U.S. patent application number 16/656322 was filed with the patent office on 2020-02-06 for activity modulator, medicinal agent comprising same, use of cd300a gene-deficient mouse, and anti-cd300a antibody.
This patent application is currently assigned to UNIVERSITY OF TSUKUBA. The applicant listed for this patent is UNIVERSITY OF TSUKUBA. Invention is credited to Syuichi Iino, Udayanga Sanath Kankanam Gamage, Haruka Miki, Tsukasa Nabekura, Chigusa Oda, Akira Shibuya, Satoko Tahara.
Application Number | 20200040077 16/656322 |
Document ID | / |
Family ID | 48469634 |
Filed Date | 2020-02-06 |
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United States Patent
Application |
20200040077 |
Kind Code |
A1 |
Shibuya; Akira ; et
al. |
February 6, 2020 |
ACTIVITY MODULATOR, MEDICINAL AGENT COMPRISING SAME, USE OF CD300A
GENE-DEFICIENT MOUSE, AND ANTI-CD300A ANTIBODY
Abstract
The present invention aims to provide: an immunostimulant useful
for maintaining, enhancing or suppressing an immune function
associated with CD300a activation signaling, or an immunomodulator
as an immunosuppressant useful for suppressing the immune function;
use of a CD300a gene-deficient mouse for pathology analysis and the
like; an anti-CD300a antibody; and the like.
Inventors: |
Shibuya; Akira;
(Tsukuba-shi, JP) ; Oda; Chigusa; (Tsukuba-shi,
JP) ; Tahara; Satoko; (Tsukuba-shi, JP) ;
Nabekura; Tsukasa; (Tsukuba-shi, JP) ; Kankanam
Gamage; Udayanga Sanath; (Tsukuba-shi, JP) ; Miki;
Haruka; (Tsukuba-shi, JP) ; Iino; Syuichi;
(Tsukuba-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF TSUKUBA |
Tsukuba-shi |
|
JP |
|
|
Assignee: |
UNIVERSITY OF TSUKUBA
Tsukuba-shi
JP
|
Family ID: |
48469634 |
Appl. No.: |
16/656322 |
Filed: |
October 17, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14359212 |
Oct 8, 2014 |
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PCT/JP2012/078898 |
Nov 7, 2012 |
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16656322 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1774 20130101;
A01K 2267/0368 20130101; A61K 38/1808 20130101; A01K 67/0276
20130101; C07K 2317/56 20130101; A61P 1/00 20180101; A61K 31/661
20130101; A61K 2039/54 20130101; A61K 2039/545 20130101; C07K
2317/76 20130101; A61K 31/04 20130101; A61K 2039/505 20130101; A61P
43/00 20180101; A61P 33/00 20180101; A61K 31/685 20130101; A61K
38/16 20130101; A61P 29/00 20180101; A61P 37/04 20180101; A01K
2227/105 20130101; A61P 31/04 20180101; C07K 16/2803 20130101; A61K
45/06 20130101; A61K 31/685 20130101; A61K 2300/00 20130101; A61K
38/16 20130101; A61K 2300/00 20130101; A61K 31/04 20130101; A61K
2300/00 20130101; A61K 31/661 20130101; A61K 2300/00 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 38/16 20060101 A61K038/16; A61K 45/06 20060101
A61K045/06; A61K 31/661 20060101 A61K031/661; A61K 31/04 20060101
A61K031/04; A61K 31/685 20060101 A61K031/685; A01K 67/027 20060101
A01K067/027; A61K 38/17 20060101 A61K038/17; A61K 38/18 20060101
A61K038/18 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 21, 2011 |
JP |
2011-254151 |
Claims
1. A method for treatment of a disease or disease state in which
inhibitory signal transduction of a cluster of differentiation 300a
(CD300a)-expressing myeloid cell is involved, comprising:
administering an effective amount of phosphatidyl serine, wherein
said phosphatidyl serine promotes inhibitory signal transduction of
a CD300a-expressing myeloid cell.
2. The method for treatment according to claim 1, wherein said
disease is an inflammatory infection, allergic disease or
autoimmune disease.
3. The method for treatment according to claim 1, wherein said
disease is celiac disease.
4. A method for carrying out pathology analysis of atopic
dermatitis, inflammatory bowel disease or asthma comprising use of
a cluster of differentiation 300a (CD300a) gene-deficient mouse to
which has been administered a substance that induces atopic
dermatitis, inflammatory bowel disease or asthma as a model mouse
that hardly develops atopic dermatitis, inflammatory bowel disease
or asthma after administration of a substance that induces atopic
dermatitis, inflammatory bowel disease or asthma.
5. The method for carrying out pathology analysis according to
claim 4, wherein said substance that induces atopic dermatitis is
mite antigen or ovalbumin, wherein said substance that induces
inflammatory bowel disease is dextran sodium sulfate, and wherein
said substance that induces asthma is mite antigen or
ovalbumin.
6. A method for screening a candidate substance for efficacy in the
treatment or prophylaxis of celiac disease, said method comprising:
i) administering said candidate substance to a cluster of
differentiation 300a (CD300a) gene-deficient mouse in which celiac
disease has been induced following the administration to said mouse
of a substance that induces celiac disease, and determining the
presence or absence of a therapeutic effect; or ii) administering
said candidate substance with a substance that induces celiac
disease to a CD300a gene-deficient mouse, and determining the
presence or absence of a prophylactic effect.
7. The method of claim 6, wherein said substance that induces
celiac disease is a gluten-derived gliadin peptide.
8. An anti-human cluster of differentiation 300a (CD300a) antibody
comprising: i) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 19 or an amino acid sequence that is the
same as the amino acid sequence of SEQ ID NO: 19 except that 1, 2,
3, 4, or 5 amino acid(s) is/are each substituted, added, inserted
or deleted in a region other than the complementarity determining
regions of SEQ ID NO: 19; and ii) an L-chain variable region having
the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence
that is the same as the amino acid sequence of SEQ ID NO: 20 except
that 1, 2, 3, 4, or 5 amino acid(s) is/are each substituted, added,
inserted or deleted in a region other than the complementarity
determining regions of SEQ ID NO: 20.
9. An anti-mouse cluster of differentiation 300a (CD300a) antibody
comprising: i) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 17 or an amino acid sequence that is the
same as the amino acid sequence of SEQ ID NO: 17 except that 1, 2,
3, 4, or 5 amino acid(s) is/are each substituted, added, inserted
or deleted in a region other than the complementarity determining
regions of SEQ ID NO: 17; and ii) an L-chain variable region having
the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence
that is the same as the amino acid sequence of SEQ ID NO: 18 except
that 1, 2, 3, 4, or 5 amino acid(s) is/are each substituted, added,
inserted or deleted in a region other than the complementarity
determining regions of SEQ ID NO: 18.
10. A method of manufacturing a pharmaceutical composition, the
method comprising: mixing the anti-human cluster of differentiation
300a (CD300a) antibody of claim 8 with a pharmaceutically
acceptable carrier.
11. An anti-human cluster of differentiation 300a (CD300a) antibody
comprising: i) an H-chain variable region having an amino acid
sequence that is the same as the amino acid sequence of SEQ ID NO:
19 except that 1, 2, 3, 4, or 5 amino acid(s) is/are each
substituted, added, inserted or deleted in a region other than the
complementarity determining regions of SEQ ID NO: 19; and ii) an
L-chain variable region having an amino acid sequence that is the
same as the amino acid sequence of SEQ ID NO: 20 except that 1, 2,
3, 4, or 5 amino acid(s) is/are each substituted, added, inserted
or deleted in a region other than the complementarity determining
regions of SEQ ID NO: 20.
12. An anti-mouse cluster of differentiation 300a (CD300a) antibody
comprising: i) an H-chain variable region having an amino acid
sequence that is the same as the amino acid sequence of SEQ ID NO:
17 except that 1, 2, 3, 4, or 5 amino acid(s) is/are each
substituted, added, inserted or deleted in a region other than the
complementarity determining regions of SEQ ID NO: 17; and ii) an
L-chain variable region having or an amino acid sequence that is
the same as the amino acid sequence of SEQ ID NO: 18 except that 1,
2, 3, 4, or 5 amino acid(s) is/are each substituted, added,
inserted or deleted in a region other than the complementarity
determining regions of SEQ ID NO: 18.
13. A method for treatment of a disease or disease state in which
inhibitory signal transduction of a cluster of differentiation 300a
(CD300a)-expressing myeloid cell is involved via an immunoreceptor
tyrosine-based inhibitory motif (ITIM) of CD300a, comprising:
administering to a subject an effective amount of a neutralizing
antibody against CD300a which inhibits binding between CD300a and
phosphatidyl serine; wherein said neutralizing antibody against
CD300a suppresses inhibitory signal transduction of the
CD300a-expressing myeloid cell, and wherein said neutralizing
antibody against CD300a is: an anti-human CD300a antibody
comprising: i) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 19 or an amino acid sequence that is the
same as the amino acid sequence of SEQ ID NO: 19 except that 1, 2,
3, 4, or 5 amino acid(s) is/are each substituted, added, inserted
or deleted in the complementarity determining regions of SEQ ID NO:
19; and ii) an L-chain variable region having the amino acid
sequence of SEQ ID NO: 20 or an amino acid sequence that is the
same as the amino acid sequence of SEQ ID NO: 20 except that 1, 2,
3, 4, or 5 amino acid(s) is/are each substituted, added, inserted
or deleted in the complementarity determining regions of SEQ ID NO:
20.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a Divisional of copending U.S.
application Ser. No. 14/359,212, filed on Oct. 8, 2014, which is a
371 of International Application No. PCT/JP2012/078898, filed Nov.
7, 2012, which claims the benefit of priority from the prior
Japanese Patent Application No. 2011-254151, filed on Nov. 21,
2011, the entire contents of all of which are incorporated herein
by reference.
TECHNICAL FIELD
[0002] The present invention relates to an activity modulator for
suppressing or promoting inhibitory signal transduction of a
CD300a-expressing myeloid cell, a medicament comprising it, use of
a CD300a gene-deficient mouse, and an anti-CD300a antibody.
BACKGROUND ART
[0003] Invasion of a pathogen (bacterium, virus, parasite or the
like) into a host (human body or animal body) or generation of an
endogenous inflammatory substance causes inflammatory reactions in
which, for example, temporary contraction of arteriolae occurs at
the site of invasion of the pathogen or the site of generation of
the inflammatory substance, and expansion and hyperemia then occur,
leading to local slowness of blood flow at the site of invasion of
the pathogen or the site of generation of the inflammatory
substance.
[0004] This causes adhesion of leukocytes to the vascular wall, and
chemical mediators released from various immunocytes then act on
the leukocytes to cause them to pass through the vascular wall by
amoeboid movement and to allow their migration. Known examples of
the chemical mediators include histamine, serotonin and
lymphokines. Mast cells, which produce and release histamine and
serotonin, are a type of lymphocytes that play a central role in
the inflammatory reaction. Similarly to mast cells, macrophages
also produce and release chemical mediators such as TNF.
[0005] The leukocytes whose migration was induced by the
inflammatory reaction are attracted by the pathogen or the like,
and this causes elimination (clearance) of the pathogen from the
body by humoral immunity accompanied by antigen-antibody reaction
and by cell-mediated immunity in which cytotoxic T cells and the
like are involved, resulting in prevention of the spread of
infection. Thus, the inflammatory reaction, and immune reactions
that occur based on the inflammatory reaction, are extremely
important for maintaining homeostasis of a living body.
[0006] On the other hand, the inflammatory reaction causes not only
the biological defense described above, but also adverse
signs/symptoms such as flare, fever, swelling, pain and
dysfunction. Specific examples of such symptoms include allergic
diseases, and various types of acute and chronic inflammations.
Also in autoimmune diseases, in which the absence of immunological
tolerance causes an autoimmune response, tissue injury occurs due
to the inflammatory reaction.
[0007] That is, for prevention of a disease accompanied by the
inflammatory reaction, it is important to kill the pathogen that
causes the inflammatory reaction using antibiotics (antimicrobial
agents), or to administer an agent that increases the immune
function in the living body (immunostimulant) to eliminate the
pathogen before an excessive inflammatory reaction occurs.
[0008] On the other hand, known examples of methods for
amelioration or treatment of a disease accompanied by the
inflammatory reaction include suppression of inflammation by
administration of an agent (anti-inflammatory agent
(immunosuppressant)) that decreases excessively activated immune
function by, for example, suppression of release of chemical
mediators.
[0009] For example, Patent Document 1 discloses, as an
immunostimulant, an activating agent for the function of dendritic
cells, which are antigen-presenting cells responsible for
activation of various immunocytes. More specifically, the agent
comprises as an effective component(s) at least one branched chain
amino acid selected from isoleucine, leucine and valine.
[0010] Patent Document 2 discloses, as an anti-inflammatory agent
(immunosuppressant), an agent comprising the SPARC (Secreted
protein which is acidic and rich in cystein) peptide and a
pharmaceutical carrier.
[0011] The following autoimmune diseases, allergic diseases and the
like are known.
[0012] Celiac disease, or coeliac disease, is an autoimmune disease
and is a progressive enteritis that is triggered by an immune
reaction to gluten, which is a protein contained in wheat, barley,
rye and the like. The incidence of this disease in the United
Kingdom, Europe and the United States is one or more per 300
individuals.
[0013] Gluten, which is a mixture of two proteins gliadin and
glutenin, is found in wheat, barley and rye. Gluten reacts with the
small intestine and activates an immune system that attacks the
fine small-intestinal epithelium, which is necessary for absorption
of nutrients and vitamins, to cause injury.
[0014] When a patient with celiac disease takes a food or the like
containing gluten, a gliadin-derived peptide, which cannot be
digested by digestive enzymes of human and is contained in wheat as
a fraction of a plant protein gluten, is deaminated by TG2 in the
duodenal submucosal tissue to produce an antigen, which then causes
production of autoantibodies.
[0015] Celiac disease occurs in genetically sensitive individuals
having any of: HLA-DQ2 (which is retained in about 90% of
individuals), which is encoded by HLA-DQA1 and HLA-DQB1; HLA-DQ2
mutants; and HLA-DQ8.
[0016] Such individuals show induction of an immune response to
peptides induced from water-insoluble proteins in flour, gluten,
and related proteins in rye and barley, which immune response is
limited to inappropriate HLA-DQ2 and/or DQ8 and mediated by
CD4.sup.+ T cells.
[0017] This immune reaction triggers an attack of the autoimmune
system on the small-intestinal epithelial tissue to cause
inflammation and then injury of villi and the like, leading to
destruction of the epithelial cells themselves. As a result,
nutrients cannot be absorbed from the small intestine, and the
patient suffers from malnutrition irrespective of the dietary
intake and the like.
[0018] Ulcerative colitis, which is a representative inflammatory
bowel disease (IBD), is a collective term for chronic diseases that
cause inflammation of unknown origin mainly in the digestive tract,
and is a chronic disease in which inflammation occurs in the large
intestine to form ulcers.
[0019] Inflammatory bowel disease (IBD) is a collective term used
for describing two gastrointestinal disorders (Crohn's disease (CD)
and ulcerative colitis (UC)) whose causes are unknown.
[0020] In Crohn's disease, a larger area is affected compared to
ulcerative colitis, and inflammation and ulcers are found in almost
the whole area of the digestive tract. Inflammatory bowel disease
(IBD) occurs worldwide, and as many as two million people are
reported to have suffered from Crohn's disease. The progression and
prognosis of IBD widely vary.
[0021] In inflammatory bowel disease (IBD), diarrhea and bloody
stool continue for a long period while the severity of symptoms
changes with time. The cause of the disease has not been
elucidated, and, in a major hypothesis, the continuous enteritis is
thought to be caused by immune reactions to foods and
enterobacteria due to disorder of immunological tolerance in the
intestinal tract.
[0022] At present, there is no fundamental therapeutic method for
the disease, and examples of therapies for inflammatory bowel
disease include dietetic treatment; and use of an antidiarrheal
(e.g., anticholinergic, loperamide or diphenoxylate),
anti-inflammatory agent (e.g., steroid drug such as aminosalicylic
acid, sulfasalazine, mesalamine, olsalazine, balsalazide or
prednisolone; or aminosalicylic acid) or immunosuppressant (e.g.,
azathioprine, mercaptopurine or cyclosporin).
[0023] Surgical operations are required over a period of 10 years
in 10% to 15% of the patients with IBD, and they have higher risk
of occurrence of intestinal cancer.
[0024] In Crohn's disease, bacteria are involved in the onset and
the progression of the disease, and intestinal inflammation in
Crohn's disease is well-known for its frequent responsiveness to
antibiotics and susceptibility to bacterial fecal flow. Common
intestinal microorganisms and novel pathogens have been suggested
to have associations with Crohn's disease, based on direct
detection or anti-microbial immune responses associated with the
disease.
[0025] Moreover, in a number of genetically susceptible models of
chronic colitis, luminal microorganisms are indispensable cofactors
for the disease, and animals kept in a microbe-free environment do
not develop colitis.
[0026] The combination of genetic factors, exogenous causes and the
endogenous microbiota may contribute to the immune-mediated injury
of the intestinal mucosa found in inflammatory bowel disease.
[0027] It is also known that development of ulcerative colitis is
associated with polymorphisms in 3 gene regions, that is, the
FCGR2A gene, which encodes a receptor protein present on the
surface of immunocytes; s8LC26A3, which encodes a transporter of
chlorine ions and hydrogen carbonate ions; and a gene in the 13q12
region whose function is unknown (Non-patent Document 3).
[0028] However, in spite of abundant direct and indirect evidence
on the role of intestinal microorganisms in Crohn's disease, no
pathogenic organism or antigen has been identified to contribute to
the impaired immunoregulation found in this disease. Tools useful
for elucidation of the pathologies of the inflammatory bowel
diseases (Crohn's disease and inflammatory enteritis), and
medicaments for treatment and the like of these diseases have been
demanded.
[0029] Atopic dermatitis is caused by entrance of an allergic
substance (antigen) into the body followed by production of
periostin due to stimulation by substances (interleukins 4 and 13)
secreted from activated immunocytes, and then binding of the
periostin to another protein "integrin" on the surface of
keratinocytes in the skin, to cause inflammation.
[0030] The binding of periostin to integrin causes production of
other inflammation-inducing substances, and the symptoms continue
even in the absence of the antigen, resulting in chronicity. It has
been shown, by an experiment using mice, that inhibition of binding
of periostin to integrin using an inhibitor prevents occurrence of
atopic dermatitis (Non-patent Document 4).
[0031] Although the major cause of atopic dermatitis has become
evident, further elucidation of the pathology of atopic dermatitis,
analysis of association of atopic dermatitis with other
inflammatory diseases, and medicaments for atopic dermatitis that
can be used in combination with the above inhibitor, are
demanded.
[0032] Bronchial asthma is a respiratory disease in which bronchial
inflammation triggered by an allergic reaction or infection with a
bacterium or virus becomes chronic to thereby cause increased
airway hyperresponsiveness and reversible airway narrowing, leading
to symptoms such as attacks of wheezing, and cough. Further,
bronchial asthma is said to be caused by the combination of airway
hyperresponsiveness, allergic diathesis and environment. Recurrent
symptoms such as wheezing, apnea, chest tightness and cough occur
especially at night or in the early morning.
[0033] A number of cells and cellular components, especially mast
cells, eosinophils, T-lymphocytes, macrophages, neutrophils and
epithelial cells play roles in inflammation of the airway.
Inflammation is associated with plasma exudation, edema, smooth
muscle enlargement, mucus plugging, and epithelial changes.
Further, inflammation causes associated increases in bronchial
hyperresponsiveness to various stimuli.
[0034] Inflammation of the airway induces atrophy of airway smooth
muscle, microvascular rupture and bronchial hyperresponsiveness. As
the responsiveness of the airway increases, the symptoms become
more severe and continuous, and daily variation of the pulmonary
function increases. The mechanism of involvement of airway
inflammation in the bronchial responsiveness is unknown, and tools
useful for elucidation of the pathology of asthma, and medicaments
and the like have been demanded.
[0035] It is known that a group of receptor molecules called MAIR
(Myeloid Associated Ig like Receptors) are expressed on the cell
membrane of myeloid (bone marrow) cells responsible for natural
immunity (Non-patent Document 1). Among these, MAIR-I, which is
also known as CD300a (also referred to as "LMIR1" or "CLM-8"), is
expressed in macrophages, mast cells, granulocytes (neutrophils)
and dendritic cells, and known to be an inhibitory receptor that
associates with phosphatase via the ITIM (Immunoreceptor
tyrosine-based inhibitory motif) sequence in the intracellular
domain to transmit an inhibitory signal (Non-patent Document 2).
However, the ligand for this receptor is unknown, and the receptor
has been the so-called orphan receptor.
PRIOR ART DOCUMENTS
Patent Documents
[0036] [Patent Document 1] JP 2007-297379 A [0037] [Patent Document
2] JP 2011-516609 A
Non-Patent Documents
[0037] [0038] [Non-patent Document 1] Yotsumoto et al., J Exp Med
198 (2), 223-233, 2003 [0039] [Non-patent Document 2] Okoshi Y et
al., Int Immunol., 17, 65-72, 2005. [0040] [Non-patent Document 3]
Asano K et al., Nature Genetics 41. 1325-1329 (2009) [0041]
[Non-patent Document 4] Miho Masuoka et al., J Clin Invest. 2012;
doi: 10. 1172/JC158978
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0042] The present invention aims to elucidate the regulatory
mechanism of activation of the innate immune response via the
inhibitory signal transduction of CD300a, and to provide means for
regulating the activity of CD300a-expressing cells involved in the
inhibitory signal transduction, means for treating or preventing
diseases or disease states in which the activity is involved, and
techniques and the like associated with these.
Means for Solving the Problems
[0043] In order to elucidate the ligand of CD300a and the function
of CD300a, the present inventors intensively studied to discover
the following.
[0044] (i) The ligand of CD300a is phosphatidyl serine (PS).
[0045] (ii) Binding of PS to CD300a on mast cells and the like
promotes inhibitory signal transduction via CD300a, and activation
of the mast cells and the like are also suppressed thereby.
[0046] (iii) Inhibition of binding of CD300a on mast cells and the
like to PS by coexistence of a phosphatidyl serine-binding
substance or CD300a-binding substance suppresses inhibitory signal
transduction of CD300a, and the active state of mast cells and the
like is maintained.
[0047] (iv) Through the suppression or maintenance of the active
state, inflammatory infections, allergic diseases, autoimmune
diseases and the like can be treated.
[0048] (v) CD300a gene-deficient mice can be a tool for performing
pathology analysis of allergic diseases and autoimmune diseases,
and screening of effective components of medicaments.
