAATGAAAAATGTGCT-3’(配列番号17) 、アンチセンス:5’-TAGTGTAAGACCGTGTTACCACGAGGGCGATCA-3’(配列番号18)
野生型BMM及びDcir-/-BMMの破骨細胞への分化を、M-CSF及びRANKLの存在下で誘導した。GlycoTech社より購入したアシアロ二本鎖N型糖鎖(NA2)を非接着性BM細胞の培養初期に加え、培地を交換するごとに継続的に添加した。破骨細胞の数をTRAP染色により数えた。NA2の生物活性を評価するため、M-CSF(20ng/ml)又はRANKL(100ng/ml)による刺激を付与する前に、BMMをNA2の存在下又は非存在下で6時間処理した。細胞溶解物をウェスタンブロット法により解析し、シグナル化合物のリン酸化のレベルを調べた。
6日間培養したBMMをCell Dissociation Solution Non-ezymatic 1×(Sigma-Aldrich社)にて回収し、TSAバッファー(2mM CaCl2、2mM MgCl2及び0.5%BSAを含むTBS)に再度懸濁させた。次いで、10万細胞あたり0.5μlのノイラミニダーゼ(Roche社)でBMMを37℃で30分間処理した。その後、細胞をTSAバッファーで洗浄し、FACS分析を行った。シアル酸の脱離が破骨細胞形成に与える影響を調べるため、1μlのノイラミニダーゼ(Roche社)の存在下で非接着性BM細胞から破骨細胞を誘導した。ノイラミニダーゼは培地を1日おきに交換する際に補充した。
骨形成の様子を調べるための動的組織形態計測学的解析は株式会社クレハ分析センターにて行った。野生型マウス及びDcir-/-マウスの8週齢における体重を測定し、16mg/kg(体重)のカルセイン(ナカライテスク株式会社)を2日おきに2回マウスの腹腔内に投与した。2度目の投与から2日後に脛骨を摘出して軟組織及び筋肉を取り除き、70%エタノールで1週間、毎日エタノールを交換して固定した。石灰化した部分が緑の線として染色された。脱灰していない骨組織はグリコールメタクリレート(GMA)の組織への浸入を促すためメチルベンゾエート中に15分間おき、次いで5%のメチルベンゾエートを含むGMA(4℃)に浸漬し、2時間の間に3回交換した。次いで、骨組織をGMAに埋め込んだ。3μm厚の脛骨断片をミクロトーム(サクラファインテックジャパン株式会社)を用いて作製し、石灰化前線における蛍光を検出した。動的組織形態計測学的解析のため、石灰化表面(石灰化表面/骨表面[MS]/[BS];%)、骨石灰化速度([MAR];μm/day)、骨形成速度([BFR]/[Bs];μm3/μm2/day)を画像解析装置(System supply社)で倍率400倍として調べた。
マウス脛骨の組織形態計測学的分析は株式会社クレハ分析センターにて行った。野生型マウス及びDcir-/-マウスの骨を、8週齢のマウス1群あたり5~6個体から取り出し、70%エタノールで固定した。骨構造の組織形態計測パラメータを測定するため、3μm厚のGMAに埋め込んだ大腿骨組織断片をトルイジンブルーで染色した。柱状骨パラメータは、成長板から0.3mmはなれた地点から末端方向に幅1.05mmの二次海綿骨の面積とした。骨幹端における柱状骨をHistometryRT CAMERAで観察した。破骨細胞数([Oc.N]/100mm)と破骨細胞表面積([Oc.S]/[BS];%)は、1以上の核を有し、柱状骨表面で吸収窩を形成している細胞を破骨細胞として計測した。骨芽細胞数([Ob.N]/100mm)及び骨芽細胞表面積([Ob.S]/[BS];%)を含む骨リモデリングのパラメータは、トルイジンブルーで染色した断面において測定した。
野生型マウスとDcir-/-マウスの骨の構造特性について、高解像度マイクロCTシステムで調べた。大腿骨骨幹端の末端における長さ1.814mmの海綿骨断面をマイクロCTシステム(Scan Xmate-L090、コムスキャンテクノ株式会社)でスキャンした。3D画像解析をTRI/3D BONソフトウェア(ラトックシステム株式会社)を用いて行った。骨梁のミネラル濃度は骨密度(骨体積[BV]/骨組織体積[TV])、骨梁幅(Tb.Th.=2×BV/骨表面積[BS])、骨梁数(Tb.N=(BV/TV)/Tb.Th.)、骨梁間隔(Tb.Sp=1/Tb.N・Tb.Th.)及び骨梁中心距離(Tb.Spac=1/Tb.N)により評価した。骨梁数は、骨梁細胞の数とした。
