JP2014094898A - MEDICINE COMPRISING ACTIVE MODULATOR OF CD300a-EXPRESSING CELL ASSOCIATED WITH INFLAMMATORY BOWEL DISEASE, CD300a GENE KNOCK-OUT MOUSE, AND USE OF ACTIVE MODULATOR OF CD300a-EXPRESSING CELL - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicine for inflammatory bowel disease, and a tool useful for the analysis of a clinical condition of the inflammatory bowel disease.SOLUTION: A medicine, which contains a substance capable of inhibiting the binding between CD300a and phosphatidyl serine and comprises, as an active ingredient, an activity modulator that can inhibit the inhibitory signal transduction of a myeloid cell capable of expressing CD300a, can be used for treating or preventing inflammatory bowel disease. A CD300a gene knock-out mouse can be used for analyzing a clinical condition of the inflammatory bowel disease or screening for a candidate substance that can act as an active ingredient for a therapeutic or prophylactic agent for the inflammatory bowel disease, as a model mouse that rarely develops the inflammatory bowel disease when a substance capable of inducing the inflammatory bowel disease is administered to the mouse.

Description

  The present invention relates to a pharmaceutical containing an activity regulator of CD300a-expressing cells related to inflammatory bowel disease, and the use of an activity regulator of CD300a gene-deficient mice and CD300a-expressing cells.

  When pathogens (bacteria, viruses, parasites, etc.) invade the host (human body or animal body) or endogenous flame retardants are generated, arterioles at the site of pathogens or the site where the flame retardants are generated After contracting temporarily, the blood reaches expansion and hyperemia, and an inflammatory reaction occurs in which the blood flow at the site where the pathogen invades or the site where the inflammatory substance is generated becomes locally slow.

  Then, leukocytes adhere to the blood vessel wall and further migrate through the blood vessel wall by amoeba-like movement by the action of chemical mediators released from various immune system cells. As chemical mediators, histamine, serotonin, lymphokine and the like are known. Mast cells that produce and release histamine and serotonin are one of the lymphocytes that play a central role in the inflammatory response. Macrophages also produce and release chemical mediators such as TNF, similar to mast cells.

  When the migrated leukocytes are attracted to a pathogen or the like by an inflammatory reaction, the pathogen is removed from the body by humoral immunity involving an antigen-antibody reaction or cytotoxic T cells involved in the pathogen ( Clearance) and spread of infection is prevented. Thus, the inflammatory response and the immune response based thereon are extremely important for maintaining the homeostasis of the living body.

  On the other hand, the inflammatory reaction shows trouble signs / symptoms such as redness, fever, swelling, pain, and dysfunction in addition to the above-described defense of the body. Specific examples of such symptoms include allergic diseases and various acute or chronic inflammations. In addition, even when an autoimmune disease in which immunological tolerance is not applied and an immune response or the like occurs against the self, tissue injury occurs due to an inflammatory reaction.

  That is, in order to prevent diseases associated with inflammatory reactions, pathogens that cause inflammatory reactions are killed by various antibiotics (antibacterial agents), or drugs that improve immune functions in the body (immunostimulants) are administered. Thus, it is important to remove the pathogen before the inflammatory response is excessively enhanced.

  On the other hand, in order to improve / treat diseases associated with inflammatory reactions, for example, administer drugs (anti-inflammatory agents (immunosuppressive agents)) that reduce excessively activated immune functions, such as suppressing the release of chemical mediators. Thus, it is known to reduce inflammation.

  For example, Patent Document 1 discloses a dendritic cell function activator responsible for activating various immune system cells as an antigen-presenting cell as an immunoactive agent, specifically selected from isoleucine, leucine and valine. And at least one branched chain amino acid as an active ingredient.

  Patent Document 2 discloses a drug containing a SECRC (Secreted protein is acid and rich in system) peptide and a pharmacological carrier as an anti-inflammatory agent (immunosuppressive agent).

  Incidentally, it is known that a receptor molecule group called MAIR (Myeloid Associated Ig like Receptors) is expressed on the cell membrane of myeloid (myeloid) cells in charge of the innate immune system (non-patented). Reference 1). Among these, MAIR-I, which is also known as CD300a (sometimes referred to as “LMIR1” or “CLM-8”), is a macrophage, mast cell, granulocyte (neutrophil), dendritic cell. It is known that it is an inhibitory receptor that associates with phosphatase through an ITIM (Immunoreceptor tyrosine-based inhibitory motif) sequence in the intracellular region and transmits an inhibitory signal (non-receptor). Patent Document 2). However, the ligand of the receptor is unknown and has become a so-called orphan receptor.

  Inflammatory bowel disease (IBD), a typical disease of inflammatory bowel disease, is a general term for chronic diseases that cause inflammation of unknown origin mainly in the digestive tract, and inflammation occurs in the large intestine to form ulcers. It is a chronic disease.

Inflammatory bowel disease (IBD) is a collective term used to describe two gastrointestinal disorders of unknown etiology (Crohn's disease (CD) and ulcerative colitis (UC)).
Crohn's disease has a wider lesion than ulcerative colitis, and inflammation and ulcers occur almost throughout the digestive tract. Inflammatory bowel disease (IBD) occurs worldwide and has been reported to affect as many as 2 million people, and the course and prognosis of IBD varies widely.

  Inflammatory bowel disease (IBD) continues with diarrhea and bloody stool over a long period of time while the quality of symptoms changes. The cause has not yet been elucidated, and a promising theory is that due to impaired tolerance of intestinal immunity, an immune response occurs against food and intestinal bacteria, causing persistent enteritis.

  Currently there is no fundamental cure, steroids such as diet, antidiarrheal drugs (such as anticholinergic drugs, loperamide or diphenoxylate) or anti-inflammatory drugs (aminosalicylic acid, sulfasalazine, mesalamine, olsalazine, balsalazide, prednisolone) Drugs or aminosalicylic acid, etc.) and immunosuppressive drugs (azathioprine, mercaptopurine, cyclosporine, etc.) are used.

10% to 15% of all people with IBD require surgery for 10 years, and the risk of developing intestinal cancer is high.
In Crohn's disease, bacteria are involved in the onset and progression of the disease, and intestinal inflammation in Crohn's disease is famous for its frequent responsiveness to antibiotics and susceptibility to bacterial fecal flow. Common intestinal microorganisms and novel pathogens have been linked to Crohn's disease by direct detection or by an antimicrobial immune response associated with the disease.

  Furthermore, in many genetically susceptible models of chronic colitis, luminal microorganisms are an essential cofactor for the disease, and animals trapped in microbial-free environments do not develop colitis.

A combination of genetic factors, exogenous triggers, and endogenous microbiota may contribute to immune-mediated damage to the intestinal mucosa seen in inflammatory bowel disease.
A person who has polymorphisms in the three gene regions of the FCGR2A gene encoding a receptor protein present on the surface of immune cells, s8LC26A3 encoding a chloride ion and bicarbonate ion transporter, and the 13q12 region gene of unknown function It is also known to be associated with the development of ulcerative colitis (Non-patent Document 3).

  However, despite much direct and indirect evidence for the role of gut microbes in Crohn's disease, no pathogenic organisms and antigens have been identified that contribute to the immune dysregulation seen in this disease, Tools useful for pathological analysis of inflammatory bowel diseases (Crohn's disease and inflammatory bowel disease) and pharmaceuticals for treatment have been desired.

JP 2007-297379 A JP 2011-516609 A

Yotsumoto et al .. J Exp Med 198 (2), 223-233, 2003 Okoshi Y et al, Int Immunol., 17, 65-72, 2005. Asano. K et al. Nature Genetics 41. 1325-1329 (2009)

  An object of the present invention is to provide a drug for inflammatory bowel disease, a tool useful for pathological analysis of inflammatory bowel disease, and the like.

As a result of intensive studies to elucidate the ligand of CD300a and the function of CD300a, the present inventor has obtained the following knowledge.
(I) The ligand of CD300a is phosphatidylserine (phosphatidylserine, phosphatidylserine) (PS).
(Ii) When PS binds to CD300a such as mast cells, the inhibitory signaling of CD300a is enhanced, and activation of mast cells and the like is also suppressed.
(Iii) When the binding of CD300a such as mast cells and PS is inhibited by the coexistence of a phosphatidylserine-binding substance or a CD300a-binding substance, suppressive signaling of CD300a is suppressed, and the activity of mast cells and the like is suppressed. Is also maintained.
(Iv) Inflammatory bowel disease can be treated through suppression or maintenance of the above activation.
(V) The CD300a gene-deficient mouse can be a tool for conducting pathological analysis of inflammatory bowel disease and the like.

By the present invention made based on these findings, for example, the inventions shown in the following [1] to [9] are provided.
[1] Inflammation characterized by containing an activity regulator as an active ingredient for inhibiting inhibitory signaling of myeloid cells expressing CD300a, which contains a substance that inhibits the binding between CD300a and phosphatidylserine. Drugs for treating or preventing sexual bowel disease.

[2] The pharmaceutical product according to [1], wherein the substance that inhibits the binding between CD300a and phosphatidylserine is a phosphatidylserine-binding substance.
[3] The phosphatidylserine-binding substance is MFG-E8, MFG-E8 mutant, T cell immunoglobulin, soluble TIM-1, soluble TIM-4, soluble stablin and soluble integrin αvβ3. The pharmaceutical according to [2], which is at least one selected from the group consisting of:

[4] The pharmaceutical product according to [1], wherein the substance that inhibits the binding between CD300a and phosphatidylserine is a CD300a-binding substance.
[5] The CD300a-binding substance has the amino acid sequence represented by SEQ ID NO: 19 or 1, 2, 3, 4 or 5 amino acid substitutions, additions, insertions or deletions relative to the amino acid sequence. H chain variable region comprising the sequence and the amino acid sequence represented by SEQ ID NO: 20, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, additions, insertions or deletions to the amino acid sequence The pharmaceutical agent according to [4], which is an anti-human CD300a antibody having an L chain variable region consisting of

  [6] The amino acid sequence wherein the CD300a binding substance has the amino acid sequence represented by SEQ ID NO: 17 or 1, 2, 3, 4 or 5 amino acid substitutions, additions, insertions or deletions relative to the amino acid sequence H chain variable region consisting of a sequence and the amino acid sequence represented by SEQ ID NO: 18 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, additions, insertions or deletions to the amino acid sequence The pharmaceutical agent according to [4], which is an anti-mouse CD300a antibody having an L chain variable region consisting of

[7] The pharmaceutical product according to [4] or [5], wherein the myeloid cells are CD11b-positive dendritic cells in the lamina propria of the large intestine.
[8] Use of a CD300a gene-deficient mouse for pathological analysis of inflammatory bowel disease, or for screening a candidate substance that can be an active ingredient of a therapeutic or prophylactic agent thereof,
Use of the CD300a gene-deficient mouse, wherein the CD300a gene-deficient mouse is used as a model mouse that hardly induces inflammatory bowel disease when a substance that induces inflammatory bowel disease is administered.

[9] Use of a CD300a gene-deficient mouse for pathological analysis of inflammatory bowel disease or for screening a candidate substance that can be an active ingredient of a therapeutic or prophylactic agent thereof,
Use of the CD300a gene-deficient mouse, wherein the CD300a gene-deficient mouse is used as a model mouse that hardly induces inflammatory bowel disease when a substance that induces inflammatory bowel disease is administered.

Moreover, the following invention is provided as another aspect of the said invention.
As another aspect of the invention according to the above [1], treatment of inflammatory bowel disease characterized by inhibiting binding of CD300a and phosphatidylserine and suppressing inhibitory signaling of myeloid cells expressing CD300a Or a method of prevention is provided. The method can be applied both in vivo (ex vivo) and ex vivo (in vitro). When applied in vivo, the species is human or non-human. Or (for example, a mammal such as a mouse).