[0049] The present invention was carried out based on these
discoveries, and provides, for example, the activity modulators,
medicaments, use of a CD300a gene-deficient mouse, anti-CD300a
antibody, and the like described in [1] to [23] below.
[0050] [1] An activity modulator for suppressing inhibitory signal
transduction of a CD300a-expressing myeloid cell, the activity
modulator comprising a substance that inhibits binding of CD300a to
phosphatidyl serine.
[0051] [2] The activity modulator according to [1], wherein the
substance that inhibits binding of CD300a to phosphatidyl serine is
a phosphatidyl serine-binding substance.
[0052] [3] The activity modulator according to [2], wherein the
phosphatidyl serine-binding substance is at least one selected from
the group consisting of MFG-E8, MFG-E8 mutants, T cell
immunoglobulin, soluble TIM-1, soluble TIM-4, soluble stabilin and
soluble integrin .alpha.v.beta.3.
[0053] [4] The activity modulator according to [1], wherein the
substance that inhibits binding of CD300a to phosphatidyl serine is
a CD300a-binding substance.
[0054] [5] The activity modulator according to [4], wherein the
CD300a-binding substance is an anti-human CD300a antibody
comprising: an H-chain variable region having the amino acid
sequence of SEQ ID NO:19 or an amino acid sequence that is the same
as the amino acid sequence except that 1, 2, 3, 4, or 5 amino
acid(s) is/are substituted, added, inserted and/or deleted; and an
L-chain variable region having the amino acid sequence of SEQ ID
NO:20 or an amino acid sequence that is the same as the amino acid
sequence except that 1, 2, 3, 4, or 5 amino acid(s) is/are
substituted, added, inserted and/or deleted.
[0055] [6] The activity modulator according to [4], wherein the
CD300a-binding substance is an anti-mouse CD300a antibody
comprising: an H-chain variable region having the amino acid
sequence of SEQ ID NO:17 or an amino acid sequence that is the same
as the amino acid sequence except that 1, 2, 3, 4, or 5 amino
acid(s) is/are substituted, added, inserted and/or deleted; and an
L-chain variable region having the amino acid sequence of SEQ ID
NO:18 or an amino acid sequence that is the same as the amino acid
sequence except that 1, 2, 3, 4, or 5 amino acid(s) is/are
substituted, added, inserted and/or deleted.
[0056] [7] An activity modulator for promoting inhibitory signal
transduction of a CD300a-expressing myeloid cell, the activity
modulator comprising a substance that promotes binding of CD300a to
phosphatidyl serine.
[0057] [8] The activity modulator according to [7], wherein the
substance that promotes binding of CD300a to phosphatidyl serine is
phosphatidyl serine.
[0058] [9] A medicament for treatment or prophylaxis of a disease
or disease state in which inhibitory signal transduction of a
CD300a-expressing myeloid cell is involved, the medicament
comprising the activity modulator according to any of [1] to
[8].
[0059] [10] The medicament according to [9], wherein the disease or
disease state in which inhibitory signal transduction of a
CD300a-expressing myeloid cell is involved is an inflammatory
infection, allergic disease or autoimmune disease.
[0060] [11] The medicament according to [10] for treatment or
prophylaxis of peritonitis or sepsis caused thereby, the medicament
comprising the activity modulator according to any of [1] to
[6].
[0061] [12] The medicament according to [10] for treatment of
inflammatory bowel disease, the medicament comprising the activity
modulator according to any of [1] to [6].
[0062] [13] The medicament according to [10] for treatment of
celiac disease, the medicament comprising the activity modulator
according to [7] or [8].
[0063] [14] The medicament according to [10] for treatment of
atopic dermatitis, the medicament comprising the activity modulator
according to any of [1] to [6].
[0064] [15] The medicament according to [10] for treatment of
asthma, the medicament comprising the activity modulator according
to any of [1] to [6].
[0065] [16] Use of a CD300a gene-deficient mouse for carrying out
pathology analysis of an allergic disease or autoimmune disease, or
for screening of a possible candidate substance for an effective
component of a therapeutic agent or prophylactic agent for the
disease.
[0066] [17] The use according to [16], comprising use of the CD300a
gene-deficient mouse as a model mouse in which inflammatory bowel
disease is hardly induced after administration of a substance that
induces inflammatory bowel disease.
[0067] [18] The use according to [16], comprising use of the CD300a
gene-deficient mouse as a model mouse that develops celiac disease
after administration of a substance that induces celiac
disease.
[0068] [19] The use according to [16], comprising the step of:
administering a candidate substance for a therapeutic agent for
celiac disease to the CD300a gene-deficient mouse that developed
celiac disease, and determining the presence or absence of a
therapeutic effect; or administering a candidate substance for a
prophylactic agent for celiac disease together with the substance
that induces celiac disease to the CD300a gene-deficient mouse
before development of celiac disease, and determining the presence
or absence of a prophylactic effect.
[0069] [20] The use according to [16], comprising use of the CD300a
gene-deficient mouse as a model mouse that hardly develops atopic
dermatitis after administration of a substance that induces atopic
dermatitis.
[0070] [21] The use according to [16], comprising use of the CD300a
gene-deficient mouse as a model mouse that hardly develops asthma
after administration of a substance that induces asthma.
[0071] [22] An anti-human CD300a antibody comprising: an H-chain
variable region having the amino acid sequence of SEQ ID NO:19 or
an amino acid sequence that is the same as the amino acid sequence
except that 1, 2, 3, 4, or 5 amino acid(s) is/are substituted,
added, inserted and/or deleted; and an L-chain variable region
having the amino acid sequence of SEQ ID NO:20 or an amino acid
sequence that is the same as the amino acid sequence except that 1,
2, 3, 4, or 5 amino acid(s) is/are substituted, added, inserted
and/or deleted.
[0072] [23] An anti-mouse CD300a antibody comprising: an H-chain
variable region having the amino acid sequence of SEQ ID NO:17 or
an amino acid sequence that is the same as the amino acid sequence
except that 1, 2, 3, 4, or 5 amino acid(s) is/are substituted,
added, inserted and/or deleted; and an L-chain variable region
having the amino acid sequence of SEQ ID NO:18 or an amino acid
sequence that is the same as the amino acid sequence except that 1,
2, 3, 4, or 5 amino acid(s) is/are substituted, added, inserted
and/or deleted.
[0073] As other aspects of the inventions described above, the
following inventions are provided.
[0074] Another aspect of the invention of [1] provides a method for
suppressing inhibitory signal transduction of a CD300a-expressing
myeloid cell, which method comprises inhibiting binding of CD300a
to phosphatidyl serine. This method is applicable either in vivo or
ex vivo/in vitro, and, in cases of in vivo application, the species
of organism may be either human or non-human (e.g., mammal such as
mouse).
[0075] Still another aspect of the invention of [1] provides use of
a substance that inhibits binding of CD300a to phosphatidyl serine
in production of an activity modulator for suppressing inhibitory
signal transduction of a CD300a-expressing myeloid cell.
[0076] As the substance that inhibits binding of CD300a to
phosphatidyl serine for carrying out the method or use, the
phosphatidyl serine-binding substance recited in [2], preferably
MFG-E8 or the like recited in [3] may be used. Similarly, as the
substance that inhibits binding of CD300a to phosphatidyl serine
for carrying out the method, the CD300a-binding substance recited
in [4], preferably the anti-human CD300a antibody comprising an
H-chain variable region and an L-chain variable region having the
prescribed amino acid sequences recited in [5], or the anti-mouse
CD300a antibody comprising an H-chain variable region and an
L-chain variable region having the prescribed amino acid sequences
recited in [6], may be used.
[0077] Another aspect of the invention of [7] provides a method for
promoting inhibitory signal transduction of a CD300a-expressing
myeloid cell, which method comprises promotion of binding of CD300a
to phosphatidyl serine. This method is applicable either in vivo or
ex vivo/in vitro, and, in cases of in vivo application, the species
of organism may be either human or non-human (e.g., mammal such as
mouse).
[0078] Still another aspect of the invention of [7] provides use of
a substance that promotes binding of CD300a to phosphatidyl serine
in production of an activity modulator for promoting inhibitory
signal transduction of a CD300a-expressing myeloid cell.
[0079] As the substance that promotes binding of CD300a to
phosphatidyl serine for carrying out the method or use, the
phosphatidyl serine recited in [8] may be used.
[0080] Another aspect of the invention of [9] provides a method for
treatment or prophylaxis of a disease or disease state in which
inhibitory signal transduction of a CD300a-expressing myeloid cell
is involved, which method comprises inhibiting or promoting binding
of CD300a to phosphatidyl serine, thereby suppressing or promoting
inhibitory signal transduction of a CD300a-expressing myeloid cell.
This method is applicable either in vivo or ex vivo/in vitro, and,
in cases of in vivo application, the species of organism may be
either human or non-human (e.g., mammal such as mouse).
[0081] Still another aspect of the invention of [9] provides use of
an activity modulator that inhibits or promotes binding of CD300a
to phosphatidyl serine to thereby suppress or promote inhibitory
signal transduction of a CD300a-expressing myeloid cell, in
production of a medicament for treatment or prophylaxis of a
disease or disease state in which inhibitory signal transduction of
a CD300a-expressing myeloid cell is involved.
[0082] Examples of the disease or disease state in the method or
use include inflammatory infection, allergic disease, and
autoimmune disease as recited in [10], and specific examples of the
disease or disease state include the peritonitis or sepsis caused
thereby, inflammatory bowel disease, celiac disease, atopic
dermatitis, and asthma as recited in [11] to [15].
[0083] Another aspect of the invention of [16] provides a method
for carrying out pathology analysis of an allergic disease or
autoimmune disease, or for screening of a possible candidate
substance for an effective component of a therapeutic agent or
prophylactic agent for the disease, by using a CD300a
gene-deficient mouse. The CD300a gene-deficient mouse used in the
method is a model mouse in which inflammatory bowel disease, atopic
dermatitis or asthma is hardly induced, or a model mouse that
develops celiac disease as recited in [17], [18], [20] or [21]
after administration of a substance that induces each allergic
disease or autoimmune disease. The method is especially preferably
a method for celiac disease, comprising the prescribed steps
recited in [19].
Effect of the Invention
[0084] The present invention enables preparation of an activity
modulator that can suppress or promote inhibitory signal
transduction of a CD300a-expressing myeloid cell, and production of
a medicament for treatment or prophylaxis of a disease or disease
state in which inhibitory signal transduction of a
CD300a-expressing myeloid cell is involved, which medicament
comprises as an effective component the activity modulator.
Further, the present invention enables use of a CD300a
gene-deficient mouse as a model mouse or the like, and production
of an anti-CD300a antibody having excellent neutralizing
action.
BRIEF DESCRIPTION OF THE DRAWINGS
[0085] FIG. 1 shows the results of flow cytometry analysis obtained
in Example 1A.
[0086] FIG. 2A shows the results of flow cytometry analysis
obtained in Example 1B.
[0087] FIG. 2B shows the results of flow cytometry analysis
obtained in Example 1B.
[0088] FIG. 2C shows the results of flow cytometry analysis
obtained in Example 1C.
[0089] FIG. 2D shows the results of flow cytometry analysis
obtained in Example 1D.
[0090] FIG. 2E shows the results of flow cytometry analysis
obtained in Example 1D.
[0091] FIG. 2F shows the results of immunoblotting analysis
obtained in Example 1E.
[0092] FIG. 3A is a schematic diagram for illustrating the
structure of the CD300a gene in the wild-type allele, the targeting
vector used for preparing a CD300a gene-deficient mouse, and the
targeted allele.
[0093] FIG. 3B is a photograph taken after electrophoresis of PCR
products from the wild-type allele and the mutant allele.
[0094] FIG. 3C shows the results of Western blotting using a
wild-type mouse and a CD300a-deficient mouse.
[0095] FIG. 3D shows the results of flow cytometry analysis of a WT
mouse and a CD300a gene-deficient mouse.
[0096] FIG. 4A shows the results of flow cytometry analysis
obtained in Example 2A.
[0097] FIG. 4B shows the results of analysis under a light
microscope obtained in Example 2C.
[0098] FIG. 4C shows the results of laser scanning confocal
microscopy obtained in Example 2C.
[0099] FIG. 4D shows the results obtained in Example 2C
illustrating the ratio of the number of cells of NIH3T3 or each
transfectant showing incorporation of a thymocyte in the
cytoplasm.
[0100] FIG. 5A shows the results of flow cytometry analysis
obtained in Example 2B.
[0101] FIG. 5B shows the results of RT-PCR analysis obtained in
Example 2B.
[0102] FIG. 6A shows the results of flow cytometry analysis
obtained in Example 3A.
[0103] FIG. 6B shows the results of analysis by the
.beta.-hexaminidase assay obtained in Example 3 A.
[0104] FIG. 7A shows the results of flow cytometry analysis
obtained in Example 3B.
[0105] FIG. 7B shows the results obtained in Example 3C on the
rates of increase in the amounts of various cytokines and
chemokines released.
[0106] FIG. 7C shows a diagram showing the results obtained in
Example 3E on the rates of increase in the amounts of various
cytokines and chemokines released.
[0107] FIG. 7D shows the results of immunoblotting analysis
obtained in Example 3F.
[0108] FIG. 7E shows a diagram showing the results of
immunoblotting analysis obtained in Example 3G.
[0109] FIG. 7F shows a diagram showing the rate of increase in
TNF-.alpha., obtained in Example 3G.
[0110] FIG. 8 shows the results of flow cytometry analysis obtained
in Example 3D.
[0111] FIG. 9A shows the results of densitometric analysis obtained
in Example 4B.
[0112] FIG. 9B shows the results of densitometric analysis obtained
in Example 4B.
[0113] FIG. 10A shows a diagram showing the results of calculation
of the CFU of aerobic bacteria, obtained in Example 4C.
[0114] FIG. 10B shows a diagram showing the numbers of neutrophils
and macrophages obtained in Example 4C after induction of CD300a
neutrophils.
[0115] FIG. 11 shows a diagram showing the rate of the number of
each type of macrophages that showed phagocytosis of E. coli.
[0116] FIG. 12A shows a diagram showing the results of flow
cytometry analysis obtained in Example 4D.
[0117] FIG. 12B shows a diagram showing the rate of survival of
each type of mice, obtained in Example 4D.
[0118] FIG. 12C shows a diagram showing the bacterial clearance in
the intestine in each type of mice in Example 4D, in terms of the
bacterial CFU.
[0119] FIG. 12D shows the results of flow cytometry analysis
obtained in Example 4E.
[0120] FIG. 12E shows a diagram showing the rate of survival of
each group of mice after administration of TX41 in Example 4F.
[0121] FIG. 12F shows a diagram showing the clearance in the
intestine after administration of TX41 in Example 4G.
[0122] FIG. 12G shows a diagram showing the result of analysis of
the change in the number of neutrophils after administration of
TX41 in Example 4G.
[0123] FIG. 13A shows a diagram illustrating the protocol for
induction of asthma with ovalbumin.
[0124] FIG. 13B shows the total cell number in the alveolar lavage
fluid from each mouse on Day 25 after the beginning of induction of
asthma in Example 5A.
[0125] FIG. 13C shows the ratio of eosinophils in the alveolar
lavage fluid from each mouse on Day 25 after the beginning of
induction of asthma in Example 5B.
[0126] FIG. 14 shows the serum IgE value in each mouse on Day 14
after the beginning of induction of asthma in Example 5B.
[0127] FIG. 15 shows a diagram showing changes in the body weight
with time due to DSS-induced enteritis in each group of mice
(Example 6A).
[0128] FIG. 16A shows a graph showing the length of the large
intestine in each type of mice on Day 6 after the beginning of
administration of DSS (Example 6B).
[0129] FIG. 16B shows a diagram showing sections of large
intestines of a WT mouse and CD300a.sup.-/- (Example 6C).
[0130] FIG. 17 shows a diagram for comparison of the degree of cell
damage in each type of mice on Day 6 of administration of DSS,
based on the clinical score (Example 6D).
[0131] FIG. 18A shows a diagram showing the amount of each cytokine
in the large intestine of each mouse on Day 9 of administration of
DSS (Example 6E).
[0132] FIG. 18B shows a diagram showing the number of each type of
immunocytes on Day 0, Day 2, Day 4 and Day 6 after the beginning of
administration of DSS in each type of mice (Example 6F).
[0133] FIG. 19 shows a diagram showing the results of flow
cytometry that was performed for identifying dendritic cells
expressing CD300a (Example 6G).
[0134] FIG. 20 shows a diagram showing the relative gene expression
levels of cytokines in the large intestine in each type of mice
(Example 6H).
[0135] FIG. 21 shows a diagram showing the relative gene expression
levels of cytokines in CD4.sup.+ T cells (Example 61).
[0136] FIG. 22 shows a diagram showing changes with time (daily
changes) in the number of times of scratching behavior per 30
minutes in each type of OVA-sensitized mouse (Example 7A).
[0137] FIG. 23 shows sections of the skin of each mouse at the end
of the 3rd week after sensitization with OVA (Example 7B).
[0138] FIG. 24 shows a diagram showing the result of toluidine blue
staining of a skin sample from each mouse at the end of the 3rd
week after sensitization with OVA (Example 7C).
[0139] FIG. 25A shows a graph showing the number of cell layers in
the epidermis in each group of mice at the end of the 3rd week
after sensitization with OVA (Example 7D).
[0140] FIG. 25B shows the numbers of eosinophils and mast cells
that showed infiltration into the dermis in skin samples of each
group of mice at the end of the 3rd week after sensitization with
OVA (Example 7E).
[0141] FIG. 26 shows the result of Langerin immunostaining of a
skin sample from each group of mice at the end of the 3rd week
after sensitization with OVA (Example 7F).
[0142] FIG. 27 shows counterstaining of each sample in FIG. 26.
[0143] FIG. 28 shows a diagram showing a state where mast cells are
interacting with Langerin-positive skin cells (Example 7H).
[0144] FIG. 29 is a diagram showing a schedule of the test for
confirming the therapeutic effect of TX41.
[0145] FIG. 30 shows the total serum IgE level in WT mice after
administration of TX74 or TX41, as measured by the ELISA
method.
[0146] FIG. 31 shows a diagram showing the number of times of
scratching behavior in WT mice after administration of TX74 or
TX41.
[0147] FIG. 32 shows a diagram showing the result of H&E
staining of a skin section of a WT mouse after administration of
TX74 or TX41.
[0148] FIG. 33 shows a diagram showing a state in which a skin
section of a WT mouse after administration of TX74 or TX41 was
counterstained by toluidine blue staining.
[0149] FIG. 34 shows a diagram showing changes in the body weight
(BW) with time monitored from the beginning of feeding with a
normal diet or high-gluten diet.
[0150] FIG. 35 shows photographs each showing the result of
histopathological analysis of the intestine of a mouse at Week 20
after the beginning of feeding with a normal diet or high-gluten
diet.
[0151] FIG. 36 shows a diagram showing the clinical score at Week
20 after the beginning of feeding with a normal diet or high-gluten
diet in each group of mice.
[0152] FIG. 37 shows a diagram showing the number of
intraepithelial lymphocytes per 100 intestinal epithelium cells at
week 20 after the beginning of feeding with a normal diet or
high-gluten diet in each group of mice.
[0153] FIG. 38 shows a diagram showing the amount of
transglutaminase 2 (TG2) in a suspension of the jejunum at week 20
after the beginning of feeding with a normal diet or high-gluten
diet in each type of mice.
[0154] FIG. 39 shows a diagram showing the results of flow
cytometry analysis of expression of CD11b and CD11c in cells of the
lamina propria gated for the CD45.sup.+PI.sup.- cell
population.
[0155] FIG. 40 shows a diagram showing the frequency of each type
of immunocytes in the jejunal lamina propria in WT mice and CD300a
gene-deficient mice kept with a normal diet or high-gluten
diet.
[0156] FIG. 41 shows a diagram showing the gene expression levels
of various cytokines and chemokines in lamina propria (LP)
macrophages in each group of mice.
[0157] FIG. 42 shows a diagram showing the result of analysis of
expression of CD300a (MAIR-I) in each type of immunocytes by flow
cytometry.
[0158] FIG. 43 shows a diagram showing the relative gene expression
levels of cytokines and chemokines in CD11b.sup.+ dendritic cells
in the lamina propria (LP) in each type of mice.
[0159] FIG. 44 shows a diagram showing changes in the expression
levels of gliadin-induced cytokines caused by addition of a
recombinant mouse MFG-E8 protein.
[0160] FIG. 45 shows a diagram showing the result of
histopathological analysis of the large intestine of a Balb/c WT
mouse or CD300a gene-deficient mouse fed with a normal diet or
high-gluten diet.
[0161] FIG. 46 shows a diagram showing the anti-gliadin IgG and IgA
antibody titers in sera derived from Balb/c WT or CD300a
gene-deficient mice.
[0162] FIG. 47 shows a diagram showing changes in the rate of
change in the body weight with time (weekly changes) in WT mice and
CD300a gene-deficient mice fed with a gluten-free diet.
[0163] FIG. 48 shows a diagram showing the expression levels of
cytokines in CD11b.sup.+ dendritic cells and macrophages in the
normal state in the lamina propria (LP) in WT mice and CD300a
gene-deficient mice.
[0164] FIG. 49 shows a diagram showing the expression levels of
cytokines in macrophages in thioglycolate-induced peritoneal
exudate cells after stimulation with the gliadin peptide
P31-43.
[0165] FIG. 50 is a diagram showing the expression levels of IL-6,
IL-15, TNF-.alpha., IFN-.beta., MCP1 and MCP5 in CD11b.sup.+
dendritic cells in the lamina propria.
[0166] FIG. 51 shows a diagram showing the expression levels of
IL-6, TNF-.alpha. and IFN-.beta. in lamina propria (LP) macrophages
in B6 WT mice or CD300a gene-deficient mice after stimulation with
gliadin.
[0167] FIG. 52 shows a diagram showing expression of cytokines and
chemokines in lamina propria (LP) macrophages (stimulated with
gliadin) derived from microbiota-depleted WT mice or CD300a
gene-deficient mice.
[0168] FIG. 53 shows a diagram showing the frequency of
phosphatidyl serine-expressing cells in isolated lamina propria
(LP) macrophages.
[0169] FIG. 54 shows a diagram showing the gene expression levels
of .alpha.v and .beta.3 integrin subunits in lamina propria (LP)
macrophages in WT mice and CD300a gene-deficient mice.
[0170] FIG. 55 shows a diagram showing the gene expression levels
of phosphatidyl serine receptors (TIM-1, TIM-4, Stabiln-2, SR-PSOX,
BAI1 and Mer).