ヘパリンを含む21ゲージ針付注射器(持田製薬株式会社)を用いて麻酔下のマウスより心臓穿刺により全血を採取した。血液は10倍量の溶血バッファーと混合し、氷上に10分間おいた。次いで1500rpmで5分間、4℃で遠心分離し、単核細胞を回収した。以上の処理を3回繰り返した。末梢血単核球(PBMC)をホルボールミリスタートアセタート(最終濃度:500ng/ml)で5時間刺激し、さらにイオノマイシン(最終濃度:50ng/ml)とブレフェルジンA(最終濃度:10μg/ml)でインキュベーションの全期間にわたって刺激した。PBMCによりIFN-γが産生されていることが細胞内分子染色とフローサイトメトリー解析より検出された。
頭蓋冠細胞を連続消化及び定法により作製した。すなわち、頭蓋冠を生後1~2日のマウス新生児より摘出し、接着性間葉組織を除去した。この頭蓋冠を0.1%コラゲナーゼ(和光純薬工業株式会社)と0.1%ディスパーゼ(ロシュ・ダイアグノスティックス株式会社)を含む分解液で10分間、37℃で撹拌しながら処理して1回目の消化を行った。デブリを含む溶液を捨て、残った頭蓋冠を上記と同じ分解液で37℃で1時間、さらに消化した。連続消化後、単離した細胞はα-MEM(10%FCS、ペニシリン100単位/ml、ストレプトマイシン100μg/ml)に懸濁させ、12ウェルプレートにウェルあたり20万個となる濃度で播種した。2日間培養後、細胞を骨形成培地(10%FBS、50μg/mlアスコルビン酸(Sigma-Aldrich社)、10mM β-グリセロリン酸(Calbiochem社)及び抗生物質を添加したα-MEM)で14日間又は21日間インキュベートした。培地は3日おきに交換した。
マウス骨芽細胞の培養及び分化は上述の方法で行った。初代頭蓋冠細胞を、マウスIFN-γ組み換え体(PeproTech社)の存在下で培養した。IFN-γは骨形成培地を交換する度に添加した。
頭蓋冠細胞は上記の方法でマウス新生児より採取し、赤血球細胞を溶血バッファーで10分間、37℃で溶解させた。残存する細胞は、1000万個の全細胞あたり90μlのautoMACS Running Buffer(Miltenyi Biotec社)に再懸濁し、1000万個の全細胞あたり10μlのCD11b MicroBeads(Miltenyi Biotec社)で15分間、4℃でインキュベートした。細胞はMACSバッファーで洗浄し、108 個の細胞あたり500μlのautoMACS Running Bufferに再懸濁し、75ナイロンメッシュ(75×75μm メッシュ)を通して細胞の凝集塊を除去した。CD11bに対し陽性の画分と陰性の画分とをauto MACS Pro separator(Milteny Biotec社)で分離し、それぞれの細胞をOsteoMacs又は初代骨芽細胞として使用した。
骨芽細胞の石灰化の形成は、フォン・コッサ法及びアリザリンレッドSによる染色で確認した。フォン・コッサ法による染色は、培養した骨芽細胞を10%ホルマリンで10分間室温で固定し、洗浄後、固定した細胞を5%硝酸銀溶液中で30分間明るい日光に曝露し、次いで5%チオ硫酸ナトリウムで洗浄することで行った。石灰化した結節は暗褐色又は黒色の斑点として可視化された。アリザリンレッドSによる染色は、培養した骨芽細胞を10%ホルマリンで10分間室温で固定し、1%アリザリンレッドS(pH4.2)で10分間室温で染色し、蒸留水で余分な染料を除去することで行った。石灰化した結節は暗赤色の斑点として可視化された。染色した細胞の画像はGT-X770イメージスキャナ(セイコーエプソン株式会社)で撮影した。
初代頭蓋冠細胞は酵素的に採取し、12ウェルプレートにウェルあたり20万個としてビタミンD3(10-8M)とプロスタグランジンE2(10-6M)を含むα-MEMとともに播種し、24時間おいた。10倍の数の非接着性骨髄細胞を、初代骨芽細胞とともに7日間共培養した。培地は3日おきに交換した。
骨芽細胞は既述の方法で5日間100mmディッシュにて培養して作製した。この骨芽細胞を酵素的に採取し、M-CSF及びRANKLの存在下、Osteo Assay プレート(Corning社)にウェルあたり2万個の細胞を再播種した。3日後、1Mアンモニア溶液中で30秒間の超音波破壊により細胞を除去した。リン酸カルシウムでコーティングされたウェルの表面全体の画像を顕微鏡(株式会社キーエンス)に取り込み、吸収された領域の面積をImageJで解析した。