  As a further aspect of the invention according to [1] above, suppressive signaling of myeloid cells expressing CD300a by inhibiting the binding of CD300a and phosphatidylserine in the manufacture of a medicament for treating or preventing inflammatory bowel disease There is provided the use of an activity modulator that inhibits

  According to the present invention, it is possible to produce a medicament for treating or preventing inflammatory bowel disease, which contains as an active ingredient an activity regulator that inhibits inhibitory signaling of CD300a-expressing cells. Further, according to the present invention, a CD300a gene-deficient mouse or the like can be used as a model mouse or the like useful for elucidating or treating the pathological condition of inflammatory bowel disease.

FIG. 1 shows the results of flow cytometry analysis obtained in Reference Example 1A. FIG. 2A shows the results of flow cytometry analysis obtained in Reference Example 1B. FIG. 2B shows the results of flow cytometry analysis obtained in Reference Example 1B. FIG. 2C shows the results of flow cytometry analysis obtained in Reference Example 1C. FIG. 2D shows the results of flow cytometry analysis obtained in Reference Example 1D. FIG. 2E shows the results of flow cytometry analysis obtained in Reference Example 1D. FIG. 2F shows the results of immunoblot analysis obtained in Reference Example 1E. FIG. 3A is a schematic diagram showing the structure of the CD300a gene in the wild-type allele, the targeting vector used to create the CD300a-deficient mouse, and the targeting allele. FIG. 3B is an electrophoretogram showing the PCR products of the wild type allele and the mutant allele. FIG. 3C shows the results of Western blot analysis using wild-type mice and CD300a-deficient mice. FIG. 3D shows the results of flow cytometry analysis of WT mice and CD300a gene-deficient mice. FIG. 4A shows the results of flow cytometry analysis obtained in Reference Example 2A. FIG. 4B shows the analysis result of the optical microscope obtained in Reference Example 2C. FIG. 4C shows the analysis result of the laser scanning confocal microscope obtained in Reference Example 2C. FIG. 4D shows the results obtained in Reference Example 2C showing the ratio of the number of cells in NIH3T3 in which thymocytes were taken into the cytoplasm or in each transformant. FIG. 5A shows the results of flow cytometry analysis obtained in Reference Example 2B. FIG. 5B shows the results of RT-PCR analysis obtained in Reference Example 2B. FIG. 6A shows the results of flow cytometry analysis obtained in Reference Example 3A. FIG. 6B shows the analysis result of the β-hexaminidase assay obtained in Reference Example 2A. FIG. 7A shows the results of flow cytometry analysis obtained in Reference Example 3B. FIG. 7B shows the results of the increase rates of the release amounts of various cytokines and chemokines obtained in Reference Example 3C. FIG. 7C is a diagram showing the results of increase rates of the release amounts of various cytokines and chemokines obtained in Reference Example 3E. FIG. 7D shows the result of immunoblotting analysis obtained in Reference Example 3F. FIG. 7E is a diagram showing the results of immunoblotting analysis obtained in Reference Example 3G. FIG. 7F is a diagram showing an increase rate of TNF-α obtained in Reference Example 3G. FIG. 8 shows the results of flow cytometry analysis obtained in Reference Example 3D. FIG. 9A shows the results of densitometric analysis obtained in Reference Example 4B. FIG. 9B shows the results of densitometric analysis obtained in Reference Example 4B. FIG. 10A is a diagram showing a calculation result of CFU of anaerobic bacteria obtained in Reference Example 4C. FIG. 10B shows the numbers of neutrophils and macrophages obtained in Reference Example 4C after induction of CD300a neutrophils. FIG. 11 is a diagram showing the cell number ratio of each macrophage phagocytosed in E. coli obtained in Reference Example 4C. FIG. 12A is a diagram showing a flow cytometry analysis obtained in Reference Example 4D. FIG. 12B is a diagram showing the survival rate of each mouse obtained in Reference Example 4D. FIG. 12C is a diagram showing bacterial clearance in the intestines of each mouse in Reference Example 4D using bacterial CFU. FIG. 12D is a diagram showing the results of flow cytometry analysis obtained in Reference Example 4E. FIG. 12E shows the survival rate of each mouse after TX41 was administered in Reference Example 4F. FIG. 12F is a diagram showing intestinal clearance after administration of TX41 in Reference Example 4G. FIG. 12G is a diagram showing the results of analyzing changes in the number of neutrophil cells after administration of TX41 in Reference Example 4G. FIG. 13 shows changes over time in the weight of each mouse due to DSS-induced enteritis (Example 1A). FIG. 14 is a graph showing the length of the large intestine of each mouse on day 6 after the start of DSS administration (Example 1B). FIG. 14A is a diagram showing a section of the large intestine of the WT mouse of FIG. 14 and CD300a ^{− / −} (Example 1C). FIG. 15 is a diagram comparing the degree of cell damage of each mouse on the 6th day of DSS administration using clinical scores (Example 1D). FIG. 16 shows the amount of each cytokine in the large intestine of each mouse on the 9th day of DSS administration (Example 1E). FIG. 16A is a graph showing the number of immune cells in each mouse on day 0, 2, 4, and 6 after the start of DSS administration (Example 1F). FIG. 17 shows the results of flow cytometry performed to identify dendritic cells expressing CD300a (Example 1G). FIG. 18 shows the relative gene expression levels of cytokines in the large intestine of each mouse (Example 1H). FIG. 19 shows the relative gene expression levels of each cytokine in CD4 + T cells (Example 1I). FIG. 20 shows the results of homology analysis of the H chain and L chain of TX41 and TX49, respectively.

  The activity regulator according to the present invention, pharmaceuticals containing it, use of CD300a gene-deficient mice, and anti-CD300a antibody will be described in detail below. Literatures used when referring to conventional knowledge and known test methods regarding the immune mechanism and CD300a are listed at the end of the examples.

[Activity regulator]
Activity regulators in the present invention include those for suppressing and enhancing inhibitory signaling of myeloid cells expressing CD300a.

  Here, “myeloid cells expressing CD300a” include mast cells (mast cells), macrophages, neutrophils, dendritic cells, and the like. CD300a is a generic term for those expressed in humans, mice, and other mammals, and the biological species is not particularly limited.

  In addition, “inhibitory signal transduction” is signal transduction that occurs when an inhibitory receptor, CD300a, associates with a dephosphorylating enzyme via an intracellular region ITIM (Immunoceptor tyrosine-based inhibitory motif) sequence. .

<First activity regulator>
The 1st activity regulator in this invention contains the component which has the effect | action which suppresses inhibitory signal transmission via CD300a. In the present invention, a substance that inhibits the binding between phosphatidylserine and CD300a, that is, a phosphatidylserine-binding substance and a CD300a-binding substance can be used as such a component. The first activity regulator may contain either one of these or both of them.

(Phosphatidylserine-binding substance)
A phosphatidylserine-binding substance, which is one of the first activity regulators, binds to phosphatidylserine (PS), which is a ligand for CD300a, and interacts with phosphatidylserine and CD300a expressed in myeloid cells. There is no particular limitation as long as it inhibits (binding).

  Specific examples of the phosphatidylserine-binding substance include MFG-E8 (Milk Fat Global Protein EGF-8), T cell immunoglobulin, soluble TIM-1, soluble TIM-4, soluble stabilizer and Examples include soluble proteins such as soluble integrin αvβ3, among which MFG-E8 is preferred.

  The phosphatidylserine-binding protein is not limited to a native protein such as MFG-E8, and one or several amino acids are deleted, substituted, or added as long as the binding to phosphatidylserine is not lost. It may be one having a modified amino acid sequence (mutant) (for example, “D89E MFG-E8” in the Examples).

These variants are made by known methods such as site-directed mutagenesis or random mutagenesis.
The above-mentioned “soluble protein” is a native protein that is insoluble in diluents and body fluids such as membrane proteins, which will be described later, and a hydrophobic domain is deleted by a known genetic recombination technique. Or a protein that has been modified so as to be soluble in a diluent or body fluid by adding a hydrophilic peptide.

(CD300a binding substance)
The CD300a-binding substance that is one of the first activity regulators is not particularly limited as long as it binds to CD300a and inhibits the interaction (binding) between CD300a expressed in myeloid cells and phosphatidylserine. .

Specific examples of the CD300a binding substance include neutralizing antibodies against CD300a. The neutralizing antibody may be a specific type of monoclonal antibody or a combination of two or more types of monoclonal antibodies (or polyclonal antibodies). Further, the neutralizing antibody may be a full-length antibody or a fragmented antibody (such as Fab fragment or F (ab ′) ₂ ).

  The neutralizing antibody can be prepared by a known technique. In the case of a monoclonal antibody, generally, an anti-CD300a monoclonal antibody can be prepared by procedures such as immunization using CD300a, preparation of a hybridoma, screening, culture, and recovery. Among them, an appropriate monoclonal antibody having a binding inhibitory ability (neutralizing action) between preferred CD300a and phosphatidylserine and exhibiting the action and effect of the present invention may be selected.

(TX41, TX49 and similar antibodies)
TX41 is an anti-mouse CD300a monoclonal antibody (rat IgG2a), and TX49 is an anti-human CD300a monoclonal antibody (mouse IgG1). Both are monoclonal antibodies prepared and used in the examples described later, and are excellent as a CD300a-binding substance in the present invention because they are excellent in the function of suppressing the signal transduction by inhibiting the binding between CD300a and phosphatidylserine. However, the anti-CD300a antibody that can be used in the present invention is not limited to TX41, TX49, and similar antibodies (having variable regions consisting of equivalent amino acid sequences).

  The TX41 heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 17, the TX41 light chain variable region has the amino acid sequence shown in SEQ ID NO: 18, and the TX49 heavy chain variable region has the SEQ ID NO: The L chain variable region of TX49 has the amino acid sequence represented by SEQ ID NO: 20. Note that these variable regions include three complementarity determining regions (CDRs) and four framework regions. FIG. 20 shows the result of analyzing the homology between the amino acid sequences of the variable regions of TX41 and TX49 (H chain and L chain, respectively).

  As a binding substance for mouse CD300a, based on TX41, the variable region of the H chain consists of an amino acid sequence represented by SEQ ID NO: 17, and the variable region of the L chain consists of an amino acid sequence represented by SEQ ID NO: 18. It is preferable to use an antibody.

  As a binding substance for human CD300a, based on TX49, the variable region of the H chain consists of the amino acid sequence represented by SEQ ID NO: 19, and consists of the amino acid sequence represented by SEQ ID NO: 20 as the variable region of the L chain. It is preferable to use an antibody.

  In addition, as a binding substance for mouse CD300a, substitution, addition, insertion or deletion of 1, 2, 3, 4 or 5 amino acids in the amino acid sequence represented by SEQ ID NO: 17 in the variable region of the H chain An amino acid sequence having an amino acid sequence, or an amino acid comprising an L chain variable region having 1, 2, 3, 4 or 5 amino acid substitutions, additions, insertions or deletions relative to the amino acid sequence represented by SEQ ID NO: 18 An antibody comprising a sequence can also be used (one of the H chain and the L chain may have the above mutation, or both of them may have the above mutation).

  As a binding substance for human CD300a, the variable region of the H chain includes 1, 2, 3, 4 or 5 amino acid substitutions, additions, insertions or deletions to the amino acid sequence represented by SEQ ID NO: 19. From an antibody comprising an amino acid sequence, or an amino acid sequence in which the variable region of the L chain has 1, 2, 3, 4 or 5 amino acid substitutions, additions, insertions or deletions relative to the amino acid sequence represented by SEQ ID NO: 20 Can be used (one of the H chain and the L chain may have the above mutation, or both of them may have the above mutation).

  It is preferable that the site causing such a mutation avoids the CDR or a site in the vicinity thereof in the variable region. Moreover, when substituting amino acids, conservative amino acid substitution performed between amino acids having similar side chain structures and / or chemical properties is preferable.