[0171] FIG. 56 shows the results of homology analysis of each of
the H-chain and L-chain of TX41 and TX49.
DESCRIPTION OF EMBODIMENTS
[0172] The activity modulator of the present invention, a
medicament comprising it, use of a CD300a gene-deficient mouse, and
an anti-CD300a antibody, are described below in detail. Literatures
used for mentioning conventional knowledge or a known test method
on the immune mechanism, CD300a or the like are listed in the end
of Examples.
[0173] [Activity Modulator]
[0174] The activity modulator of the present invention includes
those for suppressing inhibitory signal transduction of a
CD300a-expressing myeloid cell, as well as those for promoting such
inhibitory signal transduction.
[0175] The "CD300a-expressing myeloid cell" herein includes a mast
cell, macrophage, neutrophil, dendritic cell and the like. CD300a
is a collective term for those expressed in mammals such as human
and mouse, and the species of organism is not limited.
[0176] The "inhibitory signal transduction" is signal transduction
that occurs by association of the inhibitory receptor CD300a with
phosphatase via the ITIM (Immunoreceptor tyrosine-based inhibitory
motif) sequence in the intracellular domain.
[0177] In the following description, since the activity modulator
for suppressing inhibitory signal transduction of a
CD300a-expressing myeloid cell may have an action to activate an
immune function as a result, it is also referred to as
"immunostimulant" in the present invention (for example, in cases
where it is used as an effective component of a medicament for an
inflammatory infection). On the other hand, since the activity
modulator for promoting inhibitory signal transduction of a
CD300a-expressing myeloid cell may have an action to suppress an
immune function as a result, it is also referred to as
"immunosuppressant" in the present invention (for example, in cases
where it is used as an effective component of a medicament for
celiac disease).
[0178] <First Activity Modulator>
[0179] The first activity modulator by the present invention
comprises a component having an action to suppress inhibitory
signal transduction via CD300a. In the present invention, as such a
component, a substance that inhibits binding of phosphatidyl serine
to CD300a, that is, a phosphatidyl serine-binding substance or
CD300a-binding substance may be used. The first activity modulator
may contain either one of these, or may contain both of these.
[0180] (Phosphatidyl Serine-Binding Substance)
[0181] The phosphatidyl serine-binding substance as a first
activity modulator is not limited as long as it binds to
phosphatidyl serine (PS), which is a ligand of CD300a, to inhibit
interaction (binding) between the phosphatidyl serine and CD300a
expressed in a myeloid cell.
[0182] Specific examples of the phosphatidyl serine-binding
substance include MFG-E8 (Milk Fat Globular Protein EGF-8); T cell
immunoglobulin; and soluble proteins such as soluble TIM-1, soluble
TIM-4, soluble stabilin and soluble integrin .alpha.v.beta.3. Among
these, MFG-E8 is preferred.
[0183] The phosphatidyl serine-binding substance is not limited to
native proteins such as MFG-E8, and may be one having an amino acid
sequence in which one or several amino acids are deleted,
substituted and/or added (mutant) (for example, "D89E MFG-E8" in
Examples) as long as the binding capacity to phosphatidyl serine is
not lost.
[0184] Such a mutant can be prepared by a known method such as
site-directed mutagenesis or random mutagenesis.
[0185] The "soluble protein" described above means a protein
prepared by modifying a native protein, such as a membrane protein,
insoluble to the later-described diluent or body fluid by, for
example, deleting a hydrophobic domain or adding a hydrophilic
peptide by a known genetic recombination technique such that the
protein becomes soluble to the diluent or body fluid.
[0186] (CD300a-Binding Substance)
[0187] The CD300a-binding substance as a first activity modulator
is not limited as long as it binds to CD300a to inhibit interaction
(binding) between the CD300a expressed in a myeloid cell and
phosphatidyl serine.
[0188] Specific examples of the CD300a-binding substance include
neutralizing antibodies against CD300a. The neutralizing antibody
may be a single particular type of monoclonal antibody, or may be a
combination of 2 or more types of monoclonal antibodies (or
polyclonal antibodies). Further, the neutralizing antibody may be
either a full-length antibody or an antibody fragment (Fab
fragment, F(ab').sub.2 fragment or the like).
[0189] The neutralizing antibody can be prepared by a known method.
In cases of a monoclonal antibody, anti-CD300a monoclonal
antibodies can be generally prepared by, for example, a procedure
comprising immunization with CD300a, preparation of hybridomas,
screening, culturing, and recovery. From the thus prepared
antibodies, an appropriate monoclonal antibody that has a desired
capacity (neutralizing action) to inhibit binding of CD300a to
phosphatidyl serine and can exert the action and effect of the
present invention may be selected.
[0190] (TX41, TX49, and Antibodies Similar to these)
[0191] TX41 is an anti-mouse CD300a monoclonal antibody (rat
IgG2a), and TX49 is an anti-human CD300a monoclonal antibody (mouse
IgG1). Both of these are monoclonal antibodies prepared and used in
the later-described Examples, and excellent in the function to
suppress signal transduction by inhibition of binding of CD300a to
phosphatidyl serine. Therefore, these are preferred as the
CD300a-binding substance in the present invention. However,
anti-CD300a antibodies that can be used in the present invention
are not limited to TX41, TX49, and antibodies similar to these
(having a variable region with an equivalent amino acid
sequence).
[0192] The variable region in the H-chain of TX41 has the amino
acid sequence of SEQ ID NO:17; the variable region in the L-chain
of TX41 has the amino acid sequence of SEQ ID NO:18; the variable
region in the H-chain of TX49 has the amino acid sequence of SEQ ID
NO:19; and the variable region in the L-chain of TX49 has the amino
acid sequence of SEQ ID NO:20. Each of these variable regions
contains 3 complementarity determining regions (CDRs) and 4
framework regions. FIG. 56 shows the results of analysis of
homology between the amino acid sequences of the variable regions
of TX41 and TX49 (for each of the H-chain and L-chain).
[0193] The binding substance for mouse CD300a is preferably an
antibody in which the variable region in the H-chain has the amino
acid sequence of SEQ ID NO:17, and the variable region in the
L-chain has the amino acid sequence of SEQ ID NO:18, according to
TX41.
[0194] The binding substance for human CD300a is preferably an
antibody in which the variable region in the H-chain has the amino
acid sequence of SEQ ID NO:19, and the variable region in the
L-chain has the amino acid sequence of SEQ ID NO:20, according to
TX49.
[0195] Further, the binding substance for mouse CD300a may also be
an antibody in which the H-chain variable region has an amino acid
sequence that is the same as the amino acid sequence of SEQ ID
NO:17 except that 1, 2, 3, 4, or 5 amino acid(s) is/are
substituted, added, inserted and/or deleted, or an antibody in
which the L-chain variable region has an amino acid sequence that
is the same as the amino acid sequence of SEQ ID NO:18 except that
1, 2, 3, 4, or 5 amino acid(s) is/are substituted, added, inserted
and/or deleted (one of the H-chain and the L-chain may have the
above-described mutation(s), or both of these may have the
above-described mutation(s)).
[0196] Further, the binding substance for human CD300a may also be
an antibody in which the H-chain variable region has an amino acid
sequence that is the same as the amino acid sequence of SEQ ID
NO:19 except that 1, 2, 3, 4, or 5 amino acid(s) is/are
substituted, added, inserted and/or deleted, or an antibody in
which the L-chain variable region has an amino acid sequence that
is the same as the amino acid sequence of SEQ ID NO:20 except that
1, 2, 3, 4, or 5 amino acid(s) is/are substituted, added, inserted
and/or deleted (one of the H-chain and the L-chain may have the
above-described mutation(s), or both of these may have the
above-described mutation(s)).
[0197] The sites of such mutations are preferably not in the CDRs
or vicinities thereof in the variable regions. Further, in cases
where an amino acid is substituted, the substitution is preferably
conservative amino acid substitution, in which substitution occurs
between amino acids having similar side-chain structures and/or
chemical properties.
[0198] The form (amino acid sequence, amino acid length) of the
constant region, that is, the Fab region excluding the
above-described variable region, and the Fc region, of the
anti-CD300a antibody may be designed as appropriate as long as the
action and effect of the present invention are not inhibited, since
the form of the constant region hardly affects the binding capacity
to CD300a, that is, the neutralizing action.
[0199] That is, the anti-CD300a antibody can be prepared as a
fusion protein composed of the above prescribed amino acid sequence
of the variable region and a known amino acid sequence of the
constant region.
[0200] For example, use of a human constant region for preparation
of an anti-human CD300a antibody as a human chimeric antibody is
one of preferred embodiments. Such an anti-CD300a antibody can be
prepared by a known method.
[0201] For example, by synthesizing a DNA encoding the above
prescribed amino acid sequence of the variable region and linking
the synthesized DNA to a DNA encoding an amino acid sequence of the
constant region and another/other necessary DNA(s) (transcription
factor(s) and/or the like), an expression vector for an anti-CD300a
antibody gene can be constructed. By introducing this vector to a
host cell and allowing expression of the gene, the anti-CD300a
antibody of interest can be produced.
[0202] The above-mentioned TX41 and TX49, and antibodies similar to
these can be potentially used also for an object other than the
action and effect of the present invention, i.e., the inhibition of
inhibitory signal transduction that occurs due to binding of
phosphatidyl serine to CD300a.
[0203] Moreover, by suppressing expression of CD300a in myeloid
cells in the affected area using an siRNA designed based on a gene
sequence of CD300a (available from DNA databases such as
DDBREMBL/GenBank=INSD), therapeutic effects for the various
diseases described above can be obtained as in the cases where the
CD300a gene is deleted or binding of CD300a to phosphatidyl serine
is inhibited. In other words, an siRNA against the CD300a gene can
also be said to be the substance that inhibits binding of CD300a to
phosphatidyl serine in the present invention.
[0204] (Use of First Activity Modulator)
[0205] The first activity modulator of the present invention can be
used for suppressing inhibitory signal transduction of a
CD300a-expressing myeloid cell. In this case, the myeloid cell may
be either a myeloid cell present in the body, or a myeloid cell
separated from the body or cultured in vitro.
[0206] By maintaining or increasing activation signaling via CD300a
of the myeloid cell by the above action, intercellular signal
transduction via chemical mediators released from the myeloid cell
is also maintained or increased, and inflammation, allergic
reaction and the like that are caused by further intercellular
signal transduction that occurs thereafter can then be influenced.
Therefore, the first activity modulator can be used as an effective
component (for example, as an immunostimulant) of the specific
medicaments described later. Further, for example, the first
activity modulator can also be used as an effective component of an
agent to be used as a comparative analysis tool for comparative
analysis performed after amelioration of the disease state of
asthma, atopic dermatitis, inflammatory bowel disease or sepsis in
a laboratory animal.
[0207] Those skilled in the art can sufficiently presume that
CD300a-binding substances (neutralizing antibodies such as TX41 and
TX49) and phosphatidyl serine-binding substances (MFG-E8 and the
like) can be therapeutic agents for diseases that have been found
to show amelioration of symptoms by deletion of the CD300a gene
(that is, by complete prevention of binding of CD300a to
phosphatidyl serine), such as atopic dermatitis and the like
described in Examples.
[0208] On the other hand, those skilled in the art can also
sufficiently presume that phosphatidyl serine can be a therapeutic
agent for diseases that have been found to show development or
exacerbation of symptoms by deletion of the CD300a gene (that is,
by complete prevention of binding of CD300a to phosphatidyl
serine), such as celiac disease described in Examples.
[0209] <Second Activity Modulator>
[0210] The second activity modulator contains a component having an
action to promote inhibitory signal transduction via CD300a (that
is, to suppress activation signaling of CD300a). In the present
invention, such a component may be a substance that promotes
binding of phosphatidyl serine to CD300a. The substance is
especially phosphatidyl serine, which is a ligand of CD300a.
[0211] Further, by performing screening using the CD300a
gene-deficient mice provided by another aspect of the present
invention, agonists (low molecular compounds, antibodies and the
like) for CD300a having the same action as phosphatidyl serine may
be discovered, and such agonists can also be used as substances
that promote binding of phosphatidyl serine to CD300a.
[0212] (Phosphatidyl Serine)
[0213] Phosphatidyl serine (PS) is a ligand for CD300a expressed in
myeloid cells, and interaction (binding) between PS and CD300a
promotes inhibitory signaling of CD300a-expressing cells. For
example, in mast cells, inflammatory reaction-associated activities
that cause release of chemical mediators such as histamine,
cytokines and chemokines are suppressed via this inhibitory
signaling. PS is industrially produced, and can be easily
obtained.
[0214] For CD300a-expressing myeloid cells placed in vitro (in a
test environment), apoptotic cells presenting PS (it is known that
PS is present inside the cell (in the cytoplasm-side layer of the
lipid bilayer) in a normal cell, but presented outside the cell
upon occurrence of apoptosis) can also be a second activity
modulator. Further, liposomes and the like having a PS-containing
lipid membrane formed in the outside can be potentially used as
second activity modulators.
[0215] (Calcium Salt)
[0216] Since the interaction between PS and CD300a in mast cells
requires calcium ions, the second activity modulator preferably
contains a calcium salt that generates a calcium ion by ionization
(e.g., calcium chloride).
[0217] The content of the calcium salt in the second activity
modulator may be determined appropriately in consideration of the
calcium ion concentration in the site of administration, the amount
of PS contained in the second activity modulator, and the like.
[0218] (Use of Second Activity Modulator)
[0219] The second activity modulator of the present invention can
be used for promoting inhibitory signal transduction of a
CD300a-expressing myeloid cell. In this case, the myeloid cell may
be either a myeloid cell present in the body, or a myeloid cell
separated from the body or cultured in vitro.
[0220] By suppressing activation signaling via CD300a of the
myeloid cell by the above action, intercellular signal transduction
via chemical mediators released from the myeloid cell is also
suppressed, and inflammation, allergic reaction and the like that
are caused by further intercellular signal transduction that occurs
thereafter can then be influenced. Therefore, the second activity
modulator can be used as an effective component (for example, as an
immunosuppressant) of the specific medicaments described later.
Further, for example, the second activity modulator can also be
used as an effective component of an agent to be used as a
comparative analysis tool for comparative analysis performed after
amelioration of celiac disease in a laboratory animal.
[0221] [Medicament]
[0222] The medicament (pharmaceutical composition) of the present
invention contains the activity modulator as described above as an
effective component (e.g., immunostimulant or immunosuppressant),
and may further contain various pharmaceutically acceptable
additives (e.g., a carrier), if necessary.
[0223] Such a medicament can be formulated for treatment or
prophylaxis of a disease or symptom (especially inflammation
reaction) in which inhibitory signal transduction of a
CD300a-expressing myeloid cell is involved, such as an inflammatory
infection, allergic disease or autoimmune disease.
[0224] The "treatment" includes not only curing of a disease or
symptom, but also amelioration (alleviation) of a disease or
symptom. The "prophylaxis" includes not only prevention of a
disease or symptom in advance, but also prevention of recurrence of
a disease or symptom after curing of the disease or symptom.
[0225] More specifically, by blending the first activity modulator
as an effective component, a medicament or the like for treatment
or prophylaxis of bacterial peritonitis or sepsis caused thereby,
inflammatory bowel disease, atopic dermatitis or asthma can be
prepared.
[0226] Further by blending the second activity modulator as an
effective component, a medicament or the like for treatment or
prophylaxis of celiac disease can be prepared.
[0227] The site of administration of the medicament is not limited,
and may be a site where excessive immune function (inflammation
reaction) is occurring, depending on the disease or disease state
to which the medicament is applied. Examples of the site include
intraperitoneal, intratracheal, subcutaneous, intradermal, and
intraurogenital sites.
[0228] Since myeloid cells that express CD300a are usually present
in submucosal tissues and connective tissues in mammals, the
medicament is preferably directly administered to the submucosal
tissue or connective tissue at the above-described site, or in the
vicinity thereof The administration may be carried out by injection
such as intravenous injection, intraarterial injection,
subcutaneous injection, intradermal injection, intramuscular
injection or intraperitoneal injection. For example, in cases of
treatment or prophylaxis of an inflammatory infection (e.g.,
bacterial peritonitis), intraperitoneal injection is preferred.
[0229] The dose per administration and the number of doses of the
medicament (or the effective component contained therein) vary
depending on the age, sex and body weight of the patient; symptoms;
degree of the therapeutic effect required; administration method;
treatment period; type of the effective component; and the like;
and may be appropriately controlled. The number of doses is, for
example, 1 to several doses per day.
[0230] In cases where the medicament contains as an effective
component a phosphatidyl serine-binding substance as the first
activity modulator, the medicament may be formulated such that the
dose per administration of the phosphatidyl serine-binding
substance is usually 3 to 15 mg, preferably 5 to 10 mg per 1 kg of
the human or animal subjected to the administration.
[0231] In cases where the medicament contains as an effective
component a CD300a-binding substance as the first activity
modulator, the medicament may be formulated such that the dose per
administration of the CD300a-binding substance is usually 50 to 150
mg, preferably 50 to 100 mg per 1 kg of the human or animal
subjected to the administration.
[0232] In cases where the medicament contains as an effective
component a second activity modulator, the medicament may be
formulated such that the dose per administration of phosphatidyl
serine is usually 3 to 150 mg, preferably 5 to 100 mg per 1 kg of
the human or animal subjected to the administration in view of
further increasing the immunosuppressive effect.
[0233] (Pharmaceutically Acceptable Carrier)
[0234] The medicament of the present invention may contain a
pharmaceutically acceptable carrier, if necessary.
[0235] The pharmaceutically acceptable carrier is not limited as
long as it does not deteriorate the purpose of the medicament, and
examples of the carrier include diluents such as aqueous diluents
and nonaqueous diluents; stabilizers/preservatives such as
antioxidants (e.g., sulfite); buffers such as phosphates;
emulsifiers such as surfactants; coloring agents; thickeners; local
anesthetics such as lidocaine; solubilizers such as glycols;
isotonic agents such as sodium chloride and glycerin; and other
additives.
[0236] For example, in cases where the dosage form of the
medicament of the present invention is an injection solution, the
effective component is preferably dissolved or dispersed in a
diluent by blending the diluent such that a desired viscosity and
desired concentrations of components are achieved.
[0237] Examples of such a diluent include aqueous diluents such as
physiological saline and commercially available distilled water for
injection; and nonaqueous diluents such as polyethylene glycol, and
alcohols including ethanol.
[0238] The medicament whose dosage form is an injection solution
may be sterilized by filtration through a filter, or may be
sterilized by blending a microbicide or the like, according to a
conventional method.
[0239] In cases where the activity modulator is administered as an
injection solution, it may be in the form of an injection solution
to be prepared at the time of use. For example, a solid dosage form
containing the activity modulator may be prepared by freeze-drying
or the like, and may then be dissolved or dispersed in a diluent to
prepare an injection solution at the time of administration.
[0240] <Medicament for Inflammatory Infection>
[0241] Inhibition of binding of phosphatidyl serine to
CD300a-expressing cells (mast cells) enables maintenance or
improvement of the activity of the mast cells. This increases the
number of neutrophils, and activates attack of neutrophils to
pathogens (bacteria, parasites and the like), thereby improving the
function to suppress the growth of pathogens, and the pathogen
clearance function. Thus, by using the first activity modulator
(phosphatidyl serine-binding substance or CD300a-binding substance)
as an effective component (immunostimulant), a medicament for an
inflammatory infection can be obtained.
[0242] <Medicament for Inflammatory Bowel Disease>
[0243] Inhibition of binding of phosphatidyl serine to
CD300a-expressing cells (CD11b-positive dendritic cells in the
large intestinal lamina propria) increases production, by CD4.sup.+
cells and the like, of IL-10, which is known to suppress growth
induction of inflammatory T cells. This suppresses activation of
inflammatory T cells, and allows maintenance of homeostasis of the
gut immune system. Thus, by using the first activity modulator as
an effective component, a medicament for inflammatory bowel disease
can be obtained.
[0244] <Medicament for Atopic Dermatitis>
[0245] Inhibition of binding of phosphatidyl serine to
CD300a-expressing cells can suppress cellular infiltration of
eosinophils, mast cells (which are known to interact with skin
Langerin-positive dendritic cells in the dermis to activate
CD4-positive T cells) and monocytes; suppress hyperplasia of
epidermis (fibroblasts); and decrease the total serum IgE level.
Thus, by using the first activity modulator as an effective
component, a medicament for atopic dermatitis can be obtained.
[0246] <Medicament for Asthma>
[0247] Inhibition of binding of phosphatidyl serine to
CD300a-expressing cells can suppress eosinophilic airway
inflammation, and decrease the total serum IgE level. Thus, by
using the first activity modulator as an effective component, a
medicament for asthma can be obtained.
[0248] <Medicament for Celiac Disease>
[0249] In cases where binding of phosphatidyl serine to
CD300a-expressing cells (macrophages in the large intestinal lamina
propria) is maintained, MyD88- and TRIF-mediated inhibitory gliadin
signaling pathways play protective roles in the progression of
celiac disease. Thus, by using the second activity modulator as an
effective component (immunosuppressant), a medicament for celiac
disease can be obtained.
[0250] [Use of CD300a Gene-Deficient Mouse]
[0251] <Uses Related to Celiac Disease>
[0252] According to a discovery obtained in the present invention,
induction of celiac disease by administration of a gluten-derived
gliadin peptide, which is known to be a substance that induces
celiac disease, to CD300a gene-deficient mice causes more severe
symptoms of celiac disease than administration to wild-type
mice.
[0253] That is, a CD300a gene-deficient mouse can be used as a
model mouse that develops celiac disease by administration of a
substance that induces celiac disease.
[0254] The model mouse of celiac disease in the present invention
is a mouse in which the CD300a gene is inactivated and release of
anti-inflammatory cytokines from mast cells and macrophages is
derepressed.
[0255] The mouse shows symptoms of celiac disease (inflammation of
the small intestine) in cases where the mouse is fed from an early
stage (from the late middle age) with a substance that induces
celiac disease. Thus, the mouse is useful for elucidation of the
cause of celiac disease, and plays an important role in development
of a therapeutic agent for celiac disease.
[0256] Symptoms of celiac disease can be confirmed by, for example,
a known method in which an inflammation-inducing substance is
administered to the mouse, and a section of the small-intestinal
epithelium of the mouse is then observed under the microscope.
[0257] The celiac disease model mouse of the present invention can
be used also for screening of therapeutic agents for celiac
disease. In particular, since the mouse securely shows symptoms of
celiac disease, the mouse is useful for development of not only
therapeutic agents to be applied after the onset of symptoms of
celiac disease, but also prophylactic agents for celiac
disease.
