Pemphigus vulgaris (PV) is an autoimmune disease with a possible fatality of the skin and mucosae which is induced by an antibody against desmoglein 3 (Dsg3). Persistent production of anti-Dsg3 IgG can be induced by adoptively transferring spleen cells of a DSG3-/- mouse immunized with rDsg3 into an RAG2-/- immunodeficient mouse expressing Dsg3 protein. This IgG in the blood binds to the Dsg3 protein in vivo, induces the breakage of intercellular adhesion of keratinocytes and thus brings about the phenotype of pemphigus vulgaris involving the formation of blisters in the oral mucosa and the disappearance of resting hair. These effects are sustained over 6 months. By using this method, active disease model animals relating to various autoimmune diseases can be constructed.

Description of the Invention

TECHNICAL FIELD

The present invention relates to autoimmune disease model animals and methods for producing them.

BACKGROUND ART

Pemphigus vulgaris (PV) is an autoimmune disease with involvement of skin and mucous membrane blistering, which is sometimes fatal, and is histologically characterized by blistering in the epidermis as well as immunopathologically characterized by the presence of autoantibody IgG to the cell surface of keratinocyte (Stanley, J. R. Pemphigus. In Dermatology in General Medicine. I. M. Freedberg, A. Z. Eisen, K. Wolff, K. F. Austen, L. A. Goldsmith, S. I. Katz, and T. B. Fitzpatrick, eds. McGraw-Hill, New York, 654-666 (1998)). Patients with pemphigus vulgaris clinically manifest diffuse flaccid blister and erosion. These can be formed in all the stratified squamous epithelia. Without appropriate therapy, the widespread lesions on the skin result in the leakage of body fluid or secondary bacterial infection, and as a result pemphigus vulgaris may be fatal. The prognosis of pemphigus can be improved by systemic administration of corticosteroid and immunosuppression therapy, but the mortality remains considerably high because of death due to complications caused by the therapy.

The target antigen for pemphigus vulgaris was first identified as a 130 kD glycoprotein through immunoprecipitation of keratinocyte extract (Stanley, J. R. et al., J. Clin. Invest. 70:281-288 (1982); Stanley, J. R. et al., J. Clin. Invest. 74:313-320 (1984)). Then, cDNA for the pemphigus vulgaris antigen was isolated via immuno-screening of a human keratinocyte expression library using affinity-purified autoantibody specific to the pemphigus vulgaris antigen (Amagai, M. et al., Cell 67:869-877 (1991)). Nucleotide sequence analysis has revealed that the pemphigus vulgaris antigen belongs to the superfamily of genes for cadherins that are intercellular adhesion molecules. The pemphigus vulgaris antigen is a membrane protein located in desmosome (Karpati, S. et al., J. Cell Biol. 122:409-415 (1993)), and it was named desmoglein 3 (Dsg3) (Amagai, M. Adv. Dermatol. 11:319-352 (1996)).

There is much evidence showing that autoantibody IgG against Dsg3 protein plays a pathogenic role in pemphigus vulgaris. Firstly, it has been reported that activity of the disease correlates to the antibody titer in blood over time by indirect fluorescent antibody technique (Sams Jr, W. M. & Jordon, R. E., Br. J. Dermatol. 84:7-13 (1971)) or ELISA (Ishii, K., et al., J. Immunol. 159:2010-2017 (1997); Amagai, M., et al., Br. J. Dermatol. 140:351-357 (1999)). Secondly, a newborn from the mother affected with pemphigus vulgaris is also transiently affected with the disease due to the IgG transferred across the placenta from the mother (Merlob, P. et al., Pediatrics 78:1102-1105 (1986)). As the IgG derived from the mother is catabolized, the symptom is remitted. Thirdly, the IgG derived from patients with pemphigus vulgaris can induce blistering in tissue-cultured skin in the absence of complement and inflammatory cell (Schiltz, J. R., & Michel, B., J. Invest. Dermatol. 67:254-260 (1976); Hashimoto, K. et al., J. Exp. Med. 157:259-272 (1983)). Fourthly, passive transfer of the IgG derived from sera of patients into newborn mice causes intraepidermal blister formation with typical histological characteristics (Anhalt, G. J. et al., N. Engl. J. Med. 306:1189-1196 (1982)). Fifthly, depletion of patient-derived serum by immuno-absorption using recombinant Dsg3 protein (rDsg3) comprising extracellular domain thereof removes pathogenicity of the serum and inhibits blistering in newborn mice (Amagai, M. et al., J. Clin. Invest. 94:59-67 (1994)). Finally, antibody affinity-purified with rDsg3 exhibits pathogenicity and thus results in the formation of blister with histological characteristics of pemphigus vulgaris in newborn mice (Amagai, M. et al., J. Clin. Invest. 90:919-926 (1992); Amagai, M. et al., J. Clin. Invest. 102:775-782 (1998)).

