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Inhibitor screening method and atopic dermatitis like symptom inducing method which utilizes induction of production of interleukin 18 by keratinocyte and utilization of same

外国特許コード F110003339
整理番号 A211-03US2
掲載日 2011年6月23日
出願国 アメリカ合衆国
出願番号 49923109
公報番号 20100003194
公報番号 8293212
出願日 平成21年7月8日(2009.7.8)
公報発行日 平成22年1月7日(2010.1.7)
公報発行日 平成24年10月23日(2012.10.23)
国際出願番号 JP2004005747
国際公開番号 WO2004104578
国際出願日 平成16年4月21日(2004.4.21)
国際公開日 平成16年12月2日(2004.12.2)
優先権データ
  • 特願2003-120630 (2003.4.24) JP
  • 10/554,301 (2004.4.21) US
  • 2004JP005747 (2004.4.21) WO
発明の名称 (英語) Inhibitor screening method and atopic dermatitis like symptom inducing method which utilizes induction of production of interleukin 18 by keratinocyte and utilization of same
発明の概要(英語) The present invention provides methods which use induction phenomenon of production of interleukin 18 (IL-18) from keratinocyte (KC), and their usages.
The methods are preferably applicable for understanding of pathogenic mechanisms of atopic dermatitis (AD) and AD-like symptoms, and for development of therapeutic drugs for AD and AD-like symptoms.
For example, by applying, on skin of mice or the like, protein A (SpA) derived from Staphylococcus aureus, or transplanting, on mice, a skin graft which has developed an inflammatory skin disease like AD, it is possible to reproduce elevation of IgE to high level in serum, which elevation is generated in an AD-like lesion.
As a result, it is possible, for example, to screen for an inhibitor which inhibits induction of production of IL-18 from KC.
従来技術、競合技術の概要(英語) BACKGROUND ART
Skin is the largest organ in a body, and a forefront for defending a living organism.
Epidermis consists of keratinocytes (KC), melanocytes, epidermis Langerhans cells (LC), intraepithelial T cells, and the like.
LC are immature dendritic cells (DC), whose function is to capture and transport locally exposed protein antigen to draining lymph nodes in which acquired immune responses are generally accomplished.
During their migration to a lymph node, LC develop into mature DC having antigen-presenting capacity.
As a result, a systemic immune response specific to the antigen carried by the LC/DC is caused.
In this way, the antigen-specific immune response in the skin is closely associated with the systemic immune response to the same antigen.
Therefore, it is thought that skin and immune organs are tightly connected with each other via circulatory trooping of LC/DC and via antigen specific immune cells.
On the other hand, KC and melanocytes reside in the skin, and do not principally participate in an acquired immune response.
However, KC might contribute to development of local innate immune response and local inflammation.
When microbes infect skin, a host develops an inflammation reaction, and then develops an acquired immune reaction in a manner localized in the skin.
At that time, KC and LC constituting the skin are closely involved in each reaction.
Therefore, it is thought that, based on their unique property of producing various cytokines upon stimulation from microbes or chemical reagents, KC has a large influence on LC, with a result that KC modify an acquired immune response (see Non-Patent Documents 1 and 2).
In consideration of these facts, it is important to determine whether KC-induced cutaneous inflammation can also affect the systemic immune response.
The inventor of the present invention have established caspase-1 transgenic mice (KCASP1Tg mice) which KC-specifically express caspase-1, and develop atopic dermatitis (AD)-like inflammatory skin lesions (pruritic chronic inflammation) in an interleukin 18 (IL-18)- and interleukin 1beta (IL-1beta )-dependent manner (see Non-Patent Documents 3 and 4).
Further, the inventor of the present invention has disclosed that IL-1beta enhances the capacity of IL-18 to induce AD-like inflammatory skin lesions (see Non-Patent Document 4).
These results suggest that KC may also contribute to the systemic immune response by producing a number of cytokines including IL-18 and IL-1beta .
The IL-18 and IL-1beta are produced as biologically inert precursors, and are released as an active form after being cleaved by appropriate intracellular enzymes such as caspase-1 (see Non-Patent Documents 5 through 9).
The IL-18 has diverse biological actions depending on the kinds of cytokines coexisting with the IL-18.
Particularly, in the presence of interleukin 12 (IL-12), IL-18 promotes inflammatory responses via induction of IFN-gamma which is a potent pro-inflammatory cytokine (see Non-Patent Document 10).
On the other hand, in the absence of IL-12, IL-18 induces atopic response via induction of production of Th2 cytokine (see Non-Patent Document 11 through 14).
The AD is an inflammatory skin lesion in response to external stimulation, and is accompanied by chronic and repetitive strong itch.
The onset of the AD has a genetic basis, and a patient of the AD has high serum levels of IgE.
However, the pathogenic mechanism of the AD is poorly understood.
Activated T cells, basophil, and mast cells are closely involved in the pathogenic mechanism of the AD.
Particularly, it is thought that as a result of activation of mast cells or basophil by an allergen, Th2 cytokines and chemical mediators are produced, and accordingly AD is developed.