[0258] Further, the model mouse is also useful for evaluation of
therapeutic agents for celiac disease that have already been
demonstrated to be therapeutically effective.
[0259] For example, the model mouse is useful for studying the
optimal concentration of a therapeutic agent that has a therapeutic
effect within a certain range of concentration but does not have
the effect at a concentration lower than the range and shows
toxicity at a concentration higher than the range.
[0260] More specifically, the celiac disease model mice are divided
into a test mouse group and a control mouse group, and a
therapeutic agent to be tested is administered to the test mouse
group. By comparing sections of the small-intestinal epithelium
between the groups, the effect of the therapeutic agent can be
evaluated.
[0261] Further, by giving the therapeutic agent to the test mouse
group at different concentrations, a therapeutically effective and
optimal concentration of the therapeutic agent can be studied.
[0262] That is, the CD300a gene-deficient mouse can be employed for
uses in which a candidate substance for a therapeutic agent for
celiac disease is administered to the CD300a gene-deficient mouse
with celiac disease to see whether the agent is therapeutically
effective or not; uses in which a candidate substance for a
prophylactic agent for celiac disease is administered together with
the substance that induces celiac disease to the CD300a
gene-deficient mouse before development of celiac disease to see
whether the agent is prophylactically effective or not; and uses
for analyzing the pathology of celiac disease.
[0263] The CD300a (MAIR-I) receptor is positioned upstream of the
signal transduction to suppress production of anti-inflammatory
cytokines. Therefore, by administration of a CD300a (MAIR-I)
agonist or signal transducer that maintains or promotes the signal
transduction to suppress production of anti-inflammatory cytokines,
symptoms of celiac disease are expected to be ameliorated.
[0264] Thus, the CD300a gene-deficient mouse can also be preferably
used in screening for finding agonists; for finding signal
transducers that complement signal transduction downstream of the
MAIR-I receptor, and substances that induce such signal
transducers; and for finding genes involved in production of such
signal transducers and the like.
[0265] This screening can be carried out by differential analysis
between the CD300a gene-deficient mice and WT mice by DNA
microarray analysis, two-dimensional protein electrophoresis or the
like.
[0266] <Uses Related to Atopic Dermatitis>
[0267] Atopic dermatitis is hypersensitivity associated with
allergic reaction, and accompanied by skin inflammation (eczema or
the like).
[0268] Atopic dermatitis is caused by entrance of an allergic
substance (antigen) into the body followed by production of
periostin due to stimulation by substances (interleukins 4 and 13)
secreted from activated immunocytes and then binding of the
periostin to another protein "integrin" on the surface of
keratinocytes in the skin, to cause inflammation.
[0269] The binding of periostin to integrin causes production of
other inflammation-inducing substances, and the symptoms continue
even in the absence of the antigen, resulting in chronicity. It has
been shown, by an experiment using mice, that inhibition of binding
of periostin to integrin prevents occurrence of atopic dermatitis
(Document 31 listed below).
[0270] According to a discovery obtained in the present invention,
administration of a substance that induces atopic dermatitis (mite
antigen, ovalbumin or the like) to CD300a gene-deficient mice
causes milder symptoms of atopic dermatitis than administration to
wild mice.
[0271] That is, the CD300a gene-deficient mouse can be used as a
model mouse that hardly develops atopic dermatitis. Further, the
CD300a gene-deficient mouse can be used for analysis of signaling
pathways involved in atopic dermatitis, pathology analysis of
atopic dermatitis, and the like in relation to IL-4 and IL-13
production.
[0272] <Uses Related to Asthma>
[0273] Bronchial Asthma is a respiratory disease in which bronchial
inflammation triggered by an allergic reaction or infection with a
bacterium or virus becomes chronic to cause increased airway
hyperresponsiveness and reversible airway narrowing, leading to
attacks of wheezing, cough, and the like. Further, bronchial asthma
is said to be caused by the combination of airway
hyperresponsiveness, allergic diathesis and environment.
[0274] According to a discovery obtained in the present invention,
administration of a substance that induces asthma (mite antigen,
ovalbumin or the like) to CD300a gene-deficient mice to induce
asthma causes milder symptoms of asthma than administration to wild
mice.
[0275] That is, the CD300a gene-deficient mouse can be used as a
model mouse that hardly develops asthma after administration of a
substance that induces asthma, and can also be used for elucidation
of signaling pathways involved in asthma, pathology analysis of
asthma, and the like.
[0276] <Uses Related to Inflammatory Bowel Disease>
[0277] According to a discovery obtained in the present invention,
induction of inflammatory bowel disease in CD300a gene-deficient
mice by a known method such as administration of an
inflammation-inducing substance (e.g., DSS) causes milder symptoms
of inflammatory bowel disease than in wild-type mice.
[0278] That is, the CD300a gene-deficient mouse can be used as a
model mouse that hardly develops an inflammatory disease after
administration of a substance that induces inflammatory bowel
disease, and can also be used for pathology analysis and the like
of inflammatory bowel disease in relation to the causative gene of
the disease.
[0279] (Method for Preparing CD300a Gene-Deficient Mouse)
[0280] The CD300a gene-deficient mouse of the present invention is
a mouse in which the CD300a gene on the chromosome is replaced by
an inactive CD300a gene and hence the function of CD300a protein is
deficient.
[0281] The "inactive CD300a gene" means a gene that is incapable of
expressing normal CD300a protein due to, for example, partial
deletion of the CD300a gene, insertion of another base sequence to
the coding region of the CD300a gene, a point mutation(s) in the
CD300a gene, or mutation in a regulatory region for expression of
the CD300a gene. Examples of the deficient CD300a gene include, but
are not limited to, genes in which at least one of the exons 1 to 6
contained in the CD300a gene is deleted.
[0282] The term "function of CD300a protein is deficient" means
that at least a part of the function of CD300a protein involved in
the inhibitory signal transduction related to the present invention
is lost (for example, the function of CD300a protein is partially
lost by replacement of one of the alleles by an inactive form, as
in a heterozygous knockout mouse), preferably means that the
function is completely lost.
[0283] A common method for obtaining a CD300a gene-deficient mouse
using a gene cassette is as follows. However, the CD300a
gene-deficient mouse in the present invention is not limited to
those obtained by this method.
[0284] A targeting vector having a targeted allele (mutant allele)
in which the exons of the wild-type allele of the CD300a gene are
replaced by an antibiotic resistance gene (marker gene) is
prepared. According to a conventional method, a chimeric mouse is
obtained using the targeting vector and ES cells. The chimeric
mouse is crossed with a wild-type mouse to obtain F1 mice
(heterozygote (+/-)), and the F1 mice are crossed with each other
to obtain F2 mice.
[0285] Genomic DNA is extracted from the F2 mice, and genomic DNA
of each mouse is subjected to PCR to investigate the
presence/absence of the wild-type allele and the mutant allele in
the genomic DNA, to obtain an F2 mouse having only the mutant
allele (homozygote (-/-)). Further, in order to confirm the absence
of expression of CD300a, cells derived from the mouse are subjected
to confirmation by Western blotting using an anti-CD300a antibody,
to provide a CD300a gene-deficient mouse.
EXAMPLES
[0286] The present invention is described below more concretely by
way of Examples.
[0287] However, the present invention is not limited by the
Examples below.
1. Preparation Example
[0288] (1) Preparation of CD300a-Fc Fusion Protein, MFG-E8 and D89E
MFG-E8
[0289] (1-1) A CD300a fusion protein having the Fc region of human
IgG (CD300a-Fc) was prepared as described in the Document 25 listed
below from a chimeric cDNA containing a cDNA of a gene encoding the
whole extracellular domain of mouse or human CD300a and a cDNA of a
gene encoding human IgG1Fc. The fusion protein in which the
extracellular domain of CD300a is derived from mouse is referred to
as "mouse CD300a-Fc", and the fusion protein in which the
extracellular domain of CD300a is derived from human is referred to
as "human CD300a-Fc".
[0290] (1-2) MFG-E8 was provided by Mr. Masato Tanaka (RCAI,
Yokohama, Japan).
[0291] (1-3) D89E MFG-E8 is a mutant of MFG-E8 obtained by
site-directed mutagenesis in the RGD motif of MFG-E8 as described
in the Document 4 below.
[0292] In the obtained D89E MFG-E8, the 89th amino acid as counted
from the N-terminus is substituted from aspartic acid to glutamic
acid. MFG-E8 (native) binds to both phosphatidyl serine (PS) and
.alpha.v.beta.3 integrin to thereby cross-link apoptotic cells to
phagocytes expressing .alpha.v.beta.3 integrin (Document 4 listed
below). On the other hand, D89E MFG-E8 does not bind to
.alpha.v.beta.3 integrin while it binds to phosphatidyl serine
(PS).
[0293] (2) Mice, and Cecal Ligation and Puncture (CLP)
[0294] The knockout mice used in the Examples were prepared or
provided as follows.
[0295] (i) CD300a Gene-Deficient (Cd300a.sup.-/-) Mouse
[0296] Using a bacterial artificial chromosome (BAC) system, the
exons 1 to 6 of the wild-type allele of the Cd300a gene were
replaced by a neomycin resistance gene cassette (PGK-GB2-neo) to
prepare a targeted allele (mutant allele) (FIG. 3A). Subsequently,
a chimeric mouse was obtained according to a conventional method,
and the chimeric mouse was crossed with a wild-type mouse to obtain
F1 mice (heterozygote (+/-)). The F1 mice were crossed with each
other to obtain F2 mice.
[0297] In order to select CD300a gene-deficient (Cd300a.sup.-/-)
mice from the F2 mice, genomic DNA was extracted from the tail of
each F2 mouse, and the genomic DNA was subjected to PCR to
investigate the presence/absence of the WT allele and the mutant
allele in the genomic DNA.
[0298] As shown in FIG. 3B, the PCR product corresponding to the WT
allele and the PCR product corresponding to the mutant allele are
detected as a band of about 540 bp and a band of about 700 bp,
respectively.
[0299] Further, in order to confirm the absence of expression of
CD300a in the CD300a gene-deficient mice, cells derived from the
CD300a gene-deficient mice were subjected to Western blot analysis
using an anti-CD300a antibody. As shown in FIG. 3C, a wild-type
mouse showed a band of about 50 kDa derived from CD300a, but this
band was not detected in a CD300a gene-deficient mouse.
[0300] (ii) C57BL/6J-Kit.sup.W-sh/W-sh Mouse
[0301] C57BL/6J-kit.sup.W-sh/W-sh mice (hereinafter referred to as
"kit.sup.W-sh/W-sh mice" or "mast cell-deficient mice") were
provided from RIKEN BioResource Center (Tsukuba, Japan). These mice
are known to show deficiency of mast cells (Document 21 listed
below), and to show incorporation of DNA by phagocytes without
apoptotic DNA fragmentation, followed by degradation of DNA in the
phagocytes (Document 12 listed below).
[0302] (iii) CAD-Deficient Mouse
[0303] The CAD (Caspase-activated DNase)-deficient mouse described
in the Document 12 listed below was used.
[0304] (v) In Vivo Removal of Macrophages and Neutrophils
[0305] According to description in the Document 30 listed below,
clodronate liposomes and control PBS liposomes (Encapsula
NanoSciences) were prepared. Subsequently, at Hour 24 after CLP,
0.5 mL of the liposomes were injected to the abdominal cavity of
the mouse to remove macrophages.
[0306] Further, at Hour 24 after CLP, an anti-Gr-1 antibody was
injected into the abdominal cavity of the mouse to remove
neutrophils.
[0307] All the operations of preparation using mice and the
evaluation tests in the Examples were carried out in accordance
with the guideline prepared by Animal Care and Use Committee of
Laboratory Animal Resource Center, University of Tsukuba.
[0308] (3) Antibodies
[0309] The manufacturer, or the method for preparation, of each
antibody is shown in the table below.
TABLE-US-00001 TABLE 1 Antibody name Manufacturer or preparation
method Control rat antibody BD Pharmingen Anti-CD11b (M1/70)
antibody BD Pharmingen Anti-F4/80 antibody BD Pharmingen Anti-c-Kit
(2B8) antibody BD Pharmingen Anti-Gr-1 (RB6) antibody BD Pharmingen
Anti-Fc.epsilon.RI (MAR-1) antibody BD Pharmingen Anti-TNF-.alpha.
antibody BD Pharmingen Human CD300a monoclonal Prepared according
to a Document antibody TX49 (mouse IgG1) (*) listed below Mouse
CD300a monoclonal Prepared according to a Document antibody TX41
(rat IgG2a) (*) listed below Anti-MCP-1 antibody BioLegend
Anti-IL-13 antibody eBiosciences Anti-SHP-1 (C-19) antibody Upstate
Biotechnology Anti-SHP-2 (C-18) antibody Upstate Biotechnology (*):
Preparation was carried out by the method described in the Document
1 below.
[0310] (4) Preparation of Cells
[0311] Cells were prepared as follows.
[0312] (i) Bone Marrow-Derived Mast Cells (BMMCs)
[0313] In complete RPMI 1649 medium supplemented with a cell growth
factor (SCF) (10 ng/mL), IL-3 (4 ng/mL) and fetal bovine serum
(FBS) (10%) placed in a 10-cm dish, 2.times.10.sup.8 mouse bone
marrow cells were cultured for not less than 5 weeks to prepare
bone marrow-derived mast cells (BMMCs). The BMMCs were subcultured
every week with fresh medium. Flow cytometry analysis showed that
more than 95% of the prepared BMMCs were
c-Kit.sup.+Fc.epsilon.RI.sup.+ cells.
[0314] (ii) Bone Marrow-derived Macrophages (BMM.phi.)
[0315] In complete RPMI 1649 medium supplemented with M-CSF (10
ng/mL) and fetal bovine serum (FBS) (10%) placed in a 10-cm dish,
2.times.10.sup.6 mouse bone marrow cells were cultured for 1 week
to prepare bone marrow-derived macrophages (BMM.phi.).
[0316] (iii) NIH3T3 Cell Transfectant
[0317] According to a conventional method, a pMX-neo retrovirus
vector plasmid containing a cDNA of Flag-tagged CD300a or cDNA of
Flag-tagged CD300d was prepared.
[0318] NIH3T3 cells were transfected with the thus prepared
plasmid, to obtain a transfectant that stably expresses CD300a or
CD300d. The transfectant that stably expresses CD300a and the
transfectant that stably expresses CD300d obtained are referred to
as "NIH3T3 transfectant (CD300a)" and "NIH3T3 transfectant
(CD300d)", respectively.
[0319] The NIH3T3 cell transfectant that stably expresses TIM-4 was
provided by Mr. T. Kitamura (University of Tokyo). This
transfectant is referred to as "NIH3T3 transfectant (TIM-4)".
[0320] [Method of Evaluation]
[0321] Conditions for the method of evaluation are described below.
In the survival test, the Kaplan-Meier log-rank test was used, and,
in other evaluation tests, the unpaired Student's t test was used
to perform statistical analysis. Statistical significance was
assumed at P<0.05.
[0322] (5) Binding Assay Etc.
[0323] (i) Binding Assay
[0324] Cells were stained for 30 minutes in phosphate buffer
supplemented with 2% FBS in the presence or absence of 1 mM
CaCl.sub.2 using CD300a or a control human IgG, and then washed
twice with the same buffer, followed by incubation with the
F(ab').sub.2 fragment of an FITC-conjugated goat anti-human IgG.
Subsequently, for staining with annexin V, the cells were incubated
in 10 mM HEPES-NaOH buffer supplemented with 140 mM NaCl and 2.5 mM
CaCl.sub.2 together with annexin V for 15 minutes at room
temperature.
[0325] (ii) Binding Inhibition Assay
[0326] Cells were preincubated with a monoclonal antibody against
mouse CD300a (TX41), control isotype antibody or MFG-E8 for 30
minutes, and then stained with CD300a-Fc as in the above binding
assay. Further, in order to analyze whether CD300a-Fc was bound to
phospholipid or not, an assay was carried out using a PIP Strip
(manufactured by Echelon Biosciences) according to the
manufacturer's instructions.
[0327] (6) Measurement of CFU (Colony Forming Unit) of Aerobic
Bacteria
[0328] Serial dilutions of mouse peritoneal perfusate were plated,
and the dilutions were cultured on plates containing brain-heart
infusion (BHI) agar at 37.degree. C. for 48 hours. Subsequently,
the CFU of aerobic bacteria was calculated by measuring the number
of colonies in 1 mL of the peritoneal perfusate as described in the
Document 27 listed below.
Example 1; Identification of CD300a Ligand
Example 1A
[0329] In order to confirm expression of the mouse CD300a ligand in
hematopoietic stem cell lines and tumor cell lines, the following
test was carried out.
[0330] The bone marrow-derived macrophages (BMM.phi.) obtained in
"1. Preparation Example", bone marrow-derived dendritic cells
(BMDCs) or IL-3-dependent hematopoietic cell line cells (BaF/3
cells) (2.times.10.sup.5 cells per each type of cells) were
incubated in PBS (phosphate buffered saline) containing CD300a-Fc
(1 .mu.g) and calcium chloride (1 mM) at 20.degree. C. for 30
minutes, and then stained using a buffer containing an
FITC-conjugated anti-human IgG antibody (0.1 .mu.g) and propidium
iodide (PI) (1 .mu.g).
[0331] The stained cells of each type were subjected to analysis
using a flow cytometer (FACSCalibur, manufactured by Becton
Dickinson; model number, "E6133").
[0332] Further, a control test was carried out in the same manner
as in the above test method except that a control human IgG (1
.mu.g) was used instead of mouse CD300a-Fc.
[0333] The results of flow cytometry analysis for BMM.phi., BMDC
and BaF/3 cells are shown in FIG. 1A, FIG. 1B and FIG. 1C,
respectively (the results of the control test are shown as "Ctrl
Ig").
[0334] As shown in FIG. 1A to FIG. 1C, it was found that, in cases
where calcium chloride is contained, mouse CD300a-Fc binds to
PI.sup.- cells (live cells) but does not bind to PI.sup.+ cells
(dead cells). That is, the mouse CD300a ligand is suggested to be
expressed in dead cells.
Example 1B
[0335] In order to test whether mouse CD300a-Fc binds to apoptotic
cells, which are a type of dead cells, the following test was
carried out.
[0336] Thymocytes derived from a C57BL/6 mouse (wild-type mouse)
were incubated with dexamethasone (manufactured by Sigma) (10
.mu.M) in RPMI medium to prepare apoptotic thymocytes.
[0337] The obtained apoptotic cells (cell number, 2.times.10.sup.5)
were incubated in a medium (PBS) containing CD300a-Fc (1 .mu.g),
APC-conjugated annexin V (manufactured by BD Pharmingen) (1 .mu.l)
and calcium chloride (1 mM) at 20.degree. C. for 30 minutes, and
stained using a buffer containing an FITC-conjugated anti-human IgG
antibody (0.1 .mu.g) and propidium iodide (PI) (1 .mu.g).
[0338] The stained cells of each type were subjected to analysis
using a flow cytometer (FACSCalibur, manufactured by Becton
Dickinson; model number, "E6133") (results: FIG. 2A).
[0339] Further, the cells were subjected to flow cytometry analysis
under the same conditions as in the above test except that a medium
supplemented with no calcium chloride was used instead of the
medium supplemented with calcium chloride (results: FIG. 2B).
[0340] According to FIG. 2A, it can be seen that, in the presence
of calcium chloride, mouse CD300a-Fc bound to thymocytes that are
stained with annexin V but did not bind to thymocytes that are not
stained with annexin V (annexin V). That is, it is suggested that
mouse CD300a-Fc binds to apoptotic thymocytes.
[0341] On the other hand, it can be seen as shown in FIG. 2B that
mouse CD300a-Fc did not bind to apoptotic thymocytes in the absence
of calcium chloride.
[0342] From these test results, it can be understood that mouse
CD300a binds to apoptotic cells dependently on calcium ions.
Example 1C
[0343] The apoptotic cells obtained in Example 1B (cell number,
2.times.10.sup.5) were incubated in a medium (PBS) supplemented
with CD300a-Fc (1 .mu.g), APC-conjugated annexin V (manufactured by
BD Pharmingen) (1 .mu.l), control human IgG1 (1 .mu.g) and calcium
chloride (1 mM) at 20.degree. C. for 30 minutes, and stained using
a buffer containing an FITC-conjugated anti-human IgG antibody (0.1
.mu.g) and propidium iodide (PI) (1 .mu.g).
[0344] The stained cells of each type were subjected to analysis
using a flow cytometer (FACSCalibur, manufactured by Becton
Dickinson; model number, "E6133") (results: FIG. 2C "HuIgGl").
[0345] Further, the cells were subjected to flow cytometry analysis
under the same conditions as in the above test method except that
"TX41", which is a monoclonal antibody, or "MFG-E8", which is a
protein expressed in the macrophage, was used instead of the
control human IgG1 (results: FIG. 2C "TX41" and "MFG-E8").
[0346] As described above, the "TX41" used herein is an anti-mouse
CD300a monoclonal antibody, and blocks binding of mouse CD300a to
the ligand.
[0347] "MFG-E8" is known to bind to both phosphatidyl serine (PS)
and .alpha.v.beta.3 integrin to thereby cross-link apoptotic cells
to phagocytes expressing .alpha.v.beta.3 integrin (Document 4
listed below).
[0348] As can be seen by comparison among the results of flow
cytometry shown in FIG. 2C, mouse CD300a did not bind to apoptotic
cells in the presence of TX41 or MFG-E8.
[0349] From this viewpoint, it is suggested that mouse CD300a-Fc
has binding capacity to PS (that is, the ligand of CD300a is
PS).
Example 1D
[0350] In order to test whether or not human CD300a binds to
apoptotic cells similarly to mouse CD300a, the following test was
carried out.
[0351] First, Jurkat cells (human T-cell line) were suspended in
RPMI 1640 medium, and the resulting medium was irradiated with UV
for 60 minutes to prepare apoptotic Jurkat cells.
[0352] Flow cytometry analysis was carried out under the same test
conditions as in Example 1A except that the "Jurkat cell-derived
apoptotic cells" were used as apoptotic cells instead of the
apoptotic cells derived from wild-type mouse thymocytes, and "human
CD300a-Fc" was used instead of the "mouse CD300a-Fc" (results: FIG.
2D).
[0353] Further, cytometry analysis was carried out under the same
test conditions as in Example 1B except that the "Jurkat
cell-derived apoptotic cells" were used as apoptotic cells instead
of the apoptotic cells derived from wild-type mouse thymocytes;
"human CD300a-Fc" was used instead of the "mouse CD300a-Fc"; and
"TX49" or "control human IgG1" was used instead of "TX41" (results:
FIG. 2E "TX49" or "HuIgGl"). "TX49" is an anti-human CD300a
antibody (monoclonal antibody), and blocks binding of CD300a to the
ligand.