  The constant region of the anti-CD300a antibody, that is, the region of the Fab region other than the variable region as described above and the form of the Fc region (amino acid sequence, amino acid length) hardly affect the binding property to CD300a, that is, the neutralizing action. Therefore, it can design suitably in the range which does not inhibit the effect of this invention.

That is, an anti-CD300a antibody can be produced as a fusion protein comprising the amino acid sequence of the predetermined variable region and amino acid sequences of various known constant regions.
For example, it is one preferred embodiment to produce an anti-human CD300a antibody as a human chimeric antibody by utilizing a human constant region. Such an anti-CD300a antibody can be prepared by a known technique.

  For example, expression of the anti-CD300a antibody gene is performed by synthesizing DNA encoding the amino acid sequence of the predetermined variable region and ligating it with DNA encoding the amino acid sequence of the constant region and other necessary DNA (such as transcription factors). A vector can be constructed. If this vector is introduced into a host cell and expressed, the target anti-CD300a antibody can be produced.

  In addition, TX41, TX49, and similar antibodies as described above may be used for purposes other than the purpose of inhibiting inhibitory signal transduction caused by the binding of phosphatidylserine and CD300a according to the effects of the present invention. .

(CD300a siRNA)
In addition, the siRNA designed based on the gene sequence of CD300a (available from DNA databases such as DDBJ / EMBL / GenBank = INSD) suppresses the expression of CD300a in myeloid cells at the affected site, thereby allowing the CD300a gene to Similar to the deficient state or the state in which the binding between CD300a and phosphatidylserine is inhibited, the therapeutic effect on the above-mentioned various diseases can be obtained. In other words, siRNA against the CD300a gene is also a kind of substance that inhibits the binding of CD300a and phosphatidylserine in the present invention.

(Use of first activity regulator)
The 1st activity regulator in this invention can be used in order to suppress the inhibitory signal transmission of the myeloid type | system | group cell which expresses CD300a. At this time, the myeloid cells may be in vivo, isolated from in vitro, or cultured in vitro.

  By the above action, by maintaining or improving the activity signaling of CD300a of myeloid cells, signal transmission between cells via chemical mediators released from the myeloid cells is also maintained or improved, and between the additional cells connected thereto It can affect inflammation and allergic reactions caused by information transmission. Therefore, the first activity regulator can be used as an active ingredient of a predetermined pharmaceutical that will be described later. Further, for example, it can be used as an active ingredient of a drug as a comparative analysis tool for improving and comparing the pathological condition of inflammatory bowel disease in experimental animals.

  For inflammatory bowel disease in which symptoms are confirmed to be alleviated by deficiency of the CD300a gene (that is, the binding between CD300a and phosphatidylserine is not completely generated), a CD300a binding substance (TX41, It can be sufficiently estimated by those skilled in the art that neutralizing antibodies such as TX49) and phosphatidylserine-binding substances (MFG-E8 etc.) can be therapeutic agents.

<Second activity regulator>
A 2nd activity regulator contains the component which has the effect | action which enhances the inhibitory signal transmission via CD300a (that is, suppresses the activity signaling of CD300a). In the present invention, as such a component, a substance that enhances the binding between phosphatidylserine and CD300a, particularly phosphatidylserine, which is a ligand of CD300a, can be used.

  In addition, by screening using a CD300a gene-deficient mouse provided by another aspect of the present invention, there is a possibility that an agonist (low molecular compound, antibody, etc.) against CD300a having the same action as phosphatidylserine may be found, They can also be used as substances that enhance the binding between phosphatidylserine and CD300a.

  In the present invention, the second activity regulator is used as a tool for comparative analysis when conducting pathological analysis on inflammatory bowel disease or screening candidate substances that can be active ingredients of therapeutic or preventive drugs thereof. Can be used.

(Phosphatidylserine)
Phosphatidylserine (PS) is a CD300a ligand expressed in myeloid cells, and the inhibitory signaling of CD300a-expressing cells is enhanced by the interaction (binding) between PS and CD300a. For example, in mast cells, the activity of mast cells related to inflammatory reactions, such as release of chemical mediators such as histamine, cytokines, and chemokines, is suppressed through this inhibitory signaling. PS is produced industrially and can be easily obtained.

  In addition, for CD300a-expressing cells in vitro (in a test environment), apoptotic cells in a state in which PS is presented (PS is the inside of cells in normal cells (the cytoplasmic side of the lipid bilayer) But also known to be presented outside the cell when apoptosis occurs) can also be a second activity regulator. In addition, liposomes or the like in which a lipid membrane containing PS is formed on the outside may be used as the second activity regulator.

(Calcium salt)
Since the interaction between PS and CD300a in mast cells requires calcium ions, the second activity regulator may contain a calcium salt (for example, calcium chloride) that generates calcium ions by ionization. preferable.

  The content of the calcium salt in the second activity regulator can be appropriately determined in consideration of the calcium ion concentration at the administration site, the amount of PS contained in the second activity regulator, and the like.

(Use of second activity regulator)
The second activity regulator according to the present invention can be used to enhance inhibitory signaling of myeloid cells expressing CD300a. At this time, the myeloid cell may be in vivo, isolated from the living body, or produced by in vitro culture.

  By suppressing the activity signaling of CD300a of myeloid cells by the above action, information transmission between cells via chemical mediators released from the myeloid cells is also suppressed, and it is caused by information transmission between further cells connected thereto. Can affect inflammation and allergic reactions. Therefore, the second activity regulator can be used, for example, as an active ingredient of a drug as a comparative analysis tool for exacerbating inflammatory bowel disease in a laboratory animal for comparative analysis and as an immunosuppressant.

[Pharmaceuticals]
The pharmaceutical product (pharmaceutical composition) according to the present invention contains the activity regulator as described above as an active ingredient, and further contains various pharmaceutically acceptable additives (for example, carriers) as necessary. Also good.

  Such a medicament can be formulated for treating or preventing a disease or symptom (particularly an inflammatory reaction) involving inhibitory signaling of myeloid cells expressing CD300a.

More specifically, a pharmaceutical for treating or preventing inflammatory bowel disease can be prepared by blending the first activity regulator as an active ingredient.
The administration site of the drug can be a site where an excessive immune function (inflammatory reaction) occurs depending on the disease or medical condition to be applied, and is not particularly limited. For example, intraperitoneal, intratracheal, subcutaneous, intradermal, genitourinary and the like.

  Since myeloid cells expressing CD300a are usually present in mammalian submucosa or connective tissue, it is preferable that the pharmaceutical is directly administered to or near the submucosa or connective tissue of the above site. . Such administration can be performed by injection administration such as intravenous injection, intraarterial injection, subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection and the like. For example, intraperitoneal injection is preferred when treating or preventing inflammatory infections (such as bacterial peritonitis).

  The dose per administration and the number of administrations of pharmaceuticals (or active ingredients contained therein) are determined based on the patient's age, sex, body weight and symptoms, degree of required therapeutic effect, administration method, treatment time, active ingredient It may vary depending on the type and may be adjusted as appropriate. The frequency of administration is, for example, once to several times per day.

  When a pharmaceutical product contains a phosphatidylserine-binding substance as an active ingredient among the first activity regulators, the pharmaceutical is usually administered at a single dose of the phosphatidylserine-binding substance per kg of human or animal to be administered. It may be formulated to be 3 to 15 mg, preferably 5 to 10 mg.

  When the pharmaceutical product contains a CD300a binding substance as the active ingredient in the first activity regulator, the pharmaceutical product usually has a dose of 50 to 300 kg per person or animal to be administered. It may be formulated to be 150 mg, preferably 50 to 100 mg.

(Pharmaceutically acceptable carrier)
The pharmaceutical product according to the present invention may contain a pharmaceutically acceptable carrier as necessary.

  The pharmaceutically acceptable carrier is not particularly limited as long as it is not contrary to the purpose of the pharmaceutical product. For example, a diluent such as an aqueous diluent or a non-aqueous diluent, an antioxidant (sulfite, etc.), etc. Stabilizers / preservatives, phosphates and other buffers, surfactants and other emulsifiers, coloring agents, thickeners, local anesthetics such as lidocaine, glycols and other solubilizing agents, sodium chloride and glycerin, etc. Examples include tonicity agents and other additives.

  For example, when the pharmaceutical dosage form according to the present invention is an injection, it is preferable to dissolve or disperse the active ingredient in the diluent by blending the diluent so as to have a desired viscosity and concentration of the contained component. .

Examples of such a diluent include aqueous diluents such as physiological saline or commercially available distilled water for injection, and non-aqueous diluents such as alcohols such as polyethylene glycol and ethanol.
A pharmaceutical product whose dosage form is an injection may be sterilized by filtration using a filter or sterilized by blending a bactericide or the like according to a conventional method.

  Moreover, when administering the pharmaceutical which concerns on this invention as an injection, it is good also as a form of preparation before use. For example, a solid preparation containing the first activity regulator can be prepared by lyophilization and the like, and an injection can be prepared by dissolving or dispersing in a diluent before use.

<Pharmaceuticals related to inflammatory infections>
Inhibiting the binding of phosphatidylserine to CD300a-expressing cells (mast cells) can maintain or improve mast cell activity. Thereby, the number of neutrophil cells can be increased, the attack by neutrophils on pathogens (bacteria, parasites, etc.) can be activated, and the pathogen growth suppression function and pathogen clearance function can be improved. Therefore, by using the first activity regulator (phosphatidylserine binding substance or CD300a binding substance) as an active ingredient, a pharmaceutical against inflammatory infection can be obtained.

<Pharmaceuticals for inflammatory bowel disease>
Inhibition of phosphatidylserine binding to CD300a-expressing cells (CD11b-positive dendritic cells in the lamina propria of the large intestine) is known to suppress the induction of proliferation of inflammatory T cells. The production amount is increased, and the activation of inflammatory T cells can be suppressed to maintain the homeostasis of the intestinal tract immune system. Therefore, a pharmaceutical agent for inflammatory bowel disease can be obtained by using the first activity regulator as an active ingredient.

[Use of CD300a gene-deficient mice]
<Uses related to inflammatory bowel disease>
According to the knowledge obtained by the present invention, when inflammatory bowel disease is induced by a known method such as administration of an inflammation inducer (DSS or the like) to CD300a-deficient mice, it is more inflammatory than wild mice. Symptoms of bowel disease have been improved.

  That is, the CD300a gene-deficient mouse can be used as a model mouse that hardly induces an inflammatory disease when a substance that induces inflammatory bowel disease is administered. It can be used for the pathological analysis etc.

(Method for producing CD300a gene-deficient mouse)
The CD300a gene-deficient mouse of the present invention is a mouse deficient in the function of the CD300a protein by replacing the CD300a gene on the chromosome with an inactive CD300a gene.

  “Inactive CD300a gene” means a partial deletion of the CD300a gene, insertion of another nucleotide sequence into the coding region of the CD300a gene, a point mutation in the CD300a gene, a mutation in the expression regulatory region of the CD300a gene, etc. Refers to a gene that cannot express normal CD300a protein. Examples of the defective CD300a gene include, but are not limited to, a gene in which at least one of exons 1 to 6 contained in the CD300a gene is deleted.

  “Deficiency of the function of the CD300a protein” means that at least a part of the function of the CD300a protein involved in inhibitory signaling involved in the present invention has been lost (for example, one allele such as a hetero knockout mouse). Only the inactive form is substituted, and the function of the CD300a protein is partially lost.), Preferably the function is completely lost.

  A general method for obtaining a CD300a gene-deficient mouse using a gene cassette is as follows. However, the CD300a gene-deficient mouse of the present invention is not limited to that obtained by this method.