The activation of mast cells or basophil by allergen is caused by crosslinking of IgE molecule bound to Fc epsilon R (Fc receptor (FcR) to IgE antibody of basophil) on these cells.
Important ones out of the Th 2 cytokines are, for example, interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 9 (IL-9), and interleukin 13 (IL-13).
Important ones out of the chemical mediators are, for example, histamine, serotonin, and leukotriene.
It is known that infection of Staphylococcus aureus exacerbates inflammation of skin of an AD patient according to environments (see Non-Patent Documents 15 and 16), and increases density of serum IL-18 of some AD patients (see Non-Patent Document 17).
Namely, it is known that the infection of Staphylococcus aureus is an inducing factor or exacerbation factor of AD.
However, it is poorly known how Staphylococcus aureus is involved in the onset of AD.
[Non-Patent Document 1]
Jamora, C. and Fuchs, E. 2002. Intercellular adhesion, signaling and the cytoskeleton.
Nat. Cell Biol. 4:E101
[Non-Patent Document 2]
Grone, A. 2002. Keratinocyte and cytokine.
Vet. Immunol. Immunopathol. 88:1.
[Non-Patent Document 3]
Yamanaka, K., Tanaka, M., Tsutsui, H., Kupper, T. S., Asahi, K., Okamura, H., Nakanishi, K., Suzuki, M., Kayagaki, N., Black, R. A., et al. 2000. Skin-specific caspase-1 transgenic mice show cutaneous apoptosis and pre-endotoxin shock condition with a high serum level of IL-18. J. Immunol. 165:997.
[Non-Patent Document 4]
Konishi, H., Tsutsui, H., Murakami, T., Yumikura-Futatsugi, S., Yamanaka, K., Tanaka, M., Iwakura, Y., Suzuki, N., Takeda, K., Akira, S., Nakanishi, K., and Mizutani, H. 2002. IL-18 contributes to the spontaneous development of an atopic dermatitis-like inflammatory skin lesion independently of IgE/stat6 under specific pathogen-free conditions.
Proc. Natl. Acad. Sci. USA. 99:11340.
[Non-Patent Document 5]
Gu, Y., Kuida, K., Tsutsui, H., Ku, G., Hsiao, K., Fleming, M. A., Hayashi, N., Higashino, K., Okamura, H., Nakanishi, K., et al. 1997. Activation of interferon-gamma inducing factor mediate by interleukin-1beta converting enzyme.
Science 275:206.
[Non-Patent Document 6]
Tsutsui, H., Kayagaki, N., Kuida, K., Nakano, H., Hayashi, N., Takeda, K., Matsui. K., Kashiwamura, S., Hada, T., Akira, S., et al. 1999. Caspase-1-independent, Fas/Fas ligand-mediated IL-18 secretion from macrophages causes acute liver injury in mice.
Immunity 11:359.
[Non-Patent Document 7]
Seki, E., Tsutsui, H., Nakano, H., Tsuji, N. M., Hoshino, K., Adachi, O., Adachi, K., Futatsugi, S., Kuida, K., Takeuchi, O., et al. 2001. LPS-induced IL-18 secretion from murine Kupffer cells independently of IL-12 and IL-1beta . J. Immunol. 169:3863.
[Non-Patent Document 8]
Dinarello, C. A. 1998. Interleukin-1beta , interleukin-18, and the interleukin-1beta converting enzyme.
Ann. NY Acad. Sci. 856:1
[Non-Patent Document 9]
Fantuzzi, G. and Dinarello, C. A. 1999. Interleukin-18 and interleukin-1beta : two cytokine substrates for ICE (caspase-1). J. Clin. Immunol. 19:1.
[Non-Patent Document 10]
Okamura, H., Tsutsui, H., Komatsu, T., Yutsudo, M., Hakura, A., Tanimoto, T., Torigoe, K., Okura, T., Nukada, Y., Hattori, K., Akita, H., Namba, M., Tanabe, F., Konishi, K., Fukada, S., and Kurimoto, M. 1995 Cloning of a new cytokine that induces INF-gamma production by T cells.
Nature 378:88.
[Non-Patent Document 11]
Yoshimoto, T., Tsutsui, H., Tominaga, K., Hoshino, K., Okamura, H, Akira, S., Paul, W. E. and Nakanishi, K. 1999. IL-18, although anti-allergic when administered with IL-12, stimulates IL-4 and histamine release by basophils.
Proc. Natl. Acad. Sci. USA 96:13962.
[Non-Patent Document 12]
Yoshimoto, T., Mizutani, H., Tsutsui, H., Noben-Trauth, N., Yamanaka, K., Tanaka, M., Izumi, S., Okamura, H., Paul, W. E. and Nakanishi, K. 2000. IL-18 induction of IgE: Dependence on CD4+ T cells, IL-4 and STAT6.
Nat. Immunol. 1:132.
[Non-Patent Document 13]
Hoshino, T., Yagita, H., Wiltrout, R. H. and Young, H. A. 2000. In vivo administration of IL-18 can induce IgE production through Th2 cytokine induction and up-regulation of CD40 ligand (CD154) expression on CD4+ T cells.