96ウェルプレートを10mg/mlのフィブロネクチン(Sigma-Aldrich社)でコーティングし、4℃で一晩おいた。ウェルをPBSで2度洗浄し、2日培養したBMMをM-CSF(20ng/ml)とともに、又はM-CSFなしで5万個播種し、2分間、5分間又は10分間経過させた。次いで、細胞をDMEMで2度洗浄し、メタノールで2分間固定し、クリスタルバイオレットの0.5%蒸留水溶液で染色した。蒸留水で5回洗浄後、100mlの1%SDSを各ウェルに添加して色素を可溶化し、595nmでの吸収度を測定した。
2日間培養したBMMを、M-CSF(20ng/ml)、RANKL(100ng/ml)とともに48ウェルプレートでウェルあたり1万個をインキュベートした。培地は2日おきに交換した。培養開始から7日後及び8日後に培地を除去し、細胞死検出キット(Cell Death Detection ELISA、Roche Molecular Biochemicals社)の細胞溶解バッファーを用いて30分間、室温で細胞溶解液を作製した。ポジティブコントロールとして、培養8日目のウェルから最後の6時間ですべてのサイトカインを除去したものを準備した。20μlの上清を用いてELISAによりアポトーシスの程度を解析した。
破骨細胞の転写産物の検出には以下のプライマーを使用した。
・Acp(TRAP) センス:5’-cagcagcccaaaatgcct-3’(配列番号19)、アンチセンス:5’-ttttgagccaggacagctga-3’(配列番号20)
・NFATc1 センス:5’-gccaagtaccagctttccag-3’(配列番号21)、アンチセンス:5’- agggtcgaggtgacactagg-3’(配列番号22)
・Calcr(Calcitonin R) センス:5’-gcctccccatttacatctgc-3’(配列番号23)、アンチセンス:5’-ctcctcgccttcgttgttg-3’ 配列番号24)
・Nfatc1 センス:5’-gccaagtaccaggtttccag-3’(配列番号25)、アンチセンス:5’-agggtcgaggtgacactagg-3’(配列番号26)
・cSrc センス:5’-gaacccgagagggaccttc-3’(配列番号27)、アンチセンス:5’-gaggcagtaggcaccttttgt-3’(配列番号28)
・Pirb センス:5’-agccagaaaacaaggctgaa-3’(配列番号29)、アンチセンス:5’-ggctgggtgtccagtagtgt-3’(配列番号30)
・Sirpa センス:5’-gtaggtgcgactgggatgtt-3’(配列番号31)、アンチセンス:5’-agtgaggccaactcagccta-3’ (配列番号32)
・Tnfsf11(RANKL) センス:5’-cagcatcgctctgttcctgta-3’(配列番号33)、アンチセンス:5’-ctgcgttttcatggagtctca-3’(配列番号34)
・Tnfsf11b(OPG) センス:5’-gggcgttacctggagatcg-3’(配列番号35)、アンチセンス:5’-gagaagaacccatctggacattt-3’(配列番号36)
mRNAの品質及び量はGAPDHを用いて正規化した。
糖鎖-タンパク質間の相互作用は、糖鎖マイクロアレイを用いたエバネッセント場蛍光励起検出システムにより検出した。すなわち、糖タンパク質やグリコシド-ポリアクリルアミド(PAA)を含む糖鎖プローブを非接触型マイクロアレイ印刷ロボット(MicroSys 4000、Genomic Solutions社)を用いてマイクロアレイグレードエポキシ活性化ガラススライド(Schott社)上に3スポットずつ固定した。このガラススライドを25℃で3時間インキュベートし、反応バッファー(0.8% NaCl、1%(v/v)Triton-X、1 mM MnCl2及び1mM CaCl2を含む25mM Tris-HCl(pH7.4))で固定化した物質を除去し、次いでTBS(0.8% NaCl及び1% BSAを含む25mM Tris-HCl(pH7.4))で20℃、1時間ブロッキングを行った。DCIR-Fc、DCIR-Fc変異体、又はFcと、Cy3-標識抗ヒトFc抗体とのタンパク質複合体を反応バッファー中で、室温、遮光条件下で20分間で作製した。