[0354] As can be seen from FIG. 2D to FIG. 2E, human CD300a-Fc
bound to annexin V.sup.+ cells but did not bind thereto in the
presence of the anti-human CD300a antibody.
[0355] That is, it is suggested that, similarly to mouse CD300a-Fc,
human CD300a also binds to apoptotic cells.
Example 1E
[0356] Liquids (test liquids) containing various phospholipids (PS,
PC, PE) (100 pmol) were spotted on a membrane (PIP-strip
(manufactured by Echelon Bioscience)) to allow adsorption of the
phospholipids on the membrane.
[0357] Subsequently, the membrane was immersed in TBST buffer (pH
8.0) containing mouse CD300a-Fc (1.5 .mu.g/mL), supplemented with
calcium chloride (1 mM) and BSA, at 20.degree. C. for 2 hours.
[0358] Thereafter, the membrane was washed 3 times with TBST buffer
that does not contain mouse CD300a-Fc (pH 8.0) to remove CD300a-Fc
unbound to the phospholipids on the membrane. Detection was then
carried out using TBST buffer (pH 8.0) containing HRP-conjugated
anti-human IgG (manufactured by Jackson Immun), supplemented with
BSA (results: FIG. 2F).
[0359] In FIG. 2F, "PE", "PC" and "PS" indicate the positions where
phosphatidyl ethanolamine, phosphatidyl choline and phosphatidyl
serine were spotted on the membrane, respectively, and "Blank"
indicates a position where no phospholipid was spotted on the
membrane.
[0360] According to FIG. 2F, CD300a bound to neither PE nor PC, and
bound specifically to PS.
[0361] From the results of Examples 1A to 1E, it can be understood
that CD300a binds to phosphatidyl serine (PS) dependently on
calcium ions (that is, the ligand of CD300a is PS).
Example 2: Functional Analysis of CD300a (1)
[0362] Some PS-binding receptors are known to be expressed in
phagocytes and to be involved in removal of apoptotic cells under
physiological and pathological conditions (Documents 4 to 9 listed
below).
[0363] PS is known to mediate the so-called "eat me" signal in
phagocytes (macrophages and the like), which are cells expressing
CD300a (Documents 10 and 11 listed below). In view of this, the
tests described in the Examples 2A to 2C below were carried out to
test whether CD300a is involved in phagocytosis of apoptotic cells
or not.
Example 2A
[0364] Thymocytes derived from a CAD-deficient mouse were treated
in the same manner as in Example 1B to prepare apoptotic
thymocytes.
[0365] Subsequently, macrophages (thioglycolate-induced peritoneal
macrophages) derived from a CD300a gene-deficient mouse
(2.times.10.sup.5 cells) were co-cultured with the apoptotic
thymocytes derived from a CAD-deficient mouse at a ratio of 1:5
(macrophages:apoptotic thymocytes (cell numbers)) in a 8-well
Lab-TeK II chamber slide (manufactured by Nalge Nunc) at 37.degree.
C. for 1 hour.
[0366] Subsequently, as described in the Documents 5 and 26 listed
below, the co-cultured macrophages were washed with cold PBS and
fixed with a fixative containing paraformaldehyde (1%). The fixed
macrophages where then subjected to TUNEL staining using a buffer
containing FITC-labeled dUTP (manufactured by Roche).
[0367] Not less than 50 cells randomly selected from the stained
macrophages were analyzed using a laser scanning confocal
microscope ("FV10i", manufactured by Olympus Corporation; product
number, 1B22358), and the number of TUNEL-positive cells (apoptotic
cells) contained per one macrophage cell was counted. The ratios of
macrophages containing apoptotic cells in the numbers of 0 to 8
(phagocytosis rates) were calculated as percentages with respect to
the total number of macrophages (results: FIG. 4A
"Cd300a.sup.-/-").
[0368] Further, the phagocytosis rates were measured under the same
conditions as in the above test except that macrophages derived
from a wild-type mouse were used instead of the macrophages derived
from a CD300a gene-deficient mouse (results: FIG. 4A "WT").
[0369] As shown in FIG. 4A, no evident difference in the
phagocytosis rate was found between the case where the macrophages
were derived from a CD300a gene-deficient mouse and the case where
the macrophages were derived from a wild-type mouse.
Example 2B
[0370] In order to test whether mast cells express known PS
receptors (TIM-1, TIM-4, stabilin 2 and integrin .alpha.v.beta.3),
the following test was carried out.
[0371] Bone marrow-derived mast cells (BMMCs) (cell number,
2.times.10.sup.5) were incubated at 20.degree. C. for 30 minutes in
a medium (PBS) containing a PE (Phycoerythrin)-conjugated TIM-1
monoclonal antibody (0.1 .mu.g), APC-conjugated TIM-4 monoclonal
antibody (0.1 .mu.g) and Alexa-conjugated anti-mouse CD300a
monoclonal antibody (TX41) (0.5 .mu.g).
[0372] Subsequently, the stained cells were washed twice using PBS,
and subjected to analysis using a flow cytometer (FACSCalibur,
manufactured by Becton Dickinson; model number, "E6133") (results:
FIG. 5A "BMMCs").
[0373] Further, flow cytometry analysis was carried out under the
same conditions as in the above test method except that peritoneal
macrophages or BM-derived macrophages were used instead of the
BMMCs (results: FIG. 5A "Peritoneal macrophages" and "BM derived
macrophages").
[0374] Further, using High Capacity cDNA Reverse Transcription Kit
(manufactured by Applied Biosystems), cDNAs were prepared from
peritoneal macrophages and BMMCs. Using each prepared cDNA, the
expression levels of stabilin 2, BA1-1, .alpha.v integrin, Cd300a
and .beta.-actin (loading control) were analyzed by RT-PCR
(results: FIG. 5B).
[0375] As can be seen from FIG. 5A and FIG. 5B, unlike the cases of
macrophages, the mast cells expressed CD300a and .alpha.v.beta.3
integrin, but showed only low levels of expression of TIM-1, TIM-4
and stabilin 2, which are PS receptors involved in
phagocytosis.
Example 2C
[0376] The NIH3T3 transfectant (CD300a) (cell number,
6.times.10.sup.4) was co-cultured with FITC-labeled cells
(apoptotic thymocytes or thymocytes (live cells)) for 2 hours, and
washed with PBS, followed by analysis under a light microscope
(BZ-9000, manufactured by Keyence) (results: FIG. 4B).
[0377] Further, the cells obtained after the co-culture and washing
were fixed with a fixative containing paraformaldehyde, Vectashield
(manufactured by Vector Laboratories), and analyzed using a laser
scanning confocal microscope (results: FIG. 4C). In FIG. 4C, green
areas (indicated by arrows) indicate phagocytosed cells (apoptotic
thymocytes or thymocytes (live cells)).
[0378] Further, analysis using a light microscope and a laser
scanning confocal microscope was carried out under the same
conditions as in the above test except that NIH3T3 untransfected
cells (negative control) or "NIH3T3 transfectant (TIM-4)" cells
(positive control) were used instead of the NIH3T3 transfectant
(CD300a).
[0379] In FIG. 4B and FIG. 4C, "NIH-3T3" and "NIH-3T3/Tim4" show
images of "NIH3T3" (untransfected cells) and "NIH3T3 transfectant
(TIM-4)", respectively, which images were obtained using a light
microscope and a laser scanning confocal microscope.
[0380] The images obtained with the laser scanning confocal
microscope were used to measure the ratios of the number of
untransfected cells and the number of cells of each transfectant
that incorporated apoptotic thymocytes into the cytoplasm
(percentages with respect to the number of co-cultured
untransfected cells or to the number of co-cultured cells of each
transfectant) (results: FIG. 4D "apoptotic").
[0381] Further, similarly, the ratios of the number of NIH3T3 cells
and the number of cells of each transfectant that incorporated
thymocytes (live cells) into the cytoplasm (percentages with
respect to the number of co-cultured untransfected cells or to the
number of co-cultured cells of each transfectant) were measured
(results: FIG. 4D "Live").
[0382] As shown in FIG. 4B, unlike NIH3T3, both of the above
transfectants adhered to apoptotic thymocytes. However, based on
FIG. 4C and FIG. 4D, it can be seen that only the NIH3T3
transfectant (TIM-4) incorporated apoptotic thymocytes to show
phagocytosis.
[0383] Although data are not shown, the NIH3T3 transfectant
(CD300a) did not show phagocytosis of live cells (thymocytes),
similarly to NIH3T3.
[0384] From the results of Examples 2A to 2C, it can be understood
that CD300a is not involved in phagocytosis of apoptotic cells by
macrophages.
Example 3: Functional Analysis of CD300a (2)
[0385] As shown in FIG. 5, mast cells express CD300a, but, unlike
macrophages, the cells do not express TIM-1, TIM-4 and stabilin,
which are PS receptors.
[0386] PS is known to bind to these PS receptors directly or
indirectly, and contribution of these receptors to incorporation of
apoptotic cells is known (Document 13 listed below). In view of
this, the tests described below in the Examples 3A to 3C were
carried out in order to test whether CD300a also has such a
function or not (whether or not there is functional overlap in
incorporation of apoptotic cells).
Example 3A
[0387] In complete RPMI 1649 medium supplemented with a cell growth
factor (SCF) (10 ng/mL), IL-3 (4 ng/mL) and fetal bovine serum
(FBS) (10%) placed in a 10-cm dish, 2.times.10.sup.8 bone marrow
cells (BM cells) derived from a CD300a gene-deficient mouse were
cultured for 4 weeks to prepare bone marrow-derived mast cells
(BMMCs) of the CD300a gene-deficient mouse. The BMMCs were
subcultured every week with fresh medium.
[0388] Subsequently, the obtained BMMCs were incubated in RPMI1649
medium containing an FITC-conjugated anti-Fc.epsilon.RI.alpha.
antibody (0.1 .mu.g) and a PE-conjugated anti-c-Kit antibody (0.1
.mu.g/mL) at 4.degree. C. for 30 minutes, and analyzed by flow
cytometry (results: FIG. 6A "CD300.sup.-/-").
[0389] Further, flow cytometry analysis was carried out under the
same test conditions as in the above test except that BMMCs were
prepared using bone marrow cells derived from a wild-type mouse
instead of the bone marrow cells derived from a CD300a
gene-deficient mouse (results: FIG. 6A "WT"). Each number in FIG.
6A indicates the ratio of the cells shown in each box. Each test
was repeated 3 times independently.
[0390] Using each type of BMMCs, a .beta.-hexosaminidase release
assay (degranulation assay) was carried out as follows (for
detailed conditions, see the Document 29 below).
[0391] First, 1.times.10.sup.5 to 2.times.10.sup.5 BMMCs of each
type in the logarithmic growth phase were cultured at 37.degree. C.
for one day and night in a 24-well plate coated with gelatin
(manufactured by Sigma), and then incubated at 37.degree. C. for 1
hour in a medium that contains a biotin-conjugated mouse
anti-trinitrophenol IgE (0.5 mg/mL) but does not contain a
supplement.
[0392] Subsequently, streptavidin was added to the medium to cause
cross-linking between the biotin-conjugated mouse
anti-trinitrophenol IgE molecules, and culture was performed at
37.degree. C. for 45 minutes, followed by collecting the
supernatant.
[0393] To the collected supernatant, a buffer (pH 4.5) containing
p-nitrophenyl-N-acetyl-.beta.-D-glucosamide (manufactured by
Sigma), citric acid (0.4 M) and sodium phosphate (0.2 M) was added,
and the resulting mixture was incubated at 37.degree. C. for 3
hours to allow hydrolysis reaction of
p-nitrophenyl-N-acetyl-.beta.-D-glucosamide by released
.beta.-hexosaminidase. This reaction was stopped by adding 0.2 M
glycine-NaOH (pH 10.7), and the absorbance at a wavelength of 415
nm, which increases as hydrolysis of
p-nitrophenyl-N-acetyl-.beta.-D-glucosamide proceeds, was measured
to quantify the amount of .beta.-hexosaminidase released. The rate
(%) of increase in the amount of .beta.-hexosaminidase released
with respect to the amount observed with untreated BMMCs of each
type is shown in FIG. 6B.
[0394] FIG. 6B shows the ratio of BMMCs that released
.beta.-hexosaminidase. As shown in FIG. 6A and FIG. 6B, no
significant difference was found in expression of
Fc.epsilon.RI.alpha. and c-Kit (marker proteins for mast cells) and
the rate of increase in the amount of .beta.-hexosaminidase
released between the case where the BMMCs were derived from a
CD300a gene-deficient mouse and the case where the BMMCs were
derived from a wild-type mouse.
[0395] That is, it can be seen that differentiation from bone
marrow cells and degranulation mediated by FceRI are not influenced
by CD300a.
Example 3B
[0396] BMMCs derived from a CD300a gene-deficient mouse and the
apoptotic cells obtained in Example 1B (BMMCs:apoptotic cells=10:1
(ratio in terms of the cell number)) were incubated in PBS
containing calcium chloride (1 mM), APC-conjugated annexin V (1
.mu.l), CD300a-Fc (1 .mu.g/mL) and MFG-E8 (5 .mu.g) at 20.degree.
C. for 30 minutes, and then stained using a buffer containing an
FITC-conjugated anti-human IgG antibody (0.1 .mu.g/mL) and
propidium iodide (PI) (1 .mu.g).
[0397] The stained cells were subjected to analysis using a flow
cytometer (FACSCalibur, manufactured by Becton Dickinson; model
number, "E6133") (results: FIG. 7A "MFG-E8").
[0398] Further, flow cytometry analysis was carried out under the
same conditions as in the above test except that a control IgG was
used instead of MFG-E8 (results: FIG. 7A "Ctrl Ig").
[0399] From the results shown in FIG. 7A, it can be seen that
CD300a-Fc bound to apoptotic cells (annexin V.sup.+) in the
presence of the control IgG, but that the binding was specifically
inhibited in the presence of MFG-E8 (PS-binding substance).
[0400] The concentrations of cytokines and chemokines in the
supernatant of this sample mixture were quantified using ELISA kits
manufactured by BD Pharmingen (TNF-.alpha. and IL-6) and R&D
Systems (MIP-2, MCP-1, IL-13 and MIP-1a). As a result, none of the
cytokines and chemokines could be detected.
Example 3C
[0401] In order to test whether or not stimulation by LPS
(lipopolysaccharide) changes the amounts of cytokines released in
the coexistence of BMMCs and apoptotic cells, the following test
was carried out.
[0402] BMMCs derived from a CD300a gene-deficient mouse and
apoptotic cells (BMMCs:apoptotic cells=10:1 (ratio in terms of the
cell number)) were incubated in RPMI containing LPS (1 .mu.g/mL)
for 4 hours, and the supernatant of the medium was then
collected.
[0403] Subsequently, the levels of cytokines and chemokines were
measured 3 times using ELISA kits manufactured by BD Pharmingen
(TNF-.alpha. and IL-6) and R&D Systems (MIP-2, MCP-1, IL-13 and
MIP-1.alpha.), and the rate of increase in the amount of each
cytokine or chemokine released with respect to the amount observed
with the BMMCs that had not been subjected to the above LPS
treatment was calculated (results: FIG. 7B "Cd300a.sup.-/-").
[0404] Further, the rate of increase in the amount of each cytokine
or chemokine was calculated under the same conditions as in the
above test except that BMMCs derived from a wild-type mouse were
used instead of the BMMCs derived from a CD300a gene-deficient
mouse (results: FIG. 7B "WT").
[0405] As shown in FIG. 7B, LPS increased the amounts of cytokines
released in both types of BMMCs. However, the BMMCs derived from a
CD300a gene-deficient mouse showed significantly larger increases
in the amounts of TNF-.alpha., IL-13 and MCP-1 than the BMMCs
derived from a wild-type mouse.
Example 3D
[0406] Further, the following test was carried out in order to test
the rates of increase in intracellular cytokines and chemokines in
BMMCs.
[0407] BMMCs derived from a CD300a gene-deficient mouse and the
apoptotic cells obtained in Example 1B (BMMCs:apoptotic cells=10:1
(ratio in terms of the cell number)) were incubated in a medium
(RPMI) containing LPS (lipopolysaccharide) (1 .mu.g/mL) for 4
hours, and the BMMCs and apoptotic cells after the incubation were
then incubated in a medium (FIX & PERM, manufactured by
Invitrogen) supplemented with fluorescently labeled antibodies
against various cytokines and chemokines and formaldehyde at
4.degree. C. for 20 minutes. The stained cells were subjected to
analysis using a flow cytometer (FACSCalibur, manufactured by
Becton Dickinson; model number, "E6133") (results: FIG. 8, arrows
(1)). Further, flow cytometry analysis was carried out under the
same conditions as in the above test except that a control antibody
was used instead of the fluorescently labeled antibodies against
various cytokines and chemokines (control test (results: FIG. 8,
arrows (2))).
[0408] Further, flow cytometry analysis was carried out under the
same conditions as in the above test except that BMMCs derived from
a wild-type mouse were used instead of the BMMCs derived from a
CD300a gene-deficient mouse (results: FIG. 8, arrows (3)). Further,
flow cytometry analysis was carried out under the same conditions
as in the above test except that a control antibody was used
instead of the fluorescently labeled antibodies against various
cytokines and chemokines (control test (results: FIG. 8, arrows
(4))).
[0409] Each graph in FIG. 8 shows the amount of increase in MFI
(mean fluorescence intensity) observed for each cytokine or
chemokine in each type of BMMCs, relative to that of LPS-untreated
BMMCs.
[0410] According to FIG. 8, in both types of BMMCs, the amounts of
cytokines and chemokines in the cytoplasm increased compared to the
case where the LPS treatment was not carried out. In particular, it
can be seen that the BMMCs derived from a CD300a gene-deficient
mouse showed significantly larger increases in the amounts of
TNF-.alpha. and the like in the cytoplasm than the BMMCs derived
from a wild-type mouse.
Example 3E
[0411] D89E MFG-E8 is a variant (mutant) of MFG-E8 and has a point
mutation (D89E) in the RGD motif. D89E MFG-E8 binds to PS, but does
not bind to .alpha.v.beta.3 integrin.
[0412] In view of this, the following test was carried out in order
to test whether or not the amounts of cytokines and chemokines
released from BMMCs change in the presence of D89E MFG-E8.
[0413] TNF-.alpha., IL-13, MCP-1 and IL-6 were quantified in the
same manner as in Example 3C except that LPS as well as D89E MFG-E8
(5 .mu.g/mL) were added to the medium containing BMMCs derived from
a CD300a gene-deficient mouse and apoptotic cells (results: FIG. 7C
"CD300a.sup.-/-").
[0414] Further, TNF-.alpha., IL-13, MCP-1 and IL-6 were quantified
under the same conditions as in the above test except that BMMCs
derived from a wild-type mouse were used instead of the BMMCs
derived from a CD300a gene-deficient mouse (results: FIG. 7C
"WT").
[0415] As shown in FIG. 7C, in the presence of D89E MFG-E8, no
significant difference was found in the concentrations of cytokines
between the case where the BMMCs were derived from a CD300a
gene-deficient mouse and the case where the BMMCs were derived from
a wild-type mouse.
Example 3F
[0416] CD300a is known to have an immunoreceptor tyrosine-based
inhibitory motif (ITIM) in the intracellular domain, and to induce
SHP-1 upon cross-linking by an anti-CD300a antibody (Document 14
listed below).
[0417] In view of this, the following test was carried out in order
to test whether CD300a interacts with SHP-1 or not. As in Example
3C, BMMCs derived from a CD300a gene-deficient mouse or wild-type
mouse were co-cultured with apoptotic cells in the presence of LPS
for 4 hours, and a homogenate of the cells was subjected to
immunoprecipitation with an anti-CD300a antibody (TX41).
[0418] Using the thus obtained immunoprecipitates, immunoblotting
with an anti-SHP-1 antibody or an anti-CD300a antibody was carried
out as described in Document 14 listed below (results: FIG.
7D).
[0419] As can be seen from these results, the BMMCs responded to
the stimulation with LPS to induce (recruit) SHP-1 when they were
co-cultured with apoptotic cells. However, CD300a did not recruit
SHP-1 in the presence of D89E MFG-E8.
[0420] That is, it is thought that induction (recruitment) of SHP-1
by CD300a in response to LPS stimulation requires binding of PS to
CD300a.
Example 3G
[0421] In order to investigate whether SHP-1 is involved in
CD300a-mediated signaling or not, first, Ptpn6 (SHP-1 gene) of
BMMCs derived from a wild-type mouse was knocked out with an siRNA
to prepare SHP-1-deficient (Ptpn6-KD) wild-type mouse-derived BMMCs
under the following conditions. Further, similarly, SHP-1-deficient
(Ptpn6-KD) CD300a gene-deficient mouse-derived BMMCs were prepared
from BMMCs derived from a CD300a gene-deficient mouse.
[0422] With 1 mL of X-treme Gene siRNA transfection reagent
(manufactured by Roche), 0.5 mM siRNA (SHP-1 siRNA) (siGENOME
SMARTpool; ThermoScientific Dharmacom) targeting the SHP-1 gene
(Ptpn6 gene) in BMMCs was mixed, and 5.times.10.sup.5 BMMCs derived
from a CD300a gene-deficient mouse were transfected therewith as
described in the Document 28 listed below, to prepare SHP-1
knockdown BMMCs derived from a CD300a gene-deficient mouse (BMMCs
(CD300a.sup.-/- Ptpn6-KD)).
[0423] Further, SHP-1 knockdown BMMCs derived from a wild-type
mouse (BMMCs (WT Ptpn6-KD)) were prepared under the same conditions
as in the above test except that BMMCs derived from a wild-type
mouse were used instead of the BMMCs derived from a CD300a
gene-deficient mouse.
[0424] Here, in order to confirm that the BMMCs of each type were
transfected with the SHP-1 siRNA and that the expression level of
SHP-1 was decreased, a lysate of BMMCs (CD300a.sup.-/- Ptpn6-KD) or
BMMCs (WT Ptpn6-KD) was subjected to immunoblotting analysis using
an anti-SHP-1 antibody, anti-SHP-2 antibody or anti-.beta.-actin
antibody (results: FIG. 7E).
[0425] As can be seen from FIG. 7E, the BMMCs transfected with the
SHP-1 siRNA showed a decreased expression level of SHP-1. "Ctrl"
shows results of immunoblotting analysis using BMMCs transfected
with a control siRNA instead of the SHP-1 siRNA.