Eur. J. Immunol. 30:1998.
[Non-Patent Document 14]
Nakanishi, K., Yoshimoto, T., Tsutsui, H. and Okamura, H., 2001. Interleukin-18 regulates both Th1 and Th2 responses.
Annu. Rev. Immunol. 19:423.
[Non-Patent Document 15]
Thestrup-Pedersen, K. 2000. Clinical aspects of atopic dermatitis.
Clin. Exp. Dermatol. 25:535.
[Non-Patent Document 16]
Wollenberg, A., Kraft, S., Oppel, T. and Bieber, T. 2000. Atopic dermatitis: pathogenetic mechanisms.
Clin. Exp. Dematol. 25:530.
[Non-Patent Document 17]
Tanaka, T., Tsutsui, H., Yoshimoto, T., Kotani, M., Masumoto, M., Fujita, A., Wang, W., Higa, S., Kishimoto, T., Nakanishi, K., et al. 2001. Interleukin-18 is elevated in the sera from patients with atopic dermatitis and from atopic dermatitis model mice, NC/Nga.
Int. Arch. Allergy Immunol. 125:236.
At present, the pathogenic mechanism of AD is poorly understood, and therefore it is very difficult to develop an effective therapeutic drug for AD.
As described above, it is known that cells constituting epidermis are involved in a variety of immune responses.
For example, it is recently reported that dendritic cells induce an acquired immune response as antigen presenting cells.
However, as for KC which are the most main cells out of cells constituting epidermis, it is not clear how KC are involved in immune response of a host.
In developing an effective therapeutic drug for a given disease, it is one of important methods to understand the pathogenic mechanism of the disease, and to perform, by use of the mechanism, screening of a substance having a pharmacological effect.
However, as for the onset of AD, there are a lot of unsolved points including involvement of KC and infection of Staphylococcus aureus.
Therefore, a technique (including screening) for applying them to the development of a therapeutic drug for AD is rarely known.
The present invention is made in view of the foregoing problems, and its object is to provide, by means of an induction phenomenon of generation of IL-18 from KC, a variety of methods preferably applicable to solution of the pathogenic mechanisms of atopic dermatitis and atopic dermatitis-like symptoms, and for development of a therapeutic drug for atopic dermatitis and atopic dermatitis-like symptoms, and usages of the methods.

特許請求の範囲(英語) [claim1]
1. A method for screening for an inhibitor which inhibits induction of production of interleukin 18 from keratinocytes, said method comprising: a step for inducing the production of interleukin 18 from keratinocytes, by applying a stimulator on the skin of a normal living organism provided as a host thereby producing an interleukin 18 mediated inflammation;
and an inhibitor identifying step for applying a candidate-substance on the inflammatory part of the living organism to which the stimulator has been applied, or orally administering the candidate-substance to the living organism to which the stimulator has been applied, and identifying, as the inhibitor, a substance which inhibits the induction of the production of interleukin 18 from the keratinocytes,
the stimulator being selected from at least one of (i) protein A derived from Staphylococcus aureus or (ii) a mutant of the protein A, the mutant being capable of stimulating keratinocyte being used, and
the mutant being a protein which (i) has an amino acid sequence with amino acids, the number of which is not less than 1 but not more than 5, replaced, deleted, inserted, and/or added, and (ii) negatively controls expression of B7-2 molecule of a surface of cells.
[claim2]
2. The method as set forth in claim 1, wherein, as the stimulator, SDS as well as protein A is used.
[claim3]
3. The method as set forth in claim 1, wherein the living organism provided as the host is a mouse.
[claim4]
4. The method as set forth in claim 1, wherein the stimulator is the protein A derived from Staphylococcus aureus.
[claim5]
5. A method for screening for an inhibitor which inhibits induction of production of interleukin 18 from keratinocytes, said method comprising:
a step for inducing the production of interleukin 18 from keratinocytes by using, as the stimulator, a skin graft on which an inflammatory skin lesion like atopic dermatitis is developed, and
transplanting the skin graft onto the skin of a living organism provided as a host thereby producing an interleukin 18 mediated inflammation;
and an inhibitor identifying step for applying a candidate-substance on the inflammatory part of the living organism provided as the host, or orally administering the candidate-substance to the living organism provided as the host, and identifying, as the inhibitor, a substance which inhibits the induction of the production of interleukin 18 from the keratinocytes, the living organism provided as the host being at least such that (i) CD4+T cells normally exist, (ii) stat6 is expressed, and (iii) NKT cells constitutively express IL-18Ralpha chain.
[claim6]
6. The method as set forth in claim 5, wherein the living organism provided as the host is a mouse.
  • 発明者/出願人(英語)
  • NAKANISHI KENJI
  • MIZUTANI HITOSHI
  • TSUTSUI HIROKO
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
国際特許分類(IPC)
米国特許分類/主・副
  • 424/9.2
  • 435/7.21
参考情報 (研究プロジェクト等) CREST Protein Structure and Functional Mechanisms -Toward Creation of Innovative Medicines, Diagnosis, and Material Production Based on Functional Mechanisms of Proteins- AREA
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