全血は麻酔したマウスから心臓穿刺で採取した。採取した血液を4℃で一晩おいて凝固させ、3000rpmで10分間遠心分離して血清を分離した。得られた血清は-80℃で保存した。IFN-γの量は、IFN-γ用ELISAキット(MABTECH社)で検出した。
Claims (10)
- 請求項1に記載の樹状細胞免疫受容体活性化剤を細胞の樹状細胞免疫受容体に接触させることを含む、樹状細胞免疫受容体活性化方法。
- 請求項3に記載の破骨細胞形成抑制剤を破骨細胞の樹状細胞免疫受容体に接触させることを含む、破骨細胞形成抑制方法。
- 請求項5に記載の樹状細胞分化・増殖阻害剤を樹状細胞の樹状細胞免疫受容体に接触させることを含む、樹状細胞分化・増殖阻害方法。
- 請求項7に記載のサイトカイン産生抑制剤を樹状細胞の樹状細胞免疫受容体に接触させることを含む、サイトカイン産生抑制方法。
- 請求項1に記載の樹状細胞免疫受容体活性化剤を骨代謝疾患、自己免疫疾患又はアレルギー疾患の患者に投与することを含む、治療方法。
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WO2023140385A1 (ja) * | 2022-01-21 | 2023-07-27 | 学校法人東京理科大学 | 炎症性腸疾患及び腸管腫瘍からなる群より選択される少なくとも1つの疾患を治療又は予防するための医薬組成物 |
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WO2018207949A1 (ja) | 2017-05-12 | 2018-11-15 | 学校法人東京理科大学 | 糖鎖修飾酵素を含む医薬組成物及び医薬組成物を用いた樹状細胞免疫受容体関連疾患の治療方法 |
JPWO2018207949A1 (ja) * | 2017-05-12 | 2020-03-12 | 学校法人東京理科大学 | 糖鎖修飾酵素を含む医薬組成物及び医薬組成物を用いた樹状細胞免疫受容体関連疾患の治療方法 |
US11324809B2 (en) | 2017-05-12 | 2022-05-10 | Tokyo University Of Science Foundation | Method of preventing aggravation of a disease involving a biological mechanism controlled by a dendritic cell immunoreceptor |
JP7103581B2 (ja) | 2017-05-12 | 2022-07-20 | 学校法人東京理科大学 | 糖鎖修飾酵素を含む医薬組成物及び医薬組成物を用いた樹状細胞免疫受容体関連疾患の治療方法 |
WO2023140385A1 (ja) * | 2022-01-21 | 2023-07-27 | 学校法人東京理科大学 | 炎症性腸疾患及び腸管腫瘍からなる群より選択される少なくとも1つの疾患を治療又は予防するための医薬組成物 |
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Publication number | Publication date |
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EP3167889A4 (en) | 2017-07-05 |
JP6674685B2 (ja) | 2020-04-01 |
US20170143754A1 (en) | 2017-05-25 |
EP3167889A1 (en) | 2017-05-17 |
JPWO2016006700A1 (ja) | 2017-06-08 |
US10792300B2 (en) | 2020-10-06 |
EP3167889B1 (en) | 2020-12-16 |
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