[0426] Subsequently, BMMCs (CD300a.sup.-/- Ptpn6-KD) or BMMCs (WT
Ptpn6-KD), and the apoptotic cells obtained in Example 1B
(BMMCs:apoptotic cells=10:1 (ratio in terms of the cell number))
were incubated in RPMI supplemented with calcium chloride (1 mM)
and LPS (lipopolysaccharide) (1 .mu.g/mL) for 4 hours, and the
amount of TNF-.alpha. released was measured in the same manner as
in Example 3B (results: FIG. 7F).
[0427] As shown in FIG. 7F (left graph), the BMMCs derived from a
CD300a gene-deficient mouse produced a significantly larger amount
of TNF-.alpha. than the BMMCs derived from a wild-type mouse. On
the other hand, as shown in FIG. 7F (right graph), in the cases
where the BMMCs derived from a wild-type mouse or a CD300a
gene-deficient mouse were transfected with the SHP-1 siRNA, the
amount of TNF-.alpha. released was almost the same between the
BMMCs derived from a wild-type mouse and the BMMCs derived from a
CD300a gene-deficient mouse, and no significant difference was
found between these.
[0428] These results suggest that binding of PS to CD300a causes
CD300a to induce SHP-1 and to thereby mediate signaling that causes
suppression of the activity of BMMCs, resulting in suppression of
secretion of TNF-.alpha..
[0429] From the results of Example 3, it can be understood that the
interaction between PS and CD300a inhibits production of
inflammation-inducing (LPS-inducing) cytokines and chemokines from
BMMCs, and that the interaction recruits SHP-1, resulting in
suppression of secretion of TNF-.alpha..
Example 4: Functional Analysis of CD300a (3)
[0430] TNF-.alpha., IL-3 and MCP-1 produced by mast cells are
chemoattractants for neutrophils, and known to play important roles
in bacterial clearance in CLP peritonitis mice (Patent Documents 15
to 19 listed below).
[0431] In view of this, in order to study whether CD300a has a
bacterial clearance function or not, the Examples 4A to 4H below
were carried out.
Example 4A
[0432] A wild-type mouse was subjected to midline incision of 1 to
2 cm on the cecum (ventral region), and the end portion was
ligated. After performing two times of puncture using a 27-gauge
needle in the ligated area, the cecum was returned to the abdomen.
Thereafter, 1 mL of sterile physiological saline was subcutaneously
injected for rehydration, and the incision site was closed by
suturing. Details of the procedure and conditions for the CLP are
described in the Document 16 listed below.
[0433] Before performing the CLP and 4 hours after performing the
CLP, peritoneal perfusate was collected. Subsequently,
APC-conjugated annexin V (1 .mu.g) and CD300-Fc (1 .mu.g) were
added to the peritoneal perfusate, and staining was performed with
an FITC-conjugated anti-human IgG and PI (propidium iodide),
followed by performing analysis by flow cytometry (results: FIG.
12A).
[0434] As can be seen from the results shown in FIG. 12A, the site
of peritonitis was a site where a number of cells were undergoing
apoptosis, as described in the Document 20 listed below.
[0435] That is, the immune regulation by mast cells in the site of
peritonitis is suggested to be influenced by CD300a.
Example 4B
[0436] In order to test the relationship between CD300a and the
immune regulation by mast cells, proteome analysis was carried out
as follows.
[0437] First, CLP was carried out in the same manner as in Example
4A using a wild-type mouse and a mast cell-deficient mouse
(kit.sup.W-sh/W-sh).
[0438] Four hours after performing the CLP, peritoneal perfusate
was collected from each mouse, and the collected peritoneal
perfusate was subjected to proteome analysis of cytokines and
chemokines using Proteome Profiler Array (manufactured by R&D
Systems) according to the manufacturer's instructions.
[0439] FIG. 9A shows the results of densitometry analysis (proteome
analysis) using the peritoneal perfusate from each of the wild-type
mouse and the mast cell-deficient mouse (kit.sup.W-sh/W-sh mouse)
(in FIG. 9A, "PC" indicates a positive control).
[0440] FIG. 9B shows the pixel densities of the signals for the
chemokines and cytokines, which were obtained from the densitometry
images shown in FIG. 9A.
[0441] As can be seen from the results shown in FIG. 9B, at Hour 4
after the CLP, the concentrations of chemokines were higher in the
kit.sup.W-sh/W-sh mouse than in the wild-type mouse. Similar
results were obtained in 2 replicates of the test.
Example 4C
[0442] In the same manner as in Example 4B, peritoneal perfusate
was collected from wild-type mice and mast cell-deficient mice
(kit.sup.W-sh/W-sh mice) (n=3 per each type of mice).
[0443] Subsequently, a dilution series of each peritoneal perfusate
was prepared, and the prepared serial dilutions of peritoneal
perfusate were plated to perform culture on plates containing
brain-heart infusion (BHI) agar at 37.degree. C. for 48 hours.
Thereafter, the CFU of aerobic bacteria was calculated by measuring
the number of colonies in 1 mL of the peritoneal perfusate as
described in the Document 27 listed below (results: FIG. 10A).
[0444] Further, the numbers of neutrophils and macrophages in each
peritoneal perfusate were also counted. The results are shown in
FIG. 10B as "neutrophil" and "macrophage", respectively.
[0445] From a wild-type mouse and mast cell-deficient mouse
(kit.sup.W-sh/W-sh), BM-derived macrophages were prepared. These
macrophages (cell number: 1.times.10.sup.6) were co-cultured for 1
hour in a medium containing fluorescein-labeled E. coli placed in a
24-well plate, and the number of each type of macrophages that
phagocytosed E. coli was counted by flow cytometry to calculate the
ratio of phagocytosing macrophages (results: FIG. 11 "BM
macrophage").
[0446] A test was carried out under the same conditions as in the
above test except that PEC macrophages derived from a wild-type
mouse or mast cell-deficient mouse (kit.sup.W-sh/W-sh mouse) were
used instead of the BM-derived macrophages derived from a wild-type
mouse or mast cell-deficient mouse (kit.sup.W-sh/W-sh mouse), to
calculate the ratio of phagocytosing macrophages (results: FIG. 11
"PEC macrophage").
[0447] As shown in FIG. 10A, it can be seen that, at Hour 4 after
the CLP, the mast cell-deficient mouse (kit.sup.W-sh/W-sh mouse)
showed a lower intraperitoneal bacterial CFU and a larger number of
neutrophils than the wild-type mouse. On the other hand, as shown
in FIG. 10B and FIG. 11, the number of macrophages and their
phagocytosis were not significantly different between the
genotypes.
Example 4D
[0448] In order to test whether CD300a is involved in induction
(recruitment) of neutrophils or not, the following Example was
carried out.
[0449] To mast cell-deficient mice (Kit.sup.W-sh/W-sh mice), PBS
buffer containing BMMCs derived from a wild-type mouse (cell
number, 1.times.10.sup.6) was administered by intraperitoneal
injection (n=20). On Day 28 after the administration, CLP was
performed in the same manner as in Example 4A, and the survival
rate of the mice was measured (results: FIG. 12B "WT
BMMCs.fwdarw.Kit.sup.W-sh/W-sh").
[0450] Further, a test was carried out under the same conditions as
in the above test except that BMMCs derived from a CD300a
gene-deficient mouse were used instead of the BMMCs derived from a
wild-type mouse, and the survival rate of mice was measured
(results: FIG. 12B "CD300a.sup.-/-
BMMCs.fwdarw.Kit.sup.W-sh/W-sh/Kit.sup.W-sh/W-sh").
"Kit.sup.W-sh/W-sh/Kit.sup.W-sh/W-sh" in FIG. 12B indicates the
survival rate of mast cell-deficient mice (Kit.sup.W-sh/W-sh mice)
subjected to CLP without administration of BMMCs.
[0451] According to FIG. 12B, the mast cell-deficient mice
(Kit.sup.W-sh/W-sh mice) subjected to administration of BMMCs
derived from a wild-type mouse showed a higher survival rate even
after the CLP, compared to the case where administration of BMMCs
was not carried out.
[0452] However, it can be seen that the mast cell-deficient mice
(Kit.sup.W-sh/W-sh mice) subjected to administration of BMMCs
derived from a CD300a gene-deficient mouse showed a significantly
higher survival rate even after CLP, compared to the mice subjected
to administration of BMMCs derived from a wild-type mouse and the
mice that had not been subjected to administration of BMMCs (FIG.
12B).
[0453] Further, as a result of measuring the bacterial CFU in the
same manner as in Example 4C using peritoneal perfusate of each
type of mice at Hour 4 after the CLP, the mast cell-deficient mice
(Kit.sup.W-sh/W-sh mice) subjected to administration of BMMCs
derived from a CD300a gene-deficient mouse showed a significantly
higher bacterial clearance than other mice (results: FIG. 12C).
Example 4E
[0454] In order to test whether or not the amount of TNF-.alpha.
released increases by intraperitoneal administration of BMMCs to a
mast cell-deficient mouse (Kit.sup.W-sh/W-sh mouse), the following
Example was carried out.
[0455] Twenty four hours before CLP, a CFSE-labeled BMMC mixture
(BMMCs derived from a CD300a gene-deficient mouse:BMMCs derived
from a wild-type mouse=1:1 (ratio in terms of the cell number)) was
administered to mast cell-deficient mice (Kit.sup.W-sh/W-sh mice)
by intraperitoneal injection.
[0456] The mice after injection of the BMMC mixture was subjected
to CLP in the same manner as in Example 4A, and, at Hour 4 after
the CLP, peritoneal perfusate was collected. Each type of BMMCs
contained in the peritoneal perfusate were subjected to analysis
using a flow cytometer (FACSCalibur, manufactured by Becton
Dickinson; model number, "E6133") (results: FIG. 12D).
[0457] As shown in FIG. 12D, it can be seen that, at Hour 4 after
the CLP, the BMMCs derived from a CD300a gene-deficient mouse
(CD300a.sup.-CFSE.sup.+ cells) showed production of a significantly
larger amount of TNF-.alpha. than the BMMCs derived from a
wild-type mouse (CD300a.sup.+CFSE.sup.+ cells).
Example 4F
[0458] In order to test the influence of administration of an
anti-CD300a monoclonal antibody (TX41) on CD300a, the following
Example was carried out.
[0459] First, CLP was carried out in the same manner as in Example
4A except that 500 .mu.g of an anti-CD300a monoclonal antibody
(TX41) (n=13) was intraperitoneally injected to wild-type mice 1
hour or 18 hours before the CLP, and the survival rate of the mice
was measured in the same manner as in Example 4D (results: FIG. 12E
"Antibody to CD300a").
[0460] Further, a test was carried out under the same conditions as
in the above test except that an isotype control antibody (n=11)
was used as a control instead of TX41 (n=13), and the survival rate
of the mice was measured (results: FIG. 12E "Control
antibody").
[0461] As shown in FIG. 12E, it was found that the survival time of
wild type mice was longer in the cases where CLP was carried out 1
hour or 18 hours after administration of TX41 by intraperitoneal
injection, compared to the cases of administration of the control
antibody.
Example 4G
[0462] From each mouse at Hour 4 after the CLP in Example 4F,
peritoneal perfusate was collected. The obtained peritoneal
perfusate was treated in the same manner as in Example 4C to
measure the bacterial CFU and the number of neutrophils (control
antibody: n=5 and anti-CD300a monoclonal antibody: n=5) (FIG. 12F
and FIG. 12G, respectively).
[0463] The administration of TX41 by intraperitoneal injection did
not cause damage to myeloid cells including mast cells in the
mice.
[0464] As shown in FIG. 12F and FIG. 12G, the administration of
TX41 to wild-type mice by intraperitoneal injection 1 hour or 18
hours before CLP resulted in a significant increase in neutrophils
and an increased bacterial clearance in the abdominal cavity.
[0465] From the results of Example 4, it can be understood that
inhibition of the interaction between PS and CD300a by TX41 or the
like allows prevention of sepsis induced by peritonitis.
[0466] Under physiological conditions, a number of cells undergo
apoptosis. In this process, PS receptors play a central role in
incorporation of the apoptotic cells, and are indispensable for
preventing the progression of autoimmune diseases (Document 22
listed below).
[0467] On the other hand, it is known that, under pathological
conditions such as microbial infection, cell death due to apoptosis
remarkably increases, and this causes inflammation reaction by mast
cells via receptors (e.g., Toll-like receptors) against
pathogen-associated molecular patterns (PAMPs) (Documents 15, 23
and 24 listed below). Further, mast cells are known to play
important roles in immune reaction against pathogens.
[0468] Thus, from the results in the above Examples, it can be
understood that PS not only provides an incorporation signal for
phagocytes via several kinds of PS receptors, but also has an
effect to effectively suppress inflammation reaction caused by mast
cells via CD300a, as newly discovered in the present invention.
[0469] It can therefore be understood that phosphatidyl
serine-binding substances (e.g., MFG-E8) and CD300a-binding
substances (e.g., neutralizing antibodies against CD300a) inhibit
the interaction between PS and CD300a in mast cells to thereby
activate the mast cells or maintain such an activity.
[0470] That is, it can be understood that these substance are
useful as effective components of immunostimulants used for
prophylaxis of various LPS-induced inflammatory infections (and
sepsis caused thereby).
[0471] Further, since PS suppresses activation signaling of CD300a
and hence suppresses activation of mast cells, PS can be understood
to be useful as, for example, an effective component of
immunosuppressants to be used for suppressing inflammation reaction
in allergic diseases and autoimmune diseases (for example, for
suppressing release of chemical mediators such as histamine) to
alleviate or treat symptoms of the allergic diseases and autoimmune
diseases (i.e., to suppress excessive immune function).
[0472] <Asthma>
[0473] (Materials and Methods)
[0474] (Mice)
[0475] C57BL/6J mice were purchased from Clea Japan, Inc. CD300a
gene-deficient mice (CD300a.sup.-/- mice) were obtained by crossing
Balb/c CD300a gene-deficient mice prepared in the inventors'
laboratory with the purchased WT C57BL/6J mice for 12 generations,
and then performing back-crossing. Male and female mice that were 8
to 10 weeks old at the beginning of induction of asthma were
used.
[0476] (OVA-Induced Asthma)
[0477] FIG. 13A shows the protocol for inducing asthma with
ovalbumin. On Days 0, 7 and 14 after the beginning of induction of
asthma, a mixture of 100 .mu.s of ovalbumin (OVA, chicken egg
albumin, manufactured by Sigma) and 100 .mu.L of aluminum hydroxide
gel (ALUM, ALhydrogel 2%, manufactured by Invitrogen) was
intraperitoneally injected to each mouse.
[0478] Further, on Days 21, 22 and 23 after the beginning of
induction of asthma, each mouse was subjected to inhalation of 10%
ovalbumin prepared by dilution with PBS, for 30 minutes using an
ultrasonic nebulizer (NE-U17, Omuron). On Day 25 after the
beginning of induction of asthma, each mouse was subjected to
bronchoalveolar lavage (BAL), and serum was collected from each
mouse.
[Example 5A] (FIG. 13A, FIG. 13B)
[0479] (Bronchoalveolar Lavage BAL)
[0480] After subjecting each mouse to tracheotomy, washing was
performed 3 times with 1 mL of 2% FBS/PBS, followed by collecting
the washing liquid and measuring the cell number. As shown in FIG.
13A and FIG. 13B, the cell number in the bronchoalveolar lavage
fluid (BAL fluid) of each mouse on Day 25 after the beginning of
induction of asthma was significantly smaller in the CD300a
gene-deficient mice than in the WT mice in terms of both the total
cell number and the number of eosinophils. This result indicates
that CD300a exacerbated eosinophilic airway inflammation caused by
ovalbumin (OVA), and that the CD300a gene-deficient mice showed
amelioration of symptoms of eosinophilic airway inflammation.
[Example 5B] (Analysis of Cells in Bronchoalveolar Lavage Fluid)
(FIG. 13C)
[0481] With CD45.2-FITC, Siglec-F-PE, CD11b-APC Cy7, CD11c-PEC Cy7
and F4/80-Alexa (all of these were purchased; BD), 1.times.10.sup.6
cells in the collected bronchoalveolar lavage fluid were stained,
and CD45.sup.+SiglecF.sup.-CD11b.sup.+CD11c.sup.-F4/80.sup.- was
analyzed as the eosinophil fraction by flow cytometry (FACS).
[0482] FACS analysis of cells in the bronchoalveolar lavage fluid
of the mice on Day 25 after the beginning of induction of asthma
showed the ratio of eosinophils among CD45-positive cells
(CD45.sup.+SiglecF.sup.-CD11b.sup.+CD11c.sup.-F4/80.sup.-). The
CD300a gene-deficient mice showed a significantly smaller ratio of
infiltrating eosinophils in the BAL fluid than the WT mice.
[Example 5C] (Measurement of Serum IgE Value) (FIG. 14)
[0483] Serum IgE was measured by ELISA using a rat anti-mouse IgE
(BD) and a biotinylated anti-mouse IgE (BD).
[0484] As shown in FIG. 14, the CD300a gene-deficient mice showed
significantly lower serum IgE values during the period of
sensitization to cause ovalbumin-induced asthma.
[0485] Based on comparison of the serum IgE value (index of the
degree of allergy) among the OVA-induced asthma model mice on
Disease Day 14, the CD300a gene-deficient mice showed significantly
lower serum IgE values than the WT mice.
[0486] It is thought that administration of an anti-CD300a
antibody, which suppresses signal transduction of CD300a, causes
significant suppression of serum IgE in ovalbumin-induced
asthma.
[0487] <Enteritis>
[0488] (Materials and Methods)
[0489] (Mice)
[0490] C57BL/6 mice (WT mice) were purchased from Clea. CD300a
gene-deficient mice established from Balb/cA-derived ES cells in
the inventors' laboratory were backcrossed with C57BL/6, and mice
of the 12th or later generation were used.
[0491] The mice used for the experiment were kept under a Specific
Pathogen-Free (SPF) environment in Laboratory Animal Resource
Center, University of Tsukuba.
[0492] (Induction of Enteritis)
[0493] Female C57BL/6 mice of 10 to 12 weeks old were made to drink
2.5% (w/v) dextran sulfate sodium salt, reagent grade (DSS, MP
Biomedicals, molecular weight (MW)=36000 to 50000) continuously for
8 days for induction of enteritis.
[0494] (Antibodies)
[0495] BD biotinylated anti-CD300a (TX41) was prepared in the
inventors' laboratory. Other antibodies, which are described below,
were purchased from Bioscience. [0496] Purified anti-mouse
TNF-.alpha. [0497] Biotin-labeled anti-mouse TNF-.alpha. [0498]
Purified mouse IL-6 [0499] Biotin-labeled anti-mouse IL-6 [0500]
Purified anti-mouse IL-10 [0501] Biotin-labeled anti-mouse IL-10
[0502] Allophycocyanin-Cy7 (APC-Cy7)-labeled anti-mouse CD11b
(M1/70) [0503] Phycoerythrin-Cy7 (PE-Cy7)-labeled anti-mouse CD11c
(HL3) [0504] Fluorescein isothiocyanate (FITC)-labeled anti-mouse
CD4 (L3T4)
[0505] (Large Intestine Tissue Culture Liquid)
[0506] After removal of the large intestine from each mouse on Day
9 after administration of DSS or water, the large intestine was
washed with phosphate buffer to remove feces. The large intestine
was longitudinally incised, and 5 3-mm tissue pieces were cut out
from the portion 3 mm distant from the anus, and the tissue pieces
were subjected to 12 hours of culture in 10% FBS RPMI 1640
(manufactured by GIBCO). The supernatant was then collected to
provide a large intestine tissue culture liquid. TNF-.alpha., IL-6
and IL-10 contained in the culture liquid were measured by sandwich
ELISA.
[0507] (Isolation of Cells of Large Intestinal Lamina Propria)
[0508] Mice were sacrificed, and the large intestine of each mouse
was collected. The large intestine was cut into 4 pieces with
scissors, and then washed with phosphate buffer to remove feces.
The remaining tissue was placed in 1 mM DTT/5 mM MEDTA/Hank's
balanced salt solution (manufactured by Sigma), and incubated in an
incubator (shaker type) at 37.degree. C. for 30 minutes.
[0509] Subsequently, the tissue was washed again with phosphate
buffer to remove detached epithelium and contaminants. The
remaining tissue was cut into small pieces 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, and incubated in an
incubator (shaker type) at 37.degree. C. for 2 hours to allow
complete lysis.
[0510] The cell suspension was passed through 70-.mu.m nylon, and
then centrifuged. The obtained cells were suspended in 40% Percoll,
and overlaid on 70% Percoll. After centrifugation, cells in the
intermediate layer were collected to be used as cells of the large
intestinal lamina propria in flow cytometry.
[0511] (Isolation of CD11b.sup.+CD11c.sup.+ Cells and CD4.sup.+ T
Cells)
[0512] The isolated cells of the large intestinal lamina propria
were labeled with APC-Cy7-labeled anti-CD 11b (M1/70),
PE-Cy7-labeled anti-CD11c (HL3) and FITC-labeled anti-mouse CD4
(L3T4). The labeled cells were sorted by FACSAria (BD) to provide
CD11b.sup.+CD11c.sup.+ cells and CD4.sup.+ T cells,
respectively.
[0513] (Measurement of mRNA Level)
[0514] The cells sorted by FACSAria were lysed with ISOGEN-LS
(Nippon Gene). From the lysed cells, mRNA was extracted using an
mRNA extraction kit, and cDNA was prepared using a reverse
transcription kit. The prepared cDNA was mixed with primers (see
SEQ ID NOs:1 to 16 in SEQUENCE LISTING) and Power Cyber Green
(Applied Biosystems), and measurement of the mRNA levels was
carried out using 7500 Fast Real Time PCR System (Applied
Biosysytems).
[Example 6A] Body Weight Change Rate (FIG. 15)
[0515] WT mice and CD300a gene-deficient mice were made to drink
2.5% (w/v) dextran sodium sulfate (DSS) continuously for 8 days to
induce enteritis, and the body weight of each mouse was measured
every day.
[0516] The mice used were female mice of 10 to 12 weeks old, and
the rate of change in the body weight was observed for 15 WT mice
and 15 CD300a gene-deficient mice. Statistical significance was
determined by Student's t-test (**: p<0.01, ***:
p<0.001).
[0517] As shown in FIG. 15, the weight loss due to enteritis
induced by drinking of 2.5% DSS was significantly milder in the
CD300a gene-deficient mice having no MAIR-I (.quadrature.) than in
the WT mice (.circle-solid.).
[Example 6B] Measurement of Length of Large Intestine (FIG.