  A targeting vector having a targeting allele (mutant allele) in which the exon of the Cd300a gene in the wild type allele is replaced with an antibiotic resistance gene (marker gene) cassette is prepared. According to a conventional method, a chimeric mouse is obtained using the targeting vector and ES cells. The chimeric mouse and the wild-type mouse are mated to obtain a child mouse (heterozygote (+/−)), and the child mice are further mated to obtain a grandchild mouse.

  Genomic DNA is extracted from the grandchild mouse, and the presence or absence of the wild type allele and the mutant allele in the genomic DNA is confirmed by PCR, and the grandchild mouse (homozygote (− /-)). Further, in order to confirm that CD300a is not expressed, the cells derived from the mouse are confirmed by Western blot analysis using an anti-CD300a antibody to obtain a CD300a gene-deficient mouse.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples do not limit the present invention.
1. Preparation Examples (1) Preparation of CD300a-Fc fusion protein, MFG-E8 and D89E MFG-E8 (1-1) A fusion protein of CD300a having an Fc site of human IgG (CD300a-Fc) is described in Reference 25 below. As described above, it was prepared from a chimeric cDNA containing the cDNA of the gene encoding the entire extracellular domain of mouse or human CD300a and the cDNA of the gene encoding human IgG1 Fc. In the above fusion protein, the CD300a extracellular domains derived from mouse and human are referred to as “mouse CD300a-Fc” and “human CD300a-Fc”, respectively.

(1-2) MFG-E8 was provided by Mr. Masato Tanaka (RCAI, Yokohama, Japan).
(1-3) D89E MFG-E8 is a variant of MFG-E8 obtained by introducing a site-specific mutation into the RGD motif of MFG-E8, as described in Reference 4 below.

  In the obtained D89E MFG-E8, the 89th amino acid from the N-terminus is substituted from aspartic acid to glutamic acid. MFG-E8 (native) binds to both phosphatidylserine (PS) and αvβ3 integrin to crosslink apoptotic cells and phagocytic cells expressing αvβ3 integrin (Reference 4 below). D89E MFG-E8 binds to phosphatidylserine (PS) but does not bind to αvβ3 integrin.

(2) Mouse and cecal ligation and puncture (CLP)
The knockout mouse used in this example was prepared or provided as follows.

(I) CD300a gene deficient (Cd300a ^{− / −} ) mouse Exons 1 to 6 of the Cd300a gene in the wild-type allele were used in a neomycin resistance gene cassette (PGK-GB2-neo) using a bacterial artificial chromosome (BAC) system. Replacement and targeting alleles (mutant alleles) were prepared (FIG. 3A). Then, according to a conventional method, a chimeric mouse is obtained, and the chimeric mouse and a wild-type mouse are further mated to obtain a child mouse (heterozygote (+/−)), and further, the child mice are mated with each other. A mouse was obtained.

In order to select a CD300a-deficient (Cd300a ^{− / −} ) mouse from among the grandchild mice, genomic DNA was extracted from the tail of each grandchild mouse, and the genomic DNA was subjected to PCR method to detect WT alleles and mutations in the genomic DNA. The presence or absence of the type allele was confirmed.

As shown in FIG. 3B, the PCR product showing the WT allele and the PCR product showing the mutant allele are detected as bands of about 540 bp and about 700 bp, respectively.
Furthermore, in order to confirm that CD300a is not expressed in CD300a gene-deficient mice, cells derived from CD300a gene-deficient mice were subjected to Western blot analysis using an anti-CD300a antibody. As shown in FIG. 3C, the wild-type mouse showed a band derived from CD300a at about 50 kDa, whereas this band was not detected in the CD300a gene-deficient mouse.

(Ii) C57BL / 6J-kit ^{W-sh / W-sh} mouse C57BL / 6J-kit ^{W-sh / W-sh} mouse (hereinafter, “kit ^{W-sh / W-sh} mouse” or “mast cell-deficient mouse”) ) Provided by RIKEN BioResource Center (Tsukuba, Japan). The mouse is a mouse deficient in mast cells (Reference 21 below), and after DNA is taken up by phagocytic cells without undergoing apoptotic DNA fragmentation, DNA is degraded in the phagocytic cells. Is known to occur (Document 12 below).

(Iii) CAD-deficient mouse A CAD (Caspase-activated DNase) -deficient mouse is described in Reference 12 below.

(V) In vivo removal of macrophages and neutrophils Based on the description in Reference 30 below, clodonic acid liposomes and control PBS liposomes (Encapsula NanoSciences) were prepared. Then, 24 hours after CLP, 0.5 mL of these liposomes were injected into the mouse abdominal cavity to remove macrophages.

Further, 24 hours after CLP, anti-Gr-1 antibody was injected into the abdominal cavity of mice to remove neutrophils.
Note that the preparation and evaluation tests using mice in this example all complied with the guidelines prepared by the Animal Ethics Committee of the University of Tsukuba Life Science Animal Resource Center.

(3) Various antibodies Table 1 below shows the suppliers and preparation methods of various antibodies.

(4) Preparation of various cells Various cells were prepared as follows.
(I) Bone marrow derived mast cells (BMMC)
Complete RPMI 1649 medium containing 2 × 10 ⁸ mouse bone marrow cells in a 10 cm dish containing cell growth factor (SCF) (10 ng / mL), IL-3 (4 ng / mL) and calf serum (FBS) (10%) Bone marrow-derived mast cells (BMMC) were prepared by culturing in them for 5 weeks or more. BMMC was passaged with a new medium every week. Of the prepared BMMCs, more than 95% were shown to be c-Kit ⁺ FcεRI ⁺ cells by flow cytometric analysis.

(Ii) Bone marrow-derived macrophages (BMMφ)
Murine bone marrow cells 2 × 10 ⁶ cells were cultured in a complete RPMI 1649 medium containing M-CSF (10 ng / mL) and calf serum (FBS) (10%) for 1 week in a 10 cm dish, and bone marrow-derived macrophages (BMMφ) ) Was prepared.

(Iii) NIH3T3 cell transformant A pMX-neo retroviral vector plasmid containing a Flag-tagged CD300a cDNA or a Flag-tagged CD300d cDNA was prepared based on a conventional method.

  The prepared plasmid was transduced (transfected) with NIH3T3 cells to obtain a transformant stably expressing CD300a or CD300d. The obtained transformant stably expressing CD300a and transformant stably expressing CD300d are referred to as “NIH3T3 transformant (CD300a)” and “NIH3T3 transformant (CD300d)”, respectively.

  In addition, NIH3T3 cell transformants that stably express TIM-4 are described in T.W. Provided by Kitamura (University of Tokyo). This transformant is referred to as “NIH3T3 transformant (TIM-4)”.

[Evaluation method]
The conditions for the evaluation method are described below. In the survival test, Kaplan-Meier log-rank test was used, and in other evaluation tests, unpaired student's test was used. Analysis was carried out. P <0.05 was considered statistically significant.

(5) Binding assay etc. (i) Binding assay Cells were stained with CD300a or human IgG for control for 30 minutes in a phosphate buffer containing 2% FBS in the presence or absence of 1 mM CaCl _2. Was washed twice with the buffer and incubated with the F (ab ′) ₂ fragment of FITC-conjugated goat anti-human IgG. Cells were then incubated with Annexin V for 15 minutes at room temperature in 10 mM HEPES-NaOH buffer containing 140 mM NaCl and 2.5 mM CaCl ₂ for staining with Annexin V.

(Ii) Binding inhibition assay Cells were preincubated with a monoclonal antibody against mouse CD300a (TX41), a control isotype antibody or MFG-E8 for 30 minutes and then stained with CD300a-Fc as in the binding assay. Further, in order to analyze whether CD300a-Fc was bound to phospholipid, a PIP strip assay (manufactured by Echelon Biosciences) was performed according to the manufacturer's instruction manual.

(6) Measurement of aerobic bacterial CFU (colony forming unit) A serial dilution of mouse peritoneal perfusate was plated, and the dilution was placed on a plate containing brain heart infusion (BHI) agar at 37 ° C. And cultured for 48 hours. Next, CFU of aerobic bacteria was calculated by measuring the number of colonies in 1 mL of peritoneal perfusate as described in the following document 27.

[Reference Example 1: Identification of CD300a ligand]
[Reference Example 1A]
In order to confirm the expression of mouse CD300a ligand in various hematopoietic stem cell lineage cell lines or tumor cell lineage cell lines, the following tests were conducted.

Bone marrow-derived macrophages (BMMφ), bone marrow-derived dendritic cells (BMDC) or IL-3-dependent hematopoietic cell lines (BaF / 3 cells) (2 × 10 ⁵ cells each) obtained in “1. Preparation Example” , CD300a-Fc (1 μg), and PBS (phosphate buffered saline) containing calcium chloride (1 mM), incubated at 20 ° C. for 30 minutes, and then FITC-conjugated anti-human IgG antibody (0.1 μg) and iodine Staining was carried out using a buffer containing propidium iodide (PI) (1 μg).

Each stained cell was subjected to analysis using flow cytometry (manufactured by Becton Dickinson, FACSCalibur, model number “E6133”).
In addition, a control test was also conducted in the same manner as the above test method except that human IgG for control (1 μg) was used instead of mouse CD300a-Fc.

  The results of flow cytometry analysis using BMMφ, BMDC, and BaF / 3 cells are shown in FIG. 1A, FIG. 1B, and FIG. 1C, respectively (the control test is shown in “Ctrl Ig”).

As shown in FIGS. 1A-1C, mouse CD300a-Fc was found to bind PI ⁻ cells (live cells) but not PI ⁺ cells (dead cells) when calcium chloride was included. . That is, it is suggested that the mouse CD300a ligand is expressed in dead cells.

[Reference Example 1B]
In order to verify whether mouse CD300a-Fc binds to apoptotic cells, which are a type of dead cell, the following test was performed.

  Thymocytes derived from C57BL / 6 mice (wild-type mice) were incubated with dexamethasone (Sigma) (10 μM) in RPMI medium to prepare thymocyte apoptotic cells.

The obtained apoptotic cells (2 × 10 ⁵ cells) were cultured at 20 ° C. in a medium (PBS) containing CD300a-Fc (1 μg), APC-conjugated annexin V (BD Pharmingen) (1 μl) and calcium chloride (1 mM). , Incubated for 30 minutes, and then stained with a buffer containing FITC-conjugated anti-human IgG antibody (0.1 μg) and propidium iodide (PI) (1 μg).

Each stained cell was analyzed using flow cytometry (Becton Dickinson, FACSCalibur, model number “E6133”) (result: FIG. 2A).
Moreover, it used for the analysis using the flow cytometry similarly to the said test conditions except having used the medium which does not contain calcium chloride instead of the medium containing calcium chloride (result: FIG. 2B).

According to FIG. 2A, in the presence of calcium chloride, mouse CD300a-Fc does not bind to annexin V (annexin V ⁻ ) thymocytes, whereas it binds to thymocytes stained with annexin V. I understand. That is, mouse CD300a-Fc is shown to bind to apoptotic thymocytes.

On the other hand, as shown in FIG. 2B, it can be seen that in the absence of calcium chloride, mouse CD300a-Fc does not bind to apoptotic thymocytes.
From this test result, it can be understood that mouse CD300a binds to apoptotic cells in a calcium ion-dependent manner.

[Reference Example 1C]
Apoptotic cells (cell number 2 × 10 ⁵ ) obtained in Reference Example 1B were prepared by using mouse CD300a-Fc (1 μg), APC-linked annexin V (manufactured by BD Pharmingen) (1 μl), control human IgG1 (1 μg) and calcium chloride. Incubate in medium (PBS) containing (1 mM) at 20 ° C. for 30 minutes, and then using a buffer containing FITC-conjugated anti-human IgG antibody (0.1 μg) and propidium iodide (PI) (1 μg) Stained.

  Each stained cell was subjected to analysis using flow cytometry (Becton Dickinson, FACSCalibur, model number “E6133”) (result: FIG. 2C “HuIgG1”).