Based on these studies, pemphigus vulgaris is one of the best-characterized autoimmune diseases with respect to the processes after the generation of autoantibody in particular. Thus pemphigus vulgaris is now an excellent disease model for tissue-specific autoimmune diseases to study cellular mechanisms underlying the production of autoantibody or destruction of self-tolerance, as well as to develop therapeutic methods specific to the diseases. As the first step toward the goals, it is demanded to develop active disease animal model for pemphigus vulgaris.

Most of experimental autoimmune disease animal models are provided by repeated injection of autoantigen with a variety of adjuvants. However, as exemplified by the case of myasthenia gravis, in which the frequency of generation of the active disease in mice immunized with acetylcholine receptor (T. californica) varies considerably depending on the strains, the success of this method is thus highly empirical (Berman, P. W. et al., Ann. N.Y. Acad. Sci. 377:237-57 (1981)).

Previously, an in vivo experimental model for pemphigus vulgaris was developed by the reconstruction of severe combined immunodeficiency (SCID) in mice using PBMC derived from patients with pemphigus vulgaris (Juhasz, I. et al., J. Clin. Invest. 92:2401-7 (1993)). With this model, lymphocytes from the patients produced circulating autoantibody at a low titer, but it was rare that active intraepidermal blistering with deposition of human IgG was found in mouse skin. When human skin was transplanted on SCID mouse, blisters similar to those in pemphigus vulgaris were recognized on the transplanted skin. However, it cannot be denied that the cause of blister formation in this model is an inflammatory response due to the tissue incompatibility with human PBMC and skin. Thus there was no established active disease model for pemphigus vulgaris.

DISCLOSURE OF THE INVENTION

The present invention provides autoimmune disease model animals and a method for producing them. More specifically, the present invention provides non-human mammals showing phenotypes of the autoimmune disease in which activation of T cells and B cells reactive to the antigen protein for the autoimmune disease followed by stable production of autoantibody are induced and provides a method for producing them. In a preferable embodiment, the model animal can be provided by the transplantation of immune cells including B cells producing antibody against the antigen protein of the autoimmune disease and/or T cells that are reactive to the antigen protein.

To achieve the above described objective, first, the present inventors aimed at the production of autoantibody in mice by employing the previously used typical method with repeated injection. Specifically, three strains of mice, BALB/c(H-2.sup.d), C3H/HeJ(H-2.sup.k), and C57BL/6N(H-2.sup.b) were immunized with human or mouse Dsg3 protein. Complete Freund's adjuvant was used in the primary immunization, and then booster immunization was carried out 3 or 7 times by using incomplete Freund's adjuvant. However, with this method, no mice produced antibody capable of reacting to mouse Dsg3 protein (Table 1, see Original Patent) and showed phenotype of pemphigus vulgaris at all.

Based on this result, the present inventors set up the hypothesis that self-tolerance to Dsg3 protein prevents the production of pathogenic antibody in mouse body. According to the hypothesis, it can be assumed that the immune system is not exposed to Dsg3 protein during the developmental stages in Dsg3-deficient mouse created by gene-targeting technique and thus the mouse does not acquire self-tolerance to Dsg3 protein.

In order to demonstrate the hypothesis, the present inventors studied whether it was possible for Dsg3-deficient mouse immunized with Dsg3 protein to produce antibody against Dsg3 protein. From the result, it was revealed that when immunized with Dsg3 protein a homozygous DSG3 gene-deficient DSG3-/- mouse much more efficiently produced the antibody than a heterozygous DSG3 gene-deficient DSG3+/- mouse (FIG. 1A, see Original Patent). In addition, the antibody produced by DSG3-/- mouse was capable of binding to mouse Dsg3 protein on the keratinocyte, but the antibody from DSG3+/- mouse was not (FIG. 1B, see Original Patent). Specifically, it was revealed that self-tolerance to DSG3 protein had not been established in DSG3-/- mouse and the produced antibody recognized mouse Dsg3 protein as an antigen.