16A)
[0518] The large intestine was collected from 7 WT mice and 7
CD300a gene-deficient mice on Day 6 of the administration of DSS,
and the length from the anal region to the cecum region was
measured. Statistical significance was determined by Student's
t-test (**: p<0.01).
[0519] As shown in FIG. 16A, the CD300a gene-deficient mice had
longer large-intestines, and large-intestinal atrophy due to the
DSS-induced enteritis was milder in the CD300a gene-deficient mice
than in the WT mice.
[Example 6C] Measurement of Length of Large Intestine (FIG.
16B)
[0520] Histological evaluation of the large intestine was
simultaneously carried out. As a result, it was observed that the
CD300a gene-deficient mice had milder histological changes in the
large intestine than the WT mice (FIG. 16B).
[Example 6D] Measurement of Cytotoxic Activity (FIG. 17)
[0521] Large-intestine tissues of the WT mice and the CD300a
gene-deficient mice on Day 6 of the DSS administration were stained
by HE (hematoxylin-eosin) staining, and scored from 0 to 5 (0: no
change; 1: hypertrophy and structural changes in crypts; 2:
remarkable decrease in goblet cells; 3: crypts are found, but their
structures are hardly maintained; 4: disappearance of crypts is
found, but no detachment of the epithelium is found; 5:
disappearance of crypts, detachment of the epithelium, and
remarkable cellular infiltration are found).
[0522] Each of the large intestines of 7 WT mice and 7 CD300a
gene-deficient mice was photographed with visual field enhancement
at 10 areas from the anal side, and the obtained images were
scored. Statistical significance was determined by Student's t-test
(***: p<0.001). The results are shown in FIG. 17.
[0523] As shown in FIG. 17, tissue injury due to the DSS enteritis
was mild in the CD300a gene-deficient mice.
[Example 6E] Measurement of IL-10 (FIG. 18A)
[0524] DSS or water (control group) was administered, and the large
intestine of each mouse was removed on Day 9 after the
administration of DSS. The large intestine was washed with
phosphate buffer, and cut into 3-mm.sup.3 pieces from the anal
side.
[0525] The minced tissue of the large intestine was cultured in 10%
FBS RPMI 1640 (GIBCO) for 12 hours, and the supernatant was then
collected. The collected culture supernatant was subjected to
measurement of the protein contents of cytokines (TNF-.alpha., IL-6
and IL-10) by sandwich ELISA. Statistical significance was
determined by Student's t-test (*: p<0.05).
[0526] As shown in FIG. 18, in the large-intestine tissue with
DSS-induced enteritis, the contents of IL-6 and TNF-.alpha. were
not different between the WT mice and the CD300a gene-deficient
mice. However, higher-level production of IL-10 was found in the
CD300a gene-deficient mice (FIG. 18A).
[0527] It is known that, in Mreg cells, IL-10 suppresses expression
of CD80 and CD86, which are proteins required for activation of
inflammatory T cells (Document 33 listed below).
[0528] As shown in FIG. 18A, the CD300a gene-deficient mice showed
an increased production of IL-10 at the 5% significance level.
Thus, it is thought that IL-10 suppresses growth induction of
inflammatory T cells and activation of inflammatory T cells,
thereby contributing maintenance of homeostasis of the gut immune
system.
[Example 6F] Measurement of Number of Cells of Each Type (FIG.
18B)
[0529] In mice on Day 0, Day 2, Day 4 and Day 6 after
administration of DSS, changes in the cell populations of CD4.sup.+
T cells, CD8.sup.+ T cells, B cells, macrophages and dendritic
cells were investigated.
[0530] As a result, as shown in FIG. 18B, no difference was found
in the number of cells of each cell population that have moved to
the large intestine between the WT mice and the CD300a
gene-deficient mice.
[Example 6G] Expression Analysis of CD300a (FIG. 19)
[0531] Cells isolated from the large intestine were stained with
APC-Cy7-labeled anti-CD11b (M1/70), PE-Cy7-labeled anti-CD11c
(HL3), and Biotin-anti-CD300a (TX41). The cells in the
CD11b.sup.-CD11c.sup.- fraction, CD11b.sup.+CD11c.sup.- fraction,
CD11b.sup.-CD11c.sup.+ fraction, and CD11b.sup.+CD11c.sup.+
fraction were subjected to measurement of expression of CD300a by
flow cytometry. The results are shown in FIG. 19.
[0532] As shown in FIG. 19, in the large intestinal lamina propria,
expression of CD300a was found in CD11b.sup.+CD11c.sup.+ cells,
which can be said to be special cells.
[Example 6H] Quantification of Expression in CD11b.sup.+CD11c.sup.+
Cells (FIG. 20)
[0533] The CD11b.sup.+CD11c.sup.+ cells isolated from the large
intestine of each mouse were sorted by FACSAria, and mRNA was then
extracted from the cells. cDNA was prepared from the extracted
mRNA, and the mRNA levels of IL-10, TNF-.alpha. and IL-6 were
measured by quantitative PCR.
[0534] As a result, as shown in FIG. 20, no difference in the mRNA
levels of IL-10, TNF-.alpha. and IL-6 could be found in the
CD11b.sup.+CD11c.sup.+ cells between the WT mice and the CD300a
gene-deficient mice.
[Example 61] Quantification of Expression in CD4.sup.+ T Cells
(FIG. 21)
[0535] CD4.sup.+ cells isolated from the large intestine of each
mouse after administration of DSS were sorted by FACSAria, and mRNA
was then extracted from the cells. cDNA was prepared from the
extracted mRNA, and the mRNA levels of IL-10, Foxp3, TGF-.beta.,
T-bet, GATA-3 and ROR.gamma.t were measured by quantitative PCR
using ABI 7500 fast. Statistical significance was determined by
Student's t-test (*: p<0.05, **: p<0.01).
[0536] As shown in FIG. 21, the CD300a gene-deficient mice showed
higher mRNA levels of IL-10, Foxp3 and Tgf.beta. in the CD4.sup.+ T
cells.
[0537] (Discussion)
[0538] As shown by the results of Examples 6A to 61, in mice with
DSS-induced enteritis, deficiency of MAIR-I causes production of
anti-inflammatory cytokines such as IL-10 (FIG. 21), leading to
amelioration of the disease state of enteritis.
[0539] It is thought that the above phenomenon is not due to an
increase in a certain cell population (FIG. 18B), but due to
regulation of IL-10 production via CD300a (MAIR-I) by the
CD11b-positive dendritic cells per se or certain cells influenced
by those dendritic cells.
[0540] That is, it can be predicted that suppression or inhibition
of signal transduction of CD300a (MAIR-I) via CD11b-positive
dendritic cells may lead to amelioration of the disease state of
enteritis.
[0541] <Atopic Dermatitis>
[0542] Involvement of CD300a in atopic dermatitis was investigated.
The materials and methods, and Examples are shown below.
[0543] (Experimental Animals)
[0544] Balb/c mice were purchased from Clea Japan, Inc., and kept
in the inventors' laboratory under approved breeding room
conditions. The mice used in this study were 8 CD300a (MAIR-I)
gene-deficient mice and 8 Balb/c wild-type (WT: Wild Type) mice.
During the experimental period, each mouse was provided with food
and water ad libitum, and kept under normal laboratory
conditions.
[0545] (Percutaneous Sensitization)
[0546] Each mouse was mildly anesthetized with isoflurane (Mylan,
Osaka, Japan), and the hair on the back was shaved with an electric
shaver. An area (1 cm.sup.2) on the back skin of each mouse was
subjected to at least 10 times of tape stripping using adhesive
cellophane tape.
[0547] On the gauze of Band Aid (registered trademark) tape, 100
.mu.g of ovalbumin (OVA) in 100 .mu.L of phosphate buffered saline
was placed, and the resulting tape was applied to the bodies of 5
mice in each group subjected to the tape stripping. To the
remaining 3 mice, PBS was applied with tape. The tape was once
replaced on Day 2, and OVA sensitization with the tape was carried
out every day for 1 week.
[0548] Each mouse was kept without OVA sensitization during Week 2.
During Week 3, the mouse was subjected again to OVA sensitization
in the same manner as described above. At the end of Week 3, each
mouse was sacrificed, and samples for histology and ELISA were
collected.
[0549] (Number of Times of Scratching Behavior)
[0550] The number of times of scratching behavior was counted by
careful observation of each mouse for 30 minutes at the end of each
of Week 1 to Week 3.
[0551] (Histology)
[0552] For histological observation, the skin of the OVA-sensitized
area in each mouse was collected. Each collected skin sample was
cut into small tissue blocks, and immersed in 4% paraformaldehyde
at 4.degree. C. for 24 hours.
[0553] After this fixation, a dehydration step was carried out. All
skin samples were then quickly frozen in acetone in a container
containing dry ice. The samples were stored at -30.degree. C. until
use.
[0554] The skin samples were then cut into sections with a
thickness of 4 .mu.m using a frozen section preparation apparatus,
Coldtome HM560E (manufactured by Carl Zeiss, Jena, Germany), and
placed on slide glasses precoated with New Silane III (Muto Pure
Chemicals, Co., Ltd., Tokyo, Japan).
[0555] The tissue sections were stained with hematoxylin-eosin
(manufactured by Sakura Finetek Japan), and then stained with
toluidine blue (manufactured by Santa Cruz Biotechnology,
Inc.).
[0556] Evaluation of histological finding of the tissue was
performed for the skin depth (cell layer); infiltration of
eosinophils, monocytes and mast cells; and the level of hyperplasia
of fibroblasts.
[Example 7A] Number of Times of Scratching Behavior (FIG. 22)
[0557] FIG. 22 shows the number of times of scratching behavior per
30 minutes in each mouse. Pruritus (a disease that causes itch
without causing eruption) is a common condition for atopic
dermatitis. Therefore, the evaluation was carried out by counting
the number of times of scratching behavior by careful observation
of each animal for 30 minutes during the OVA sensitization.
[0558] As shown in FIG. 22, the OVA-sensitized WT mice
(.diamond-solid.) showed a severe condition of scratching behavior
after the OVA sensitization, and a larger number of times of
scratching behavior than the OVA-sensitized CD300a gene-deficient
mice (.tangle-solidup.). The largest number of times of scratching
behavior was observed at the end of the 4th OVA sensitization.
[0559] During the OVA sensitization, the most severe symptom of
scratching behavior was observed in the WT mice (FIG. 22).
Appearance of a severe symptom of scratching behavior is one of the
most common pathological features of atopic dermatitis. Thus,
involvement of CD300a (MAIR-I)-positive cells in an important role
in atopic dermatitis might become clear.
[Example 7B] Observation (see FIG. 23)
[0560] The skin of each OVA-sensitized mouse was observed.
[0561] FIG. 23(A) shows the skin of a WT mouse that had not
undergone OVA sensitization. In untreated WT mice, which had not
been subjected to ovalbumin sensitization, cellular infiltration
was not found in the dermis, and a thin epidermis could be
observed. The lower panels in FIG. 23 show magnified views of the
rectangular areas in the upper panels. The length of each thick bar
corresponds to 10 .mu.m.
[0562] On the other hand, as shown in FIG. 23(B), the skin of the
OVA-sensitized WT mice showed hyperplasia of the epidermis and
hyperplasia of fibroblasts. Obvious infiltration of eosinophils
(see white arrowheads) and monocytes was found in the dermis of the
skin in the WT mice subjected to sensitization with ovalbumin.
[0563] As shown in FIG. 23(C), in the epidermis of the CD300a
gene-deficient mice that had not been subjected to OVA
sensitization, cellular infiltration was not found, and a thin
epidermis could be observed similarly to FIG. 23(A).
[0564] Surprisingly, as shown in FIG. 23(D), the skin of the
OVA-sensitized CD300a gene-deficient mice showed an increased
thickness of the epidermis, but did not show hyperplasia of the
epidermis. Further, the dermis showed neither cellular infiltration
nor hyperplasia of fibroblasts.
[Example 7C] Staining of Mast Cells (See FIG. 24)
[0565] As shown in FIG. 24, all mouse skin samples were subjected
to toluidine blue staining. Toluidine blue staining is a staining
method that preferentially stains intracellular granules in mast
cells.
[0566] FIG. 24(A) shows the skin of a WT mouse that had not been
subjected to OVA sensitization. FIG. 24(B) shows the skin of an
OVA-sensitized WT mouse. FIG. 24(C) shows the skin of a CD300a
gene-deficient mouse that had not been subjected to OVA
sensitization. FIG. 24(D) shows the skin of an OVA-sensitized WT
mouse. Stained mast cells are indicated by open arrowheads.
[0567] As shown in FIG. 24, after the OVA sensitization, an
increased number of mast cells were found in the skin of the WT
mouse. Moreover, the epidermis in the mouse was thicker than that
in the CD300a gene-deficient mouse.
[Example 7D] Measurement of Number of Cell Layers (FIG. 25A)
[0568] All mouse skin samples were subjected to counting of the
number of cell layers in the epidermis (see FIG. 25A). After the
OVA sensitization, the WT mice showed the largest number of cell
layers. On the other hand, in the OVA-sensitized CD300a
gene-deficient mice, the number of cell layers was less than half
of this number.
[Example 7E] Counting of Number of Eosinophils and Number of Mast
Cells (FIG. 25B)
[0569] As shown in FIG. 25B, all mouse epidermal samples were
subjected to counting of the number of eosinophils and the number
of mast cells showing infiltration into the dermis, which are
indices for atopic dermatitis. The largest cell numbers were
observed in the epidermis of the WT mice after the sensitization
with ovalbumin.
[0570] On the other hand, in the OVA-sensitized CD300a
gene-deficient mice, the increases in the number of eosinophils and
the number of mast cells were milder than those in the WT mice.
After the OVA sensitization, hyperplasia of epidermis and
hyperplasia of fibroblasts appeared most severely in the WT mice
(FIG. 23 and FIG. 25A). Hyperplasia of epidermis and fibroblasts is
another major pathological feature of atopic dermatitis.
[0571] Further, a high level of infiltration of eosinophils, mast
cells and monocytes was found in the skin of the OVA-sensitized WT
mice (FIG. 23 to FIG. 25B). Interaction between infiltrating
eosinophils and hyperplasia of fibroblasts causes secretion of
IL-31. IL-31 is an itch-inducing cytokine (Document 34 listed
below: Wong C K et al., 2012).
[0572] Therefore, after OVA sensitization, WT mice show more severe
features of atopic dermatitis than CD300a gene-deficient mice.
[0573] (Immunohistology)
[0574] In order to detect CD300a (MAIR-I) and the Langerin antigen
on serial sections of the skin, the single-step or two-step method
of enzyme immunohistochemistry was carried out. First, all sections
were rinsed 3 times with phosphate-buffered saline supplemented
with 0.05% Tween (TPBS, pH 7.4). The sections were then immersed
for 30 minutes in each of cold absolute methanol and 0.5%
H.sub.2O.sub.2.
[0575] After the washing in TPBS, the sections were subjected to
blocking treatment using the Blocking One Histo reagent (Nacalai
Tesque, Inc., Kyoto, Japan) for 10 minutes, and then washed with
TPBS. The skin sections were cultured at 4.degree. C. for 18 hours
in the presence of anti-Langerin goat IgG (Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif., US) and biotinylated
anti-CD300a (MAIR-I) rat IgG 2a.lamda. (prepared in the inventors'
laboratory), and then at room temperature for 1 hour in the
presence of biotinylated donkey anti-goat IgG as the secondary
antibody for Langerin.
[0576] Finally, these were cultured in the presence of DAB/Metal
Concentrate (Thermo Scientific, Waltham, Mass., US), and
counterstained with hematoxylin. Negative control sections were
cultured in the presence of TPBS or an isotype control antibody
instead of the primary antiserum.
[Example 7F] Langerin Immunostaining (FIG. 26)
[0577] The epidermis of each mouse was investigated to see whether
or not it is positive for Langerin, which is a dendritic cell
marker in immunostaining. Langerin is a C-type lectin expressed in
Langerhans cells, and also expressed in part of dermal dendritic
cells. Langerin is involved in recognition and incorporation of
antigens, and formation of Birbeck granules, which are responsible
for intracellular antigen delivery.
[0578] (Results)
[0579] FIG. 26(A) shows the epidermis of an untreated WT mouse
(mouse without OVA sensitization). FIG. 26(B) shows the epidermis
of an OVA-sensitized WT mouse. FIG. 26(C) shows the epidermis of an
untreated CD300a gene-deficient mouse. FIG. 26(D) shows the
epidermis of an OVA-sensitized CD300a gene-deficient mouse. The
panels a, b, c and d are magnified views of the rectangular areas
in the upper panels.
[0580] After the OVA sensitization, a significantly larger number
of Langerin-positive cells were found in the epidermis of WT mice
than in the epidermis of CD300a gene-deficient mice. The arrowheads
indicate Langerin-positive cells. Each scale bar represents 10
.mu.m.
[Example 7G] Counterstaining after Langerin Immunostaining (FIG.
27)
[0581] Immunopositivity of the Langerin antibody was evaluated by
counterstaining with toluidine blue. The skin of the WT mouse
showed an increased number of Langerin-positive cells in the
dermis. In the dermis, interaction of several Langerin-positive
cells with mast cells was found. Mast cells were stained in purple.
Each scale bar represents 10 .mu.m.
[0582] Langerhans cells and skin dendritic cells are major
antigen-presenting cells in the skin. Langerhans cells are positive
for the Langerin antigen in their cell membranes, and
Langerin-positive cells are also present among skin dendritic cells
(Document 35 listed below: Nakajima S. et al, 2012).
[0583] Skin Langerin-positive dendritic cells interact with mast
cells to activate CD4-positive T cells (Document 36 below: Otsuka
A. et al., 2011). In the OVA-sensitized model, the numbers of mast
cells (FIG. 24 and FIG. 25B) and Langerin-positive cells (see FIG.
26) largely increased in the skin of the OVA-sensitized WT mice
(see white arrowheads in FIG. 26 for comparison).
[0584] In the skin of the OVA-sensitized WT mice, interaction
between mast cells and Langerin-positive cells was found (FIG.
27).
[0585] (Discussion)
[0586] Thus, it can be deduced that atopic dermatitis more severely
appears in the skin of WT mice than the skin of CD300a
gene-deficient mice. These results suggest that CD300a (MAIR-I)
plays an important role in atopic dermatitis.
[0587] In the dermis of the WT skin, CD300a (MAIR-I)-positive cells
largely increased after the OVA sensitization (FIG. 27). This
result further confirms that CD300a (MAIR-I)-positive cells play an
important role in atopic dermatitis.
[0588] (Treatment of Atopic Dermatitis)
[0589] (Treatment with Anti-CD300a (MAIR-I) Antibody)
[0590] In the present experiment, 6 Balb/c mice of 7 weeks old were
used. According to the protocol shown in FIG. 29, 3 animals out of
the 6 animals were subjected to intravenous injection of
anti-CD300a (MAIR-I) rat IgG 2a.lamda. (TX41), and the remaining 3
animals were subjected to intravenous injection of a rat IgG
2a.lamda. control antibody (TX74). Both of these antibodies were
prepared and checked in the inventors' laboratory. "TX74" is an
isotype control antibody of TX41, and does not have a neutralizing
action as described below. Each antibody was diluted with sterile
PBS to an antibody concentration of 1600 .mu.g/mL, and 150 .mu.L of
the dilution was injected at once.
[0591] <Blocking of CD300a (MAIR-I) Antigen by Injection of
CD300a (MAIR-I) Antibody>
[0592] FIG. 29 shows the procedure to block the CD300a (MAIR-I)
antigen in a Balb/c WT mouse. Each thick line indicates the period
of OVA sensitization. The arrowheads indicate the schedule of
injection of the antibodies.
[0593] Total serum IgE was evaluated at the end of each week of
sensitization. Histological samples were collected after the
continuous sensitization. The anti-rat CD300a (MAIR-I) antibody IgG
2a.lamda. (TX41), and the control antibody (TX74), which is an
isotype thereof, were used for intravenous injection.
[0594] (ELISA)
[0595] Peripheral blood was collected from the retro-orbital cavity
using a plain glass hematocrit tube (Drummond Scientific Company,
Broomall, Pa., US), and centrifuged at 12000 rpm for 5 minutes.
[0596] Serum was collected by cutting the tube, and the whole serum
was diluted with a blocking serum before use in ELISA. The ELISA
experiment was carried out according to the standard protocol for
total IgE, recommended by BD Biosciences, California, US.
[Example 7H] Immunoreaction with Anti-CD300a Antibody (Confirmation
of Presence of Receptors) (FIG. 28)
[0597] Immunopositivity of an anti-CD300a antibody was evaluated
with a skin sample of each mouse. FIG. 28(A) shows untreated
epidermis of a WT mouse, and (B) shows OVA-sensitized epidermis of
a WT mouse.
[0598] The epidermis of WT mice after OVA sensitization showed
cells that are significantly immunopositive. The epidermis of
CD300a gene-deficient mice did not show immunopositive reaction.
The arrowheads in (B) indicate CD300a (MAIR-I)-positive cells. Each
scale bar represents 10
[Example 71] Treatment by Administration of CD300a Antibody (FIG.
30)
[0599] (ELISA)
[0600] As shown in FIG. 29, each of TX41 and TX74 was administered
to WT mice according to the above-described procedure, and the IgE
level, which is an index of atopic dermatitis, was measured.
[0601] FIG. 30 shows the total serum IgE level measured by ELISA.
The IgE level after OVA sensitization was higher in the mice to
which TX74 was injected than in the mice to which TX41 was
injected.
[Example 7J] Treatment by Administration of Anti-CD300a Antibody
(FIG. 31)
[0602] As shown in FIG. 31, after the OVA sensitization, the WT
mice to which TX74 was injected showed a more severe scratching
behavior than the WT mice to which TX41 was injected.
[Example 7K] H&E Staining (FIG. 32)
[0603] As shown in FIG. 32, skin sections of the WT mice in Example
71 were subjected to H&E staining. After the OVA sensitization,
the skin of the WT mice to which TX74 was injected showed higher
levels of hyperplasia of the epidermis and infiltration of
monocytes than the skin of the WT mice to which TX41 was injected.
In FIG. 32, the scale bar in the lower right corner of each
photograph represents 10
[Example 7L] Toluidine Blue Staining (FIG. 33)
[0604] As shown in FIG. 33, the skin of each WT mouse was subjected
to toluidine blue staining. After the OVA sensitization, the skin
of the WT mice to which TX71 was injected showed more mast cells
than the skin of the WT mice to which TX41 was injected. Each scale
bar represents 10 .mu.m, similarly to FIG. 32.