  Further, in place of the control human IgG1, a monoclonal antibody “TX41” or a protein “MFG-E8” expressed in macrophages was used in the same manner as in the above test conditions, except that the protein expressed in macrophages “MFG-E8” was used. The results were analyzed using flow cytometry (Results: FIG. 2C “TX41” and “MFG-E8”).

As described above, “TX41” used herein is an anti-mouse CD300a monoclonal antibody, which blocks the binding between mouse CD300a and a ligand.
In addition, “MFG-E8” binds to both phosphatidylserine (PS) and αvβ3 integrin, and is known to crosslink apoptotic cells and phagocytes expressing αvβ3 integrin ( Document 4) below.

As can be seen by comparing the results of each flow cytometry in FIG. 2C, it can be seen that mouse CD300a does not bind to apoptotic cells in the presence of TX41 or MFG-E8.
In view of this point, it is suggested that mouse CD300a-Fc has a binding property to PS (the ligand of CD300a is PS).

[Reference Example 1D]
In order to verify whether human CD300a also binds to apoptotic cells in the same manner as mouse CD300a, the following test was performed.

  First, Jurkat cells (human T cell lineage) were suspended in RPMI 1640 medium, and the medium was irradiated with ultraviolet rays for 60 minutes to prepare Jurkat cell apoptotic cells.

  As the apoptotic cells, the above “Jurkat cell-derived apoptotic cells” were used instead of wild-type mouse thymocyte-derived apoptotic cells, and “human CD300a-Fc” was used instead of “mouse CD300a-Fc”. In the same manner as in the test conditions of Reference Example 1A, analysis was performed using flow cytometry (result: FIG. 2D).

  In addition, as the apoptotic cells, the above-mentioned “Jurkat cell-derived apoptotic cells” are used instead of wild-type mouse thymocyte-derived apoptotic cells, “human CD300a-Fc” is used instead of “mouse CD300a-Fc”, and , Except that “TX49” or “control human IgG1” was used instead of “TX41”, analysis was performed using flow cytometry in the same manner as in the test conditions of Reference Example 1B (Result: FIG. 2E “TX49” Or “HuIgG1”). “TX49” is an anti-human CD300a antibody (monoclonal antibody) that blocks the binding between CD300a and a ligand.

As shown in FIGS. 2D-2E, it can be seen that human CD300a-Fc also binds to annexin V ⁺ cells, but not in the presence of anti-human CD300a antibody.
That is, it is suggested that human CD300a binds to apoptotic cells as well as mouse CD300a-Fc.

[Reference Example 1E]
A liquid (sample liquid) containing various phospholipids (PS, PC, PE) (100 pmol) is spotted on a membrane (PIP-strip (manufactured by Echelon Bioscience)), and various phospholipids are adsorbed on the membrane. It was.

  Next, the membrane was immersed in a TBST buffer solution (pH 8.0) supplemented with calcium chloride (1 mM) containing mouse CD300a-Fc (1.5 μg / mL) and BSA at 20 ° C. for 2 hours.

  After soaking, the cells were washed 3 times with TBST buffer (pH 8.0) not containing mouse CD300a-Fc to remove unbound CD300a-Fc on each phospholipid on the membrane, and then HRP-conjugated anti-human IgG Detection was performed using a TBST buffer (pH 8.0) containing BSA containing (Jackson Immun) (result: FIG. 2F).

  In FIG. 2F, “PE”, “PC”, and “PS” indicate portions where phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine are spotted on the membrane, respectively, and “Blank” indicates any phospholipid on the membrane. The part that is not spotted.

FIG. 2F shows that CD300a did not bind to either PE or PC, but specifically bound to PS.
From the results of Reference Examples 1A to 1E, it can be understood that CD300a binds to phosphatidylserine (PS) in a calcium ion-dependent manner (the ligand of CD300a is PS).

[Reference Example 2: Functional analysis of CD300a (2)]
Some PS-coupled receptors are expressed in phagocytic cells and are known to be involved in the removal of apoptotic cells under physiological and pathological conditions (References 4-9 below).

  In addition, PS is known to mediate a so-called “eat me” signal in phagocytic cells (macrophages and the like) that are cells expressing CD300a (the following documents 10 to 11). In view of this point, the tests described in Reference Examples 2A to 2C below were performed to verify whether CD300a is involved in the phagocytosis of apoptotic cells.

[Reference Example 2A]
Apoptotic thymocytes were prepared from thymocytes derived from CAD-deficient mice in the same manner as in Reference Example 1B.

Next, macrophages derived from CD300a gene-deficient mice (thioglycolate-induced peritoneal macrophages) (2 × 10 ⁵ cells) were apoptotic derived from CAD-deficient mice in an 8-well Lab-TeKII chamber slide (manufactured by Nalge Nunc). The cells were co-cultured with thymocytes at a ratio of 1: 5 (macrophages: apoptotic thymocytes (number of cells)) at 37 ° C. for 1 hour.

  Next, as described in Reference 5 or 26 below, the co-cultured macrophages were washed with cold PBS, fixed with a fixative containing paraformaldehyde (1%), and then FITC-labeled dUTP (Roche). The sample was subjected to TUNEL staining using a buffer solution containing

The stained macrophages of 50 cells or more are randomly analyzed with a laser scanning confocal microscope (Olympus, “FV10i”, model number: 1B22358), and the number of TUNEL positive cells (apoptotic cells) contained in one macrophage cell Was measured. The ratio (phagocytosis rate) of macrophages containing 0 to 8 apoptotic cells was calculated with the total number of macrophages as 100% (result: FIG. 4A “Cd300a ^{− / −} ”).

  In addition, the phagocytosis rate was measured in the same manner as in the above test conditions except that a macrophage derived from a wild type mouse was used instead of a macrophage derived from a CD300a gene-deficient mouse (result: FIG. 4A “WT”).

  As shown in FIG. 4A, there was no clear difference in the phagocytosis rate regardless of whether the macrophage was derived from a CD300a gene-deficient mouse or a wild-type mouse.

[Reference Example 2B]
In order to verify whether mast cells express known PS receptors (TIM-1, TIM-4, stabilin 2, integrin αvβ3), the following test was performed.

Bone marrow-derived mast cells (BMMC) (2 × 10 ⁵ cells), PE (Phycoerythrin) -conjugated anti-TIM-1 monoclonal antibody (0.1 μg), APC-conjugated anti-TIM-4 monoclonal antibody (0.1 μg) and Alexa Incubation was carried out at 20 ° C. for 30 minutes in medium (PBS) containing conjugated anti-mouse CD300a monoclonal antibody (TX41) (0.5 μg).

  Next, after washing twice with PBS, each stained cell was subjected to analysis using flow cytometry (Becton Dickinson, FACSCalibur, model number “E6133”) (Result: FIG. 5A “ BMMCs ").

  In addition, flow cytometry analysis was also performed in the same manner as in the above test method except that peritoneal macrophages and BM-derived macrophages were used instead of BMMC (results: FIG. 5A “Peritoneal macrophages” and “BM derived macrophages”). .

Furthermore, cDNA was prepared from peritoneal macrophages and BMMC using a High Capacity cDNA Reverse Transcription Kit (manufactured by Applied Biosystems), and each of the prepared cDNAs was used for stabilin 2, BAl-1, αv integrin, Cd300a and β The expression level of actin (loading control) was analyzed by RT-PCR (result: FIG. 5B).
Referring to FIGS. 5A and 5B, unlike macrophages, mast cells express CD300a and αvβ3 integrin, but are PS receptors involved in phagocytosis TIM-1, TIM-4 and stabilin 2 Is expressed only at a low level.

[Reference Example 2C]
NIH3T3 transformant (CD300a) (6 × 10 ⁴ cells) was co-cultured with FITC-labeled cells (apoptotic thymocytes or thymocytes (live cells)) for 2 hours, washed with PBS, and then light microscope ( Keyence (BZ-9000) (result: FIG. 4B).

  Further, the cells after co-culture and washing were fixed with a fixative Vectashield (produced by Vector Laboratories) containing paraformaldehyde and analyzed by a laser scanning confocal microscope (result: FIG. 4C). In FIG. 4C, a green part (indicated by an arrow) indicates an encapsulated cell (apoptotic thymocyte or thymocyte (live cell)).

  Moreover, it is the same as that of the said test conditions except having used the NIH3T3 non-transformed cell (negative control) or the "NIH3T3 transformant (TIM-4)" (positive control) instead of the NIH3T3 transformant (CD300a). Then, analysis was performed using an optical microscope and a laser scanning confocal microscope.

  “NIH-3T3” and “NIH-3T3 / Tim4” in FIG. 4B and FIG. 4C represent “NIH3T3” (untransformed cells) and “NIH3T3 transformant (TIM-4)” respectively. An image of a laser scanning confocal microscope is shown.

  In addition, from the image of the laser scanning confocal microscope, the ratio of the number of untransformed cells or each transformant in which apoptotic thymocytes were taken into the cytoplasm (co-cultured untransformed cells or each transformant) (Result: FIG. 4D “apoptotic”).

  Similarly, the ratio of the number of cells of NIH3T3 or each transformant in which thymocytes (live cells) are taken into the cytoplasm (the number of co-cultured untransformed cells or each transformant is 100%). (Result: FIG. 4D “Live”).

  As shown in FIG. 4B, unlike NIH3T3, both of the above transformants attached to apoptotic thymocytes. However, judging from FIGS. 4C and 4D, it can be seen that only the NIH3T3 transformant (TIM-4) took up apoptotic thymocytes and exerted phagocytosis.

Further, although no data is shown, it was observed that the NIH3T3 transformant (CD300a) did not phagocytose live cells (thymocytes), similar to NIH3T3.
From the results of Reference Examples 2A to 2C, it can be understood that CD300a is not involved in the phagocytosis of apoptotic cells by macrophages.

[Reference Example 3: Functional analysis of CD300a (2)]
As shown in FIG. 5, mast cells express CD300a, but do not express PS receptors TIM-1, TIM-4, and stabilin, unlike macrophages.

  These PS receptors are known to bind directly or indirectly to PS, and are known to contribute to the uptake of apoptotic cells (Reference 13 below). Therefore, in order to verify whether CD300a also has such a function (whether there is a functional duplication that takes in apoptotic cells), tests shown in Reference Examples 3A to 3C below were performed.

[Reference Example 3A]
Bone marrow cells (BM cells) of CD300a gene-deficient mice (2 × 10 ⁸ ) in a 10 cm dish, cell growth factor (SCF) (10 ng / mL), IL-3 (4 ng / mL) and calf serum (FBS) (10 %) Was cultured for 4 weeks in complete RPMI1649 medium to prepare bone marrow-derived mast cells (BMMC) of CD300a gene-deficient mice. BMMC was passaged with a new medium every week.

Next, the obtained BMMC was incubated at 4 ° C. for 30 minutes in RPMI1649 medium containing FITC-conjugated anti-FcεRIα antibody (0.1 μg) and PE-conjugated anti-c-Kit antibody (0.1 μg / mL). The results were analyzed by flow cytometry (result: FIG. 6A “CD300 ^{− / −} ”).

  Further, except that bone marrow cells derived from wild type mice were used instead of bone marrow cells derived from CD300a gene-deficient mice, BMMC was prepared in the same manner as in the test conditions described above (results: FIG. 6A “WT”) The numbers in FIG. 6A refer to the ratio of cells in the box, and each test was performed three times independently.

Moreover, (beta) -hexosaminidase release assay (degranulation assay) was implemented as follows using each BMMC (detailed conditions are described in the following literature 29).
First, each BMMC 1 × 10 ⁵ cells to 2 × 10 ⁵ cells in the logarithmic growth phase were cultured overnight at 37 ° C. in a 24-well plate coated with gelatin (manufactured by Sigma), and then biotin-conjugated mouse anti-trinitrophenol was obtained. Incubation was carried out at 37 ° C. for 1 hour in a medium containing IgE (0.5 mg / mL) and no supplement.