[0605] (Discussion)
[0606] The total IgE and the number of scratching behavior were
higher in the mice to which TX74 was injected than the mice to
which TX41 was injected (see FIG. 30 and FIG. 31). The epidermal
thickness, number of infiltrating cells, number of fibroblasts and
number of mast cells were also higher in the mice to which TX74 was
injected than the mice to which TX41 was injected (see FIG. 32 and
FIG. 33).
[0607] From these results, it could be further confirmed that
CD300a (MAIR-I) plays an important role in atopic dermatitis, and
the effect of TX41, which is an anti-CD300a antibody, as a
therapeutic agent for atopic dermatitis could be confirmed.
[0608] <Celiac Disease>
[0609] Celiac disease (CD) is a progressive enteritis caused by
immune response to dietary gluten protein. The adaptive immune
response specific to the gliadin peptide derived from gluten is
involved in the progression of celiac disease. The innate immune
response as the fundamental cause of the disease has not been
completely elucidated.
[0610] Here, we demonstrate that CD300a (MAIR-I), which is
expressed in lamina propria macrophages (macrophages in the lamina
propria) and is a member of the bone marrow-associated
immunoglobulin-like receptor family, plays a regulatory role in the
progression of dietary gluten-induced intestinal diseases.
[0611] CD300a gene-deficient mice, which lack CD300a (MAIR-I), fed
with a high-gluten diet showed celiac disease-like symptoms.
Compared to wild-type (WT) mice fed with a high-gluten diet, each
CD300a gene-deficient mouse showed a mild increase in the body
weight, high clinical score, large amount of transglutaminase 2,
and accumulation of lamina propria macrophages in the jejunum.
[0612] Compared to the WT mice fed with a high-gluten diet, lamina
propria macrophages of each CD300a gene-deficient mouse fed with a
high-gluten diet showed higher expression of IL-6, IL-15,
TNF-.alpha., IFN-.beta., MCP1 and MCP5.
[0613] After in vitro stimulation with the toxic gliadin peptide
(P31-43), lamina propria macrophages of each CD300a gene-deficient
mouse showed higher-level expression of the above-described
cytokines.
[0614] Enhanced expression of IL-6, TNF-.alpha. and IFN-.beta.
occurred in lamina propria macrophages of CD300a gene-deficient
mice, which lack CD300a (MAIR-I) and have a MyD88-deficient and
TRIF-deficient genetic background.
[0615] Further, blocking of binding of phosphatidyl serine, which
is the ligand of CD300a (MAIR-I) on apoptotic cells, to CD300a
(MAIR-I) promoted production of cytokines in lamina propria
macrophages of WT mice.
[0616] In summary, these results indicate that the interaction
between macrophages in the intestinal lamina propria and CD300a
(MAIR-I) on apoptotic cells plays a protective role in progression
of celiac disease, by MyD88- and TRIF-mediated inhibitory gliadin
signaling pathways.
[0617] (Materials and Methods)
[0618] (Mice)
[0619] Male Balb/c wild-type (WT) mice of 9 to 14 weeks old, and
littermates of Balb/c CD300a gene-deficient mice, which do not have
CD300a (MAIR-I), were provided. These mice were used in an
experiment in which they were fed with a normal diet (ND),
high-gluten diet (HGD) or gluten-free diet (GFD).
[0620] For in vitro experiments, Balb/c WT mice; and CD300a
gene-deficient Balb/c or C57BL/6 B6 mice, which have no CD300a
(MAIR-I); were used. These mice had the same sex and age.
MyD88-gene deficient B6 mice and TRIF gene-deficient B6 mice were
purchased from OrientalBio Service, Inc. (Kyoto, Japan). All mice
were kept under specific pathogen-free conditions.
[0621] (Feeding Test)
[0622] WT mice and CD300a (MAIR-I) mice were kept with normal
powder diet containing less than 2% gluten (MF, Oriental Yeast Co.,
Ltd., Tokyo, Japan) (ND). WT mice and CD300a gene-deficient mice
were kept with a high-gluten diet (HGD), which is the same as MF
except that gluten is contained at 30% (Sigma-Aldrich, St. Louis,
Mo.). In several experiments, mice were fed with pellets of ND (MF)
or a gluten-free diet (GFD; AIN-76A, Research Diets, Inc., New
Brunswick, N.J.).
[0623] (Depletion of Microbiota)
[0624] Depletion of the microbiota was carried out as in the
[Document 37 listed below]. Depletion of the microbiota was
confirmed by observing colony forming units in feces from each
mouse using an agarose plate containing brain-heart infusion
medium.
[0625] (Histopathological Analysis)
[0626] Histopathological changes in mice were observed in the
later-described specific weeks after feeding with ND, HGD or GFD.
Samples of the jejunum and colon were isolated and fixed with
formalin, followed by staining with hematoxylin-eosin.
[0627] The standard for the clinical score for the jejunum was
defined as in the [Document 38 listed below]. Briefly, the score of
the ratio between crypts and villi was calculated according to the
average depths of crypts and villi (score 0 to 3). Further, the
score (score 0 to 3) of infiltration of monocytes was calculated
according to the average diameters of the lamina propria of villi,
and crypts. The final clinical score was represented as the total
of 0 to 6.
[0628] The number of intraepithelial lymphocytes in intestinal
epithelial cells (IECs) of the jejunum was counted, and represented
as the IEL number per 100 intestinal epithelial cells ([Document 39
listed below]). Quantification of transglutaminase 2 (TG2) in the
jejunum was carried out using a tissue suspension of the jejunum.
The test was carried out using TG2-CovTest (Zedira, Darmstadt,
Germany) according to the manufacturer's instructions.
[0629] (Titration of IgG and IgA Antibodies Against Gliadin)
[0630] The serum antibody titers of IgG and IgA against gliadin in
mice were determined by an enzyme immunoassay. This was carried out
by the method described in the [Document 40 listed below] with some
modifications.
[0631] An anti-mouse IgG antibody labeled with horseradish
peroxidase (HRP) (GE Healthcare, Little Chalfont, UK), and an
anti-mouse IgA antibody labeled with HRP (Southern Biotech,
Birmingham, Ala.) were used as anti-gliadin IgG and IgA detection
antibodies, respectively. OPD Reagent (Sigma-Aldrich) was used as
the substrate of HRP in colorimetric analysis.
[0632] (Isolation of Lamina Propria Macrophages and Dendritic
Cells)
[0633] Lamina propria macrophages (LP M.phi.) and dendritic cells
(DCs) were isolated according to the [Document 41 listed below]
with some modifications.
[0634] After removal of the mesentery and Peyer patches, the
jejunum was cut into small pieces. The pieces were washed a total
of 3 times with PBS buffer supplemented with 2 mM EDTA (Sigma
Aldrich) and 20% FCS, with shaking at 37.degree. C. for 15
minutes.
[0635] The remaining tissue was homogenized, and then digested in
PBS buffer supplemented with 1.5 mg/mL type VIII collagenase
(Sigma-Aldrich) and 20% FCS, with shaking at 37.degree. C. for 20
minutes.
[0636] Lamina propria macrophages and lamina propria dendritic
cells expressing CD45 in the tissue suspension were concentrated
using biotinylated anti-mouse CD45 (30-F11, BD Biosciences,
Franklin Lakes, N.J.) and streptavidin particles plus IMAG (BD
Biosciences).
[0637] The CD45-expressing cells in the lamina propria were stained
with a fluorescein isothiocyanate-conjugated anti-mouse CD11b
(M1/70), phycoerythrin-conjugated CD11c (HL3), propidium iodide,
Alexa 647-labeled TX41 (anti-mouse CD300a (anti-mouse MAIR-I), rat
IgG2a) or Alexa 647-labeled TX74 (anti-FLAG, isotype control for
TX41), biotinylated anti-mouseCD45, and streptavidin
allophycoerythrin Cy7 (allophycoerythrin-Cy7).
[0638] All fluorescently labeled antibodies and streptavidin
allophycoerythrin-Cy7 were purchased from BD Biosciences.
[0639] CD11b.sup.+CD11c.sup.low cells and CD11c.sup.+ cells gated
based on the number of CD45.sup.+PI.sup.- cells were isolated as
lamina propria macrophages and lamina propria dendritic cells using
FACSAria (BD Biosciences).
[0640] An important principle of flow cytometry data analysis is to
selectively visualize the particles of interest while removing
unnecessary particles (dead cells, residues and the like). This
operation is called gating (gate).
[0641] (Flow Cytometry Analysis)
[0642] Macrophages, CD11b.sup.+ dendritic cells and CD11b.sup.-
dendritic cells among the cells of lamina propria, which are
CD45.sup.+PI.sup.-, were gated based on the numbers of
CD11b.sup.+CD11c.sup.low, CD11b.sup.+CD11c.sup.+ and
CD11b.sup.-CD11c.sup.+, respectively.
[0643] Intraepithelial lymphocytes and intestinal epithelial cells
were obtained by washing small pieces of jejunum with PBS buffer
supplemented with 2 mM EDTA and 20% FCS. The intraepithelial
lymphocytes and intestinal epithelial cells contained in the
suspension were gated based on the number of CD45.sup.+PI.sup.-
cells and the number of CD45.sup.-PI.sup.- cells.
[0644] Expression of CD300a (MAIR-I) in macrophages, CD11b.sup.+
dendritic cells and CD11b.sup.- dendritic cells in the lamina
propria, intraepithelial lymphocytes, and intestinal epithelial
cells were analyzed with Alexa 647-labeled TX41 (anti-mouse MAIR-I,
rat IgG2a) or Alexa 647-labeled TX74 (anti-FLAG of TX41, isotype
control).
[0645] Apoptotic cells presenting phosphatidyl serine (PS) were
analyzed using allophycocyanin-conjugated annexin V. Flow cytometry
analysis was carried out using FACSAria (BD Biosciences).
[0646] (Quantitative RT-PCR Method)
[0647] In Isogen LS (Nippon Gene, Tokyo, Japan), 10000 to 20000
lamina propria macrophages and lamina propria dendritic cells that
were stimulated with or not stimulated with the toxic gliadin
peptide P31-43 were resuspended, and total RNA was isolated from
the resulting suspension according to the manufacturer's
instructions.
[0648] Single-stranded DNA was synthesized from total RNA using a
cDNA reverse transcription kit (Applied Biosystems, Foster City,
Calif.). The primer sets used for quantitative reverse
transcriptase-mediated polymerase chain reaction (Q-RT-PCR) were
designed by PrimerBank (http://pga.mgh.harvard.edu/primerbank/)
[Document 42 listed below].
[0649] Q-RT-PCR was carried out using Platinum SYBR Green Super Mix
UDG (Invitrogen, Carlsbad, Calif.) and ABI 7500 Fast (Applied
Biosystems). The data were analyzed by the DDCT (delta delta CT)
method.
[0650] Control samples expressing all genes tested in this study
were prepared from spleen cells stimulated with lipopolysaccharide
(1000 ng/mL, 6 hours), and were single-stranded DNA. The results
were shown based on comparative quantification with samples showing
expression of the respective genes.
[0651] (Gliadin Stimulation)
[0652] The toxic .alpha.-gliadin peptide P31-43 derived from gluten
[Document 43 listed below] and the ovalbumin peptide P323-339 as a
control were synthesized by Operon Biotechnologies (Huntsville,
Ala.).
[0653] The purities of these peptides were not less than 95%, and
the endotoxin unit/mL was less than 0.001. This was confirmed with
Limulus Color KY Test Wako (Wako Pure Chemical Industries, Ltd.,
Osaka).
[0654] Macrophages in thioglycolate-induced peritoneal exudate
cells (PECs) prepared from a B6 mouse were stimulated using 100
.mu.g/mL gliadin peptide P31-43 or ovalbumin P323-339 as described
in the [Document 44 listed below].
[0655] In wells of a 96-well flat-bottom plate, 10000 to 20000
lamina propria macrophages and CD11b.sup.+ dendritic cells derived
from WT mice or CD300a gene-deficient mice were cultured in RPMI
1640 medium supplemented with 10% FCS,
glutamine/streptomycin/penicillin, HEPES and non-essential amino
acids.
[0656] Lamina propria macrophages of Balb/c mice or B6 mice were
stimulated for 10 hours or 3 hours with 100 .mu.g/mL gliadin
peptide P31-43. In several experiments, the lamina propria
macrophages were treated or not treated with MFG-E8 (final
concentration, 5 .mu.g/mL) of a recombinant mouse at 4.degree. C.
for 30 minutes, and further, stimulated with the gliadin peptide
P31-43 at 37.degree. C. for 3 hours.
[0657] (Statistical Analyses)
[0658] All statistical analyses were carried out using the
Mann-Whitney U test. Statistical significance was judged at
P<0.05.
[Example 8A] Body Weight (BW) Change (FIG. 34)
[0659] In order to investigate whether or not CD300a (MAIR-I) is
involved in the progression of celiac disease (CD), CD300a
gene-deficient Balb/c mice or wild-type (WT) mice were kept with a
normal diet containing less than 2% gluten (ND) or a high-gluten
diet containing 30% gluten (HGD). After starting feeding with the
normal diet or high-gluten diet, changes in the body weight (BW)
were monitored.
[0660] ".smallcircle." represents "WT mice, n=7"; ".circle-solid."
represents "CD300a gene-deficient mice, normal diet, n=9";
".quadrature." represents "WT mice, high-gluten diet, n=10"; and
".box-solid." represents "CD300a gene-deficient mice, high-gluten
diet, n=14".
[0661] The CD300a gene-deficient mice kept with the high-gluten
diet (HGD) showed a significantly smaller degree of increase in the
body weight (BW) than the WT mice kept with the high-gluten diet
(HGD) (FIG. 34). The CD300a gene-deficient mice were confirmed to
show progression of celiac disease-like enteropathy after feeding
with the high-gluten diet. These results suggest that CD300a
(MAIR-I) plays a protective role in the progression of the
intestinal disease in the jejunum after feeding with a high-gluten
diet.
[Example 8B] H&E Staining (FIG. 35)
[0662] Histopathological analysis of the intestines of mice at Week
20 after feeding with a normal diet or a high-gluten diet (HGD) was
carried out. The jejunum of each mouse was fixed with formalin,
followed by staining with hematoxylin-eosin. FIG. 35 shows a
representative result obtained for a mouse in each group.
[0663] The CD300a gene-deficient mice kept with a high-gluten diet
(HGD) showed a more advanced intestinal disease in the jejunum
compared to the WT mice kept with a high-gluten diet (HGD) (see
FIG. 35)
[Example 8C] Number of Intraepithelial Lymphocytes (FIG. 36, FIG.
37)
[0664] According to the above-described method, the clinical score
and the number of intraepithelial lymphocytes (IELs) per 100
intestinal epithelial cells (IECs) in the jejunum of each mouse
were counted at Week 20 after feeding with the normal diet or the
high-gluten diet. "* and **" represent p<0.05 and p<0.01,
respectively.
[0665] As described above, the CD300a gene-deficient mice showed
atrophy of villi in the jejunum (FIG. 35), and significantly higher
clinical scores (see FIG. 36). Further, an increase in
intraepithelial lymphocytes (IELs) in small-intestinal epithelial
cells (IECs) was found as compared to the CD300a gene-deficient
mice kept with FD as well as the WT mice kept with a high-gluten
diet (HGD) (see FIG. 37).
[Example 8D] TG2-CovTest (Colorimetric Analysis for Quantitation of
Specific TG2 Cross-Linking Activity) (FIG. 38)
[0666] The level of transglutaminase 2 (TG2) is known to be
influenced by intestinal inflammation. TG2 is a key enzyme for the
progression of celiac disease. WT mice and CD300a gene-deficient
mice were fed with a normal diet or high-gluten diet for at least
20 weeks, and the level of transglutaminase 2 (TG2) in a jejunum
suspension of each mouse was quantified with TG2-CovTest.
[0667] As a result, the TG2 level in the intestine was highest in
the CD300a gene-deficient mice kept with a high-gluten diet (HGD)
among the groups of mice (see FIG. 38).
[Example 8E] Flow Cytometry (FIG. 39)
[0668] Expression of CD11b and CD11c in lamina propria cells gated
for the CD45.sup.+PI.sup.- cell population was analyzed by flow
cytometry. Lamina propria (LP) cells of WT mice or CD300a
gene-deficient mice of the Balb/c strain fed with the normal diet
or high-gluten diet were analyzed by flow cytometry.
CD45-expressing LP cells were concentrated using the I-Mag
technology (cell separation system using magnetic beads).
[0669] CD11b.sup.+CD11c.sup.low cells are lamina propria
macrophages, and CD11b.sup.+CD11c.sup.+ and CD11b.sup.-CD11c.sup.+
are lamina propria (LP) dendritic cells (DCs).
[Example 8F] Quantitative and Qualitative Changes in Immunocytes
(FIG. 40)
[0670] In each of WT mice and CD300a gene-deficient mice, the
frequency of lamina propria (LP) macrophages (open bar), the
frequency of lamina propria (LP) CD11b.sup.+ dendritic cells
(closed bar), and the frequency of lamina propria (LP) CD11b.sup.-
dendritic cells (shaded bar) were measured.
[0671] That is, quantitative and qualitative changes in the
immunocytes in the lamina propria of the jejunum of WT mice and
CD300a gene-deficient mice kept with a normal diet (ND) or
high-gluten diet (HGD) were investigated.
[0672] The frequency of lamina propria macrophages expressing
CD11b.sup.+CD11c.sup.low (lp Mf) was significantly higher in the
CD300a gene-deficient mice kept with a high-gluten diet (HGD) than
in the WT mice kept with a high-gluten diet (HGD) or in the CD300a
gene-deficient mice kept with a normal diet (ND) (see and compare
the cell populations 5.8 in the lower right panel, 2.3 in the lower
left panel, and 2.2 in the upper right panel of FIG. 39; and see
FIG. 40).
[0673] Lamina propria macrophages in the intestine are cells
differentiated from inflammatory monocytes expressing CCR2 in
peripheral blood [Document 45 listed below].
[0674] In view of the fact that CCR2 is a receptor of MCP1 and
MCP5, the increased expression of these chemokines in lamina
propria macrophages after stimulation with gliadin is consistent
with the high frequency of lamina propria macrophages in the CD300a
gene-deficient mice kept with a high-gluten diet (HGD) (see FIG.
40).
[Example 8H] Gene Expression Levels of Cytokines and Chemokines
(FIG. 41)
[0675] The gene expression levels of cytokines and chemokines in
lamina propria (LP) macrophages isolated from each mouse were
analyzed by quantitative RT-PCR. In FIG. 41, the expression level
of each gene is expressed as the relative level with respect to the
level in WT mice fed with a normal diet, which is taken as "1" to
be used as a standard.
[0676] Lamina propria macrophages in the CD300a gene-deficient mice
kept with a high-gluten diet (HGD) showed significantly higher
levels of inflammatory cytokines (IL-6, IL-15, TNF-.alpha., IFN-b
and MCP1) and some chemokines such as MCP5 compared to the WT mice
kept with a high-gluten diet (HGD) (see FIG. 41).
[0677] The enhanced expression of inflammatory cytokines and
chemokines from lamina propria macrophages in the CD300a
gene-deficient mice kept with a high-gluten diet (HGD) is thought
to be due to the intestinal disease observed in the mice.
[0678] Further, interestingly, lamina propria macrophages in the
CD300a gene-deficient mice kept with a normal diet (ND) showed
higher expression of IL-15 and p19 compared to the WT mice kept
with ND (FIG. 48).
[Example 81] (FIG. 42)
[0679] Expression of CD300a (MAIR-I) in lamina propria macrophages,
CD11b.sup.+ dendritic cells, CD11b.sup.- dendritic cells,
intestinal epithelial cells and intraepithelial lymphocytes was
analyzed by flow cytometry.
[0680] CD300a (MAIR-I) was detected using TX41 (anti-MAIR-I mouse
monoclonal antibody). Lamina propria macrophages (M.phi.),
CD11b.sup.+ dendritic cells and CD11b dendritic cells were gated
based on the number of CD45.sup.+PI.sup.- lamina propria cells.
[0681] Intraepithelial lymphocytes (IELs) and intestinal epithelial
cells (IECs) were gated into the CD3.sup.+CD45.sup.+PI.sup.- and
CD45.sup.-PI.sup.- fractions, respectively.
[0682] CD300a (MAIR-I) is expressed in most of bone marrow cells
including macrophages, granulocytes, mast cells and dendritic cells
([Document 46 listed below]).
[0683] As described above, the present inventors analyzed
expression of CD300a (MAIR-I) in macrophages on the lamina propria,
CD11b.sup.+ inflammatory dendritic cells in the lamina propria,
CD11b.sup.- tolerogenic dendritic cells, intestinal epithelial
cells (IELs) and intraepithelial lymphocytes (IECs), and lamina
propria macrophages, and further investigated the CD300a
(MAIR-I)-expressing subpopulations of lamina propria CD11b.sup.+
dendritic cells and lamina propria CD11b.sup.- dendritic cells. As
a result, no expression of CD300a (MAIR-I) was found in the
intraepithelial lymphocytes (IELs) and intraepithelial lymphocytes
(IECs) (FIG. 42).
[Example 8J] (FIG. 43)
[0684] Although the receptor of the gliadin peptide has not been
identified, earlier studies reported that the gliadin P31-43
peptide accumulates in early endosomes and inhibits their
maturation (Documents 47 and 48 listed below).
[0685] Lamina propria (LP) CD11b.sup.+ dendritic cells were
isolated from Balb/c WT mice or CD300a gene-deficient mice, and
stimulated in vitro with 100 mg/mL toxic gliadin peptide P31-43 for
10 hours.
[0686] The gene expression levels of cytokines and chemokines
(IL-6, IL-15, TNF-.alpha., IFN-.beta., MCP1 and MCP5) in lamina
propria (LP) CD11b.sup.+ dendritic cells in the WT mice and the
CD300a gene-deficient mice after the gliadin stimulation were
analyzed by quantitative RT-PCR. The expression level of each gene
is expressed as the relative level with respect to the level in
untreated WT mice, which is taken as "1" to be used as a
standard.
[0687] As a result of the investigation of the gene expression
levels, the expression levels of IL-6, IL-15, TNF-.alpha.,
IFN-.beta., MCP1 and MCP5 after stimulation with the gliadin
peptide P31-43 were significantly higher in lamina propria
macrophages of CD300a gene-deficient mice than in lamina propria
macrophages of WT mice (FIG. 43).
[0688] Inflammatory lamina propria CD11b.sup.+ dendritic cells of
CD300a gene-deficient mice kept with a normal diet (ND) showed
higher expression of IL-15 as compared to WT mice kept with a
normal diet (ND).
[Example 8K] Suppression of Gliadin-Induced Expression of Cytokines
(FIG. 44)
[0689] The present inventors tested whethe
References