  Next, streptavidin was added to the above medium to crosslink the biotin-conjugated mouse anti-trinitrophenol IgEs and incubated at 37 ° C. for 45 minutes, and then the supernatant was collected.

  To the collected supernatant, p-nitrophenyl-N-acetyl-β-D-glucosamide (manufactured by Sigma) and a buffer solution (pH 4.5) containing citric acid (0.4M) and sodium phosphate (0.2M). ) And incubated at 37 ° C. for 3 hours to hydrolyze p-nitrophenyl-N-acetyl-β-D-glucosamide with the released β-hexosaminidase, and the reaction was conducted with 0.2 M glycine. After stopping by adding NaOH (pH 10.7), the absorbance at a wavelength of 415 nm generated by hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosamide was measured to release β-hexosaminidase The amount was quantified. Furthermore, the increase rate (%) of the release amount of β-hexosaminidase with respect to each BMMC not subjected to the above treatment is shown in FIG. 6B.

FIG. 6B shows the ratio of BMMC that released β-hexosaminidase. As shown in FIG. 6A and FIG. 6B, the expression of FcεRIα and c-Kit (mast cell marker protein), β-hex and BMMC are derived from CD300a gene-deficient mice and wild-type mice. There was no significant difference in the increase rate of sosaminidase release.
That is, it can be seen that CD300a does not affect differentiation from bone marrow cells or degranulation mediated by FceRI.

[Reference Example 3B]
BMMC derived from a CD300a gene-deficient mouse and apoptotic cells obtained in Reference Example 1B (BMMC: apoptotic cells = 10: 1 (cell number ratio)), calcium chloride (1 mM), APC-linked annexin V (1 μl), Incubation in PBS containing CD300a-Fc (1 μg / mL) and MFG-E8 (5 μg) for 30 minutes at 20 ° C., followed by FITC-conjugated anti-human IgG antibody (0.1 μg / mL) and propidium iodide (PI ) (1 μg) containing buffer solution.

  Stained cells were subjected to analysis using flow cytometry (Becton Dickinson, FACSCalibur, model number “E6133”) (result: FIG. 7A “MFG-E8”).

  Further, a flow cytometry analysis was performed in the same manner as in the above test conditions except that control IgG was used instead of MFG-E8 (result: FIG. 7A “Ctrl Ig”).

From the results shown in FIG. 7A, CD300a-Fc binds to apoptotic cells (annexin V ⁺ ) in the presence of control IgG, whereas in the presence of MFG-E8 (PS-binding substance) It can be seen that the binding is specifically inhibited.

  In the supernatant of this mixed sample, the concentrations of cytokine and chemokine were determined by BD Pharmingen (TNF-α and IL-6) and R & D Systems (MIP-2, MCP-1, IL-13 and MIP-). When an attempt was made to quantify cytokines and chemokines using the 1α) ELISA kit, no cytokines or chemokines were detected.

[Reference Example 3C]
In the case where BMMC and apoptotic cells coexist, the following test was conducted in order to verify whether or not the release amount of various cytokines was changed by stimulation with LPS (lipopolysaccharide).

  BMMC and apoptotic cells derived from CD300a gene-deficient mice (BMMC: apoptotic cells = 10: 1 (cell number ratio)) were incubated in RPMI containing LPS (1 μg / mL) for 4 hours, and then the medium supernatant was collected. did.

Cytokine and chemokine concentrations were then determined using ELISA kits from BD Pharmingen (TNF-α and IL-6) and R & D Systems (MIP-2, MCP-1, IL-13 and MIP-1α). The measurement was performed three times, and the increase rate of the release amount of each cytokine and chemokine of BMMC not subjected to the above LPS treatment was calculated (result: FIG. 7B “Cd300a ^{− / −} ”).

  In addition, the increase rates of cytokines and chemokines were calculated in the same manner as in the above test conditions except that BMMC derived from wild-type mice was used instead of BMMC derived from CD300a gene-deficient mice (Result: FIG. 7B “WT” ).

  As shown in FIG. 7B, in any BMMC, the amount of various cytokines released was increased by LPS. However, in BMMC derived from CD300a gene-deficient mice, TNF-α, IL-13, and MCP-1 were significantly increased compared to BMMC derived from wild-type mice.

[Reference Example 3D]
Furthermore, in order to verify the increase rate of various cytokines and chemokines in the cells of each BMMC, the following tests were conducted.

  BMMC derived from a CD300a gene-deficient mouse and the apoptotic cells obtained in Reference Example 1B (BMMC: apoptotic cells = 10: 1 (cell number ratio)) were treated with a medium containing LPS (lipopolysaccharide) (1 μg / mL) ( Incubated in RPMI) for 4 hours, the incubated BMMC and apoptotic cells were cultured at 4 ° C. in a medium (FIX & PERM, manufactured by Invitrogen) containing various fluorescently labeled antibodies against various cytokines and chemokines and formaldehyde. The cells that had been incubated for 20 minutes and stained were subjected to analysis using flow cytometry (manufactured by Becton Dickinson, FACSCalibur, model number “E6133”) (result: arrow (1) in FIG. 8). In addition, the use of a control antibody instead of various fluorescently labeled antibodies against various cytokines and chemokines was carried out under the same conditions as in the above-mentioned test conditions (control test (result: FIG. 8 arrow (2 ))).

  Further, flow cytometry analysis was performed in the same manner as the above test conditions except that BMMC derived from a wild type mouse was used instead of BMMC derived from a CD300a gene-deficient mouse (result: arrow (3) in FIG. 8). . Furthermore, instead of using various fluorescently labeled antibodies against various cytokines and chemokines, a control antibody was used in the same manner as the above test conditions, and flow cytometric analysis was performed (control test (result: arrow (4 in FIG. 8)). ))).

  Further, the graph of FIG. 8 shows the amount of increase in MFI with respect to BMMC not treated with LPS by measuring MFI values (mean fluorescence intensity) for each cytokinin and chemokine in each BMMC.

  FIG. 8 shows that the amount of cytokinin and chemokine increased in the cytoplasm of any BMMC compared to the case where LPS was not treated. In the cytoplasm of BMMC derived from a CD300a gene-deficient mouse, It can be seen that TNF-α and the like were significantly increased as compared to the cytoplasm of BMMC derived from wild type mice.

[Reference Example 3E]
D89E MFG-E8 is a variant (mutant) of MFG-E8 containing a point mutation (D89E) in the RGD motif, and has the binding property of binding to PS but not αvβ3 integrin.

Therefore, the following test was performed to verify whether the amount of cytokine and chemokine released from each BMMC changes in the presence of D89E MFG-E8.
In the same manner as Reference Example 3C, except that D89E MFG-E8 (5 μg / mL) was added together with LPS in a medium containing BMMC and apoptotic cells derived from CD300a gene-deficient mice, TNF-α, IL-13, MCP-1 and IL-6 were quantified (result: FIG. 7C “CD300a ^{− / −} ”).

  Further, TNF-α, IL-13, MCP-1 and IL-6 were quantified in the same manner as in the above test conditions except that BMMC derived from a wild type mouse was used instead of BMMC derived from a CD300a gene-deficient mouse. (Result: FIG. 7C "WT").

  As shown in FIG. 7C, in the presence of D89E MFG-E8, significant differences were observed in the concentrations of various cytokines when BMMC was derived from a CD300a gene-deficient mouse and from a wild-type mouse. There wasn't.

[Reference Example 3F]
CD300a has an immunoreceptive inhibitory tyrosine motif (ITIM) at the intracellular site, and it is known that SHP-1 is induced when crosslinked with an anti-CD300a antibody (Reference 14 below).

  Therefore, in order to verify whether or not CD300a and SHP-1 interact, the following test was performed. As in Test Example 3C, BMMC cell debris derived from CD300a gene-deficient mice or wild-type mice co-cultured with apoptotic cells in the presence of LPS for 4 hours was immunoprecipitated with an anti-CD300a antibody (TX41).

Using the obtained immunoprecipitate, immunoblotting with an anti-SHP-1 antibody or an anti-CD300a antibody was performed as described in Reference 14 below (result: FIG. 7D).
As can be seen from this result, it is understood that when BMMC is co-cultured with apoptotic cells, SHP-1 is induced (recruited) in response to LPS stimulation. However, in the presence of D89E MFG-E8, CD300a did not recruit SHP-1.
That is, it is considered that PS is required to bind to CD300a in order to induce (recruit) SHP-1 by CD300a in response to LPS stimulation.

[Reference Example 3G]
In order to examine whether SHP-1 is involved in CD300a-mediated signaling, first, in BMMC derived from a wild-type mouse, Ptpn6 (SHP-1 gene) was knocked out with siRNA, and SHP-1 deficiency ( BMMC derived from (Ptpn6-KD) wild type mouse was prepared under the following conditions. Similarly, a BMMC derived from a SHP-1 deficient (Ptpn6-KD) CD300a gene deficient mouse was prepared from a BMMC derived from a CD300a gene deficient mouse.

SiRNA targeting SHP-1 gene (Ptpn6 gene) in BMMC (SHP-1 siRNA) (siGENOME SMARTpool; Thermo Scientific Dharmacom) 0.5 mM, mixed by X-treme Gene siRNA tranfection refraction ratio 1 As described in the following document 28, BMMC derived from CD300a gene-deficient mice in which SHP-1 was knocked down by transfecting (transfecting) BMMC derived from CD300a gene-deficient mice of 5 × 10 ⁵ cells. (BMMC (CD300a ^{− / −} · Ptpn6-KD)) was produced.

  In addition, BMMC derived from a wild type mouse in which SHP-1 was knocked down (BMMC) was used in the same manner as in the above test conditions except that BMMC derived from a wild type mouse was used instead of BMMC derived from a CD300a gene-deficient mouse. (WT · Ptpn6-KD)) was produced.

Here, in order to confirm that SHP-1 siRNA was transduced into each BMMC and the expression level of SHP-1 was reduced, BMMC (CD300a ^{− / −} · Ptpn6-KD) or BMMC (WT · Ptpn6) -KD) lysate was used for immunoblotting analysis with anti-SHP-1 antibody, anti-SHP-2 antibody and anti-β-actin antibody (result: FIG. 7E).

  As shown in FIG. 7E, it can be confirmed that the expression level of SHP-1 is reduced in BMMC transduced with SHP-1 siRNA. “Ctrl” indicates the result of immunoblotting analysis using BMMC transduced with control siRNA instead of SHP-1 siRNA.

Next, BMMC (CD300a ^{− / −} · Ptpn6-KD) or BMMC (WT · Ptpn6-KD) and the apoptotic cells obtained in Reference Example 1B (BMMC: apoptotic cells = 10: 1 (cell number ratio)), After incubation in RPMI containing calcium chloride (1 mM) and LPS (lipopolysaccharide) (1 μg / mL) for 4 hours, the amount of TNF-α released was measured in the same manner as in Reference Example 3B (result: FIG. 7F). .

  As shown in FIG. 7F (left graph), BMMC derived from CD300a gene-deficient mice produced TNF-α significantly more than BMMC derived from wild-type mice. On the other hand, as shown in FIG. 7F (right graph), BMMCs derived from wild mice or CD300a gene-deficient mice, when SHP-1 siRNA was transduced, both wild-derived BMMCs and CD300a gene-deficient mouse-derived BMMCs, The amount of TNF-α released was similar, and no significant difference was observed between the two.

  These results suggest that, when PS binds to CD300a, CD300a induces SHP-1 and mediates a BMMC activation suppression signal, resulting in suppression of TNF-α secretion. .

  From the results of Reference Example 3, the interaction between PS and CD300a inhibits the production of pro-inflammatory (LPS-induced) cytokines and chemokines from BMMC, and this interaction recruits SHP-1. It is understood that it inhibits the secretion of TNF-α.

[Reference Example 4: Functional analysis of CD300a (3)]
TNF-α, IL-3, and MCP-1 produced by mast cells are neutrophil chemoattractants and are known to play an important role in bacterial clearance in CLP peritonitis mice (the following literature) 15-19).
Therefore, in order to examine whether CD300a has a bacterial clearance function, the following Reference Examples 4A to 4H were performed.

[Reference Example 4A]
A midline incision of 1 to 2 cm was performed on the cecum (ventral region) of a wild-type mouse, and its end was ligated. Next, after puncturing twice using a 27 gauge needle at the ligation site, the cecum was returned to the abdomen, and 1 mL of sterile saline was subcutaneously injected to replenish the water, and then the incision was sutured to close the incision. The detailed contents and conditions of the above CLP are described in Reference 16 below.

  Peritoneal perfusate was collected before CLP or at 4 hours after CLP. Subsequently, APC-conjugated annexin V (1 μg) and CD300-Fc (1 μg) were added to the peritoneal perfusate, followed by staining with FITC-conjugated anti-human IgG and PI (propidium iodide) and analyzed by flow cytometry. (Result: FIG. 12A).

FIG. 12A shows the result that it can be confirmed that the peritonitis site is a site where many cells lead to apoptosis, as described in Reference 20 below.
That is, it is suggested that CD300a affects the immune control of mast cells at the membrane inflammation site.

[Reference Example 4B]
In order to verify the relationship between CD300a and mast cell immune control, proteome analysis was performed as follows.

First, CLP was performed on wild-type mice and mast cell-deficient mice (kit ^{W-sh / W-sh} mice) in the same manner as in Reference Example 4A.
After 4 hours from the CLP, the peritoneal perfusate of each mouse was collected, and the collected peritoneal perfusate was collected using the Proteome Profiler Array (manufactured by R & D Systems) according to the manufacturer's instructions. It was used for proteome analysis of chemokines.

FIG. 9A shows the results of densitometric analysis (proteomic analysis) using peritoneal perfusate of wild-type mice and mast cell-deficient mice (kit ^{W-sh / W-sh} mice) (“PC” in FIG. 9A represents Positive control is shown.)

FIG. 9B shows the pixel density of each chemokine and cytokine signal obtained from the densitometric image of each signal shown in FIG. 9A.
As shown in FIG. 9B, at 4 hours after CLP, the concentration of chemokine in the peritoneal cavity is higher in kit ^{W-sh / W-sh} mice than in wild-type mice. In addition, the same result was obtained even if this test was implemented twice.

[Reference Example 4C]
In the same manner as in Reference Example 4B, peritoneal perfusate was collected from wild type mice and mast cell-deficient mice (kit ^{W-sh / W-sh} mice) (n = 3 for each).

  Next, a serial dilution series was prepared from each peritoneal perfusate, each serial dilution of the prepared peritoneal perfusate was plated, and then on a plate containing brain heart infusion (BHI) agar at 37 ° C. for 48 hours. Cultured. Next, as described in Reference 27 below, the number of colonies in 1 mL of peritoneal perfusate was counted to calculate the CFU of aerobic bacteria (result: FIG. 10A).

In addition, the numbers of neutrophils and macrophages in each peritoneal perfusate were counted, and the results are shown in “neutropil” and “macrophage” in FIG. 10B, respectively.
Furthermore, BM-derived macrophages were prepared from wild-type mice or mast cell-deficient mice (kit ^{W-sh / W-sh} mice). These macrophages (cell number: 1 × 10 ⁶ ) were co-cultured in a medium containing fluorescein-labeled E. coli for 1 hour in a 24-well plate, and the number of cells of each macrophage that phagocytosed E. coli was measured by flow cytometry. The ratio of macrophages that were in contact was calculated (result: FIG. 11 “BM macrophage”).

Instead of a wild-type mice or mast cell-deficient mice ^{(kit W-sh / W-} sh mice) from BM-derived macrophages, wild-type mice or mast cell-deficient mice ^{(kit W-sh / W-} sh mice) from the PEC The ratio of phagocytic macrophages was calculated in the same manner as in the above test conditions except that macrophages were used (result: FIG. 11 “PEC macrophage”).

As shown in FIG. 10A, mast cell-deficient mice (kit ^{W-sh / W-sh} mice) 4 hours after CLP have less bacterial CFU in the abdominal cavity than wild-type mice, and neutrophils It turns out that there are many cells. In contrast, as shown in FIG. 10B and FIG. 11, the number of cells and phagocytosis of macrophages were not significantly different between the two genotypes.

[Reference Example 4D]
In order to verify whether CD300a is involved in neutrophil induction (recruitment), the following examples were performed.

Mast cell-deficient mice (Kit ^{W-sh / W-sh} mice) were administered by intraperitoneal injection with PBS buffer containing BMMC derived from wild-type mice (cell number 1 × 10 ⁶ ) (n = 20). Then, after 28 days from the administration, CLP was performed in the same manner as in Reference Example 4A, and the survival rate of the mice was measured (result: FIG. 12B “WT BMMCs → Kit ^{W-sh /} Kit ^W-sh ”). .

Further, the survival rate of the mice was measured in the same manner as in the above test conditions except that BMMC derived from a CD300a gene-deficient mouse was used instead of BMMC derived from a wild type mouse (result: FIG. 12B “CD300a ⁻ // ^{- BMMCs → Kit W-sh /} Kit W-sh "). Note that “Kit ^{W-sh /} Kit ^W-sh ” in FIG. 12B indicates the survival rate of mast cell-deficient mice (Kit ^{W-sh / W-sh} mice) in which CLP was performed without administration of BMMC.

According to FIG. 12B, even after CLP, mast cell-deficient mice (Kit ^{W-sh / W-sh} mice) provided with BMMC derived from wild-type mice were compared with the case where BMMC was not administered. A high survival rate was shown.

However, after CLP, in mast cell-deficient mice (Kit ^{W-sh / W-sh} mice) provided with BMMC derived from CD300a gene-deficient mice, BMMC derived from wild-type mice and BMMC not administered In comparison, it can be seen that the survival rate was significantly higher even after CLP (FIG. 12B).

Furthermore, using the peritoneal perfusate of each mouse 4 hours after CLP, the bacterial CFU was measured in the same manner as in Reference Example 4C. As a result, mast cell-deficient mice provided with BMMC derived from CD300a gene-deficient mice ( Kit ^{W-sh / W-sh} mice) showed greater bacterial clearance than other mice (result: FIG. 12C).

[Reference Example 4E]
In order to verify whether or not the amount of TNF-α released increases when BMMC is administered intraperitoneally to mast cell-deficient mice (Kit ^{W-sh / W-sh} mice), the following examples are performed. It was.

24 hours before CLP, CFSE-labeled mixed BMMC (BMMC derived from CD300a gene-deficient mouse: BMMC derived from wild mouse = 1) were injected into the abdominal cavity of mast cell-deficient mice (Kit ^{W-sh / W-sh} mice). 1 (cell number ratio)) was administered by intraperitoneal injection.

  CLP was performed on mice injected with the mixed BMMC in the same manner as in Reference Example 4A, and peritoneal perfusate was collected 4 hours after CLP. Each BMMC contained in the peritoneal perfusate was subjected to analysis using flow cytometry (Becton Dickinson, FACSCalibur, model number “E6133”) (result: FIG. 12D).

As shown in FIG. 12D, at 4 hours after CLP, BMMC derived from CD300D gene-deficient mice (CD300a ⁻ CFSE ⁺ cells) was significantly larger than BMMC derived from wild-type mice (CD300a ⁺ CFSE ⁺ cells). It turns out that TNF- (alpha) is produced.

[Reference Example 4F]
In order to verify what effect CD300a has when the anti-CD300a monoclonal antibody (TX41) is administered, the following examples were performed.

  First, CLP was administered in the same manner as in Reference Example 4A, except that 500 μg of anti-CD300a monoclonal antibody (TX41) (n = 13) was intraperitoneally injected into wild-type mice 1 hour or 18 hours before CLP. The survival rate of the mice was measured in the same manner as in Reference Example 4D (result: FIG. 12E “Antibody to CD300a”).

  Further, as a control, the survival rate of mice was measured in the same manner as in the above test conditions except that an antibody for isotype control (n = 11) was used instead of TX41 (n = 13) (result: FIG. 12E "Control antibody").

  As shown in FIG. 12E, when CLP was performed after intraperitoneal injection of wild type mice 1 or 18 hours before CLP, survival time was compared to when control antibody was administered. Was found to be longer.

[Reference Example 4G]
Peritoneal perfusate was collected from each mouse 4 hours after CLP in Reference Example 4F. Using the obtained peritoneal perfusate, the numbers of bacterial CFU and neutrophil cells were measured in the same manner as in Reference Example 4C (control antibody: n = 5 and anti-CD300a monoclonal antibody: n = 5) ( FIG. 12F and FIG. 12G, respectively.

In addition, even when TX41 was administered by intraperitoneal injection, myeloid cells including mast cells of each mouse were not lost.
As shown in FIGS. 12F and 12G, when TX41 was administered by intraperitoneal injection to wild-type mice 1 hour or 18 hours before CLP, the number of neutrophil cells increased significantly in the peritoneal cavity. Bacterial clearance was also improved.

From the results of Reference Example 4, it is understood that peritonitis-induced sepsis can be prevented by blocking the interaction between PS and CD300a with, for example, TX41.
It should be noted that many cells have undergone apoptosis under physiological conditions. Here, PS receptors play a central role in the uptake of apoptotic cells, and are essential for preventing the development of autoimmune diseases (Reference 22 below).

  On the other hand, in pathological situations such as microbial infection, cell death due to apoptosis is markedly increased, causing an inflammatory response by mast cells via receptors (for example Toll-like receptors) against pathogen-related molecular patterns (PAMPs). It is known (Documents 15, 23 and 24 below). Mast cells are also known to play an important role in the immune response against pathogens.

  From the above, the results in the above examples show that PS not only provides an uptake signal to phagocytic cells via several types of PS receptors but also a mast via CD300a as obtained as a new finding this time. It can be understood that it has the effect of effectively suppressing the inflammatory reaction by cells.

  Therefore, a phosphatidylserine-binding substance (such as MFG-E8) or a CD300a-binding substance (such as a neutralizing antibody against CD300a) blocks the interaction between PS and CD300a in mast cells and activates mast cells, or It is understood that the activity is maintained.

  That is, it can be understood that these substances are useful as active ingredients of immunostimulating agents used for prevention of various inflammatory infections induced by LPS (and sepsis caused thereby), for example.

  In addition, PS suppresses mast cell activation by suppressing CD300a activity signaling, and thus suppresses inflammatory reactions in allergic diseases and autoimmune diseases (for example, release of chemical mediators such as histamine). It can be understood that it is useful as an active ingredient of an immunosuppressive agent used for alleviating or treating symptoms of allergic diseases and autoimmune diseases (suppressing excessive immune function).

<Enteritis>
(Materials and methods)
(mouse)
C57BL / 6 mice (WT mice) were purchased from Clea. As a CD300a gene-deficient mouse, a mouse established from Balb / cA-derived ES cells in this laboratory was backcrossed to C57BL / 6, and a 12th generation or later mouse was used.
The mice used in the experiments were bred in a specific pathogen-insensitive (SPF) environment at the University of Tsukuba Life Science Animal Resource Center.

(Induction of enteritis)
10-12 weeks old C57BL / 6 female mice were allowed to drink dextran sulfate sodium salt reagent grade (DSS, MPBiomedicals, molecular weight (MW) = 36000-50000) for 2.5 days (w / v) for 8 consecutive days. Triggered.

(antibody)
The BD biotin anti-CD300a (TX41) produced in our laboratory was used.
The following other antibodies were purchased from Bioscience.
Purified anti-mouse TNF-α,
・ Biotin-labeled anti-mouse TNF-α,
Purified mouse IL-6,
Biotin-labeled anti-mouse IL-6,
Purified anti-mouse IL-10,
Biotin-labeled anti-mouse IL-10,
Allophycocyanin-Cy7 (APC-Cy7) labeled anti-mouse CD11b (M1 / 70),
Phycoerythrin-Cy7 (PE-Cy7) labeled anti-mouse CD11c (HL3),
Fluorescein isothiocyanate (FITC) labeled anti-mouse CD4 (L3T4)

(Colon tissue culture)
The large intestine was removed from the mouse 9 days after administration of DSS or water, and then washed with a phosphate buffer to remove feces. After dissecting the large intestine in the long axis, 5 tissue pieces were cut from the anus 3 mm each from a 3 mm portion, cultured for 12 hours with 10% FBSRPMI 1640 (GIBCO), and the supernatant was recovered. It was. TNF-α, IL-6, and IL-10 contained in the culture solution were measured by a sandwich ELISA method.

(Isolation of colonic lamina propria cells)
Mice were sacrificed and their large intestine collected. First, the large intestine was cut into four pieces with scissors, washed with a phosphate buffer, and feces were removed. The remaining tissue was placed in 1 mM DTT / 5 mM MEDTA / Hank's balanced salt solution (manufactured by Sigma) and incubated in a 37 ° C. constant temperature bath (shaker type) for 30 minutes.

  Thereafter, the tissue was washed again with a phosphate buffer to remove the peeled epithelium and contaminants. The remaining tissue was minced with scissors and placed in 1 mg / mL collagenase type 3 (manufactured by Worthington) /0.1 mg / mL DNase (manufactured by Worthington) / 5% Fetal bovine serum / Hanks's balanced salt solution. It was incubated for 2 hours in a thermostatic chamber (shaker type) and completely thawed.

  The cell suspension was passed through 70 μm nylon and centrifuged. The obtained cells were suspended in 40% Percoll, layered on 70% Percoll, and the cells in the intermediate layer obtained after centrifugation were collected and used in the flow cytometry method as colonic lamina propria cells.

(Isolation of CD11b + CD11c + cells, CD4 + T cells)
Isolated colon lamina propria cells were labeled with APC-Cy7-labeled anti-CD11b (M1 / 70), PE-Cy7-labeled anti-CD11c (HL3) and FITC-labeled anti-mouse CD4 (L3T4). Labeled cells were sorted with FACSAria (BD) to obtain CD11b + CD11c + cells and CD4 + T cells, respectively.

(Measurement of mRNA amount)
Cells sorted with FACSAria were thawed with ISOGEN-LS (Nippon Gene). MRNA was extracted from the thawed cells using an mRNA extraction kit, and cDNA was prepared using a reverse transcription kit. The prepared cDNA was mixed with various primers (see SEQ ID Nos. 1 to 16 in the Sequence Listing) and Power Cyber Green (Applied Biosystems), and the amount of mRNA was measured using 7500 Fast Real Time PCR System (Applied Biosystems).

Example 1A Weight change rate (FIG. 13)
WT mice and CD300a gene-deficient mice were allowed to drink 2.5% (w / v) dextran sodium sulfate (DSS) for 8 consecutive days, respectively, to induce enteritis, and body weight was measured daily.

  As the mice, females aged 10 to 12 weeks were used, and the weight change rates of 15 WT mice and 15 CD300a gene-deficient mice were observed. Significant differences were determined using Student's t test (**: p <0.01, ***: p <0.001).

  As shown in FIG. 13, the weight loss due to enteritis induced by drinking 2.5% DSS was caused by the weight of CD300a gene-deficient mice (□) not having MAIR-I compared with WT mice (●). The decrease was significantly mild.

[Example 1B] Measurement of the length of the large intestine (FIG. 14)
On day 6 of DSS administration, the large intestine was collected from 7 mice each of WT mice and CD300a gene-deficient mice, and the length from the anus to the cecum was measured. Significant differences were determined using Student's t test (**: p <0.01).

  As shown in FIG. 14, the colon of CD300a gene-deficient mice was longer, and colonic atrophy due to enteritis induced by DSS was milder in CD300a gene-deficient mice than in WT mice.

[Example 1C] Measurement of the length of the large intestine (FIG. 14A)
When the histological evaluation of the large intestine was performed at the same time, it was observed that the histological changes in the CD300a gene-deficient mice were milder than those in the WT mice (FIG. 14A).

[Example 1D] Measurement of cytotoxic activity (FIG. 15)
After staining the colon tissue of WT mice and CD300a gene-deficient mice on day 6 after DSS administration with HE (hematoxylin and eosin) staining, scores 0 to 5 (0: no change, 1: crypt enlargement and structure) Change: 2: Significant reduction of goblet cells; 3: Crypt (Crypt) is observed, but little structure is maintained; 4: Crypt disappearance is observed, but epithelial detachment is not observed 5: Disappearance of crypt (Crypt), epithelial detachment, and significant cell infiltration were observed).

For the large intestine of 7 mice each of WT mice and CD300a gene-deficient mice, 10 sections taken from the anal side with a strong visual field were scored, and a significant difference was obtained using Student's t test (***: p <0.001). The result is shown in FIG.
As shown in FIG. 15, tissue damage due to DSS enteritis was mild in the CD300a gene-deficient mice.

[Example 1E] Measurement of IL-10 (FIG. 16)
DSS or water (control group) was administered, the large intestine was taken out from each mouse 9 days after the DSS administration, washed with a phosphate buffer, and then cut into 3 mm ³ pieces from the anal side.

  After culturing the minced colon tissue with 10% FBS RPMI1640 (GIBCO) for 12 hours, the supernatant was collected, and the culture supernatant was subjected to sandwich ELISA to each cytokine (TNF-α, IL-6, The amount of IL-10) protein was measured. Significant differences were determined using Student's t test (*: p <0.05).

  As shown in FIG. 16, in the large intestine tissue in which enteritis was induced by DSS induction, IL-6 and TNF-α did not show a difference between WT mice and CD300a gene-deficient mice, but production of IL-10 was CD300a. It was revealed that the value was high in the gene-deficient mice (FIG. 16).

It is known that IL-10 suppresses the expression of proteins CD80 and CD86 necessary for the activation of inflammatory T cells in Mreg cells (Reference 33 below).
As shown in FIG. 16, in the CD300a gene-deficient mice, the production amount of IL-10 is increased with a significant difference of 5%, so that the induction of proliferation of inflammatory T cells is suppressed and inflammatory T cells are suppressed. It is considered that it contributes to the maintenance of homeostasis of the intestinal tract immune system.

[Example 1F] Measurement of the number of cells (FIG. 16A)
Changes in cell population were examined for CD4 + T cells, CD8 + T cells, B cells, macrophages and dendritic cells for each mouse on DSS administration, Day 0, Day 2, Day 4 and Day 6.
As a result, as shown in FIG. 16A, there was no difference in the number of cell populations that gathered in the large intestine between WT mice and CD300a gene-deficient mice.

[Example 1G] Expression analysis of CD300a (FIG. 17)
Cells isolated from the large intestine were stained with APC-Cy7 labeled antiCD11b (M1 / 70), PE-Cy7 labeled antiCD11c (HL3), and Biotin-antiCD300a (TX41). The expression of CD300a was measured by the flow cytometry method on the cells of CD11b-CD11c- fraction, CD11b + CD11c- fraction, CD11b-CD11c + fraction, CD11b + CD11c + fraction. The result is shown in FIG.
As shown in FIG. 17, in the lamina propria of the large intestine, CD300a was expressed in CD11b + CD11c + cells, which are special cells.

[Example 1H] Expression quantification in CD11b + CD11c + cells (FIG. 18)
After CD11b + CD11c + cells isolated from the large intestine of each mouse were sorted by FACSAria, mRNA was extracted from the cells. CDNA was prepared from the extracted mRNA, and the amounts of IL-10, TNF-α, and IL-6 mRNA were measured by quantitative PCR.

  As a result, as shown in FIG. 18, in CD11b + CD11c + cells, there was no difference in IL-10, TNF-α, and IL-6 mRNA levels between WT mice and CD300a gene-deficient mice.

[Example 11] Expression quantification in CD4 + T cells (Fig. 19)
CD4 + cells isolated from the large intestine of each mouse administered with DSS were sorted with FACSArAia, and then mRNA was extracted from the cells. CDNA was prepared from the extracted mRNA, and the amount of mRNA of IL-10, Foxp3, TGF-β, T-bet, GATA-3, and RORγt was measured by quantitative PCR using ABI7500fast. Significant differences were determined using Student's t test (*: p <0.05, **: p <0.01).
As shown in FIG. 19, the levels of IL-10, Foxp3, and Tgfβ mRNA in CD4 + T cells were high in CD300a gene-deficient mice.

(Discussion)
From the results of Examples 1A to 6I, when MAIR-I is deficient in mice in which enteritis is induced by DSS, various anti-inflammatory cytokines such as IL-10 are produced (FIG. 19), and the pathology of enteritis is improved. .

  This is not an increase in some cell population (FIG. 16A), but CD11b-positive dendritic cells themselves, or these dendritic cells act on either cell to produce IL-10, and CD300a (MAIR-I) It is thought that it is controlling via.

  That is, it is predicted that the pathological condition of enteritis is improved by suppressing or inhibiting the signal transmission of CD300a (MAIR-I) via CD11b-positive dendritic cells.

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Claims (9)

  Inflammatory bowel disease characterized by containing as an active ingredient an active regulator for suppressing inhibitory signaling of myeloid cells expressing CD300a, which contains a substance that inhibits the binding between CD300a and phosphatidylserine Drugs for treating or preventing.   The pharmaceutical product according to claim 1, wherein the substance that inhibits the binding between CD300a and phosphatidylserine is a phosphatidylserine-binding substance.   The phosphatidylserine binding substance is a group consisting of MFG-E8, MFG-E8 mutant, T cell immunoglobulin, soluble TIM-1, soluble TIM-4, soluble stabilizer and soluble integrin αvβ3. The pharmaceutical product according to claim 2, which is at least one selected from the group consisting of:   The pharmaceutical product according to claim 1, wherein the substance that inhibits the binding between CD300a and phosphatidylserine is a CD300a-binding substance.   The CD300a binding substance consists of the amino acid sequence represented by SEQ ID NO: 19 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, additions, insertions or deletions relative to the amino acid sequence. L consisting of a heavy chain variable region and an amino acid sequence represented by SEQ ID NO: 20 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, additions, insertions or deletions relative to the amino acid sequence The pharmaceutical product according to claim 4, which is an anti-human CD300a antibody having a chain variable region.   The CD300a binding substance consists of the amino acid sequence represented by SEQ ID NO: 17 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, additions, insertions or deletions relative to the amino acid sequence. L consisting of a heavy chain variable region and an amino acid sequence represented by SEQ ID NO: 18 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, additions, insertions or deletions relative to the amino acid sequence The pharmaceutical product according to claim 4, which is an anti-mouse CD300a antibody having a chain variable region.   The pharmaceutical product according to any one of claims 1 to 6, wherein the myeloid cells are CD11b-positive dendritic cells in the lamina propria of the large intestine. Use of a CD300a gene-deficient mouse for pathological analysis of inflammatory bowel disease, or for screening a candidate substance that can be an active ingredient of a therapeutic or prophylactic agent thereof,
Use of the CD300a gene-deficient mouse, wherein the CD300a gene-deficient mouse is used as a model mouse that hardly induces inflammatory bowel disease when a substance that induces inflammatory bowel disease is administered. As a tool for comparative analysis when conducting pathological analysis of inflammatory bowel disease, or screening candidate substances that can be active ingredients of therapeutic or preventive drugs,
Use of an activity regulator for enhancing inhibitory signaling of myeloid cells expressing CD300a, which contains a substance that enhances the binding between CD300a and phosphatidylserine.