Thus, the present inventors next aimed at the production of antibody against Dsg3 protein and expression of phenotype of pemphigus vulgaris in RAG2-/- immunodeficiency mouse by collecting splenocytes (which have capability of producing antibody against DSG3 protein) from DSG3-/- mouse immunized with Dsg3 protein and adoptively transferring them into the immunodeficiency mouse. Such RAG2-/- mice express Dsg3 protein, but the mice have neither mature T cells nor B cells because they are deficient in rearrangement of T cell receptor genes and immunoglobulin genes (namely, they are immunodeficient).

As a result, in RAG2-/- mice in which splenocytes from DSG3-/- mouse had been transplanted, the encounter of Dsg3 protein-specific lymphocytes among splenocytes with endogenous Dsg3 protein resulted in permanent production of the antibody against Dsg3 protein (FIG. 2A, see Original Patent). In addition, it was found that RAG2-/- mice having the immunized DSG3-/- splenocytes showed nearly identical phenotype of DSG3-/- mouse (Koch, P. J., et al., J. Cell Sci. 111:2529-2537 (1998); Koch, P. J., et al., J. Cell Biol. 137:1091-1102 (1997)). All of the mice exhibited erosive lesions in mucous membranes with epidermal separation just above the basal cell layer and telogen hair loss (FIG. 3, see Original Patent). The presence of nearly identical phenotype reproduced by adoptive transfer of DSG3-/- splenocytes in RAG2-/- recipient mice demonstrated that the produced antibody was specific and pathogenic.

The specificity of the antibody can also be verified by the fact that the in vivo deposition is not detectable in other simple epithelia expressing Dsg2 protein (Schafer, S. et al., Exp. Cell Res. 211:391-9 (1994)) and upper part of epidermis expressing Dsg1 protein (FIG. 3G, see Original Patent) (Amagai, M. et al., J. Invest. Dermatol. 106:351-355 (1996)).

Thus, the present invention provides the first disease mouse model for pemphigus and a method for producing them. The method of the present invention, because of the nature thereof, can be widely applicable to the preparation of model animals for other autoimmune diseases in which associated autoimmune targets have been identified.

Accordingly the present invention relates to autoimmune disease model animals and a method for producing them, more specifically relates to:

(1) a non-human mammal showing a phenotype of autoimmune disease through production of an antibody reacting to an antigen protein for an autoimmune disease or T cell activation;

(2) the non-human mammal of (1), wherein immune cells from a non-human mammal lacking an antigen gene for the autoimmune disease have been transplanted to the non-human mammal;

(3) the non-human mammal of (1), wherein immune cells from a non-human mammal that lacks the antigen gene for the autoimmune disease and that has been immunized with the antigen protein have been transplanted to the non-human mammal;

(4) the non-human mammal of (2) or (3), wherein the immune cells are transplanted to an immunodeficient non-human mammal;

(5) the non-human mammal of (4), wherein the immunodeficient non-human mammal is a non-human mammal that lacks the RAG2 gene;

(6) the non-human mammal of any one of (2) to (5), wherein the immune cells are splenocytes;

(7) the non-human mammal of any one of (1) to (6), wherein the autoimmune disease is pemphigus vulgaris;

(8) the non-human mammal of (7), wherein the antigen protein is desmoglein 3 protein;

(9) the non-human mammal of any one of (1) to (8), wherein the non-human mammal is a rodent;

(10) the non-human mammal of (9), wherein the rodent is a mouse;

(11) a method for producing a non-human mammal showing a phenotype of autoimmune disease through production of an antibody reacting to an antigen protein for an autoimmune disease or T cell activation, which comprises the steps of: (a) immunizing, with the antigen protein for the autoimmune disease, a non-human mammal that lacks the antigen gene for the autoimmune disease, (b) preparing immune cells from the non-human mammal, and (c) transplanting the immune cells to a non-human mammal having the antigen protein;

(12) the method of (11), wherein the immune cells are transplanted to an immunodeficient non-human mammal;

(13) the method of (12), wherein the immunodeficient non-human mammal is a non-human mammal that lacks the RAG2 gene;

(14) the method of any one of (11) to (13), wherein the immune cells are splenocytes;

(15) the method of any one of (11) to (14), wherein the autoimmune disease is pemphigus vulgaris;

(16) the method of (15), wherein the antigen protein is desmoglein 3 protein;

(17) the method of any one of (11) to (16), wherein the non-human mammal is a rodent; and

(18) the method of (17), wherein the rodent is a mouse.

The model animal of the present invention can show phenotype of autoimmune disease through the stable production of antibody reacting to the antigen protein for the autoimmune disease or sustained activation of T cell.

There is no particular restriction on the type of objective disease for which model animals are to be prepared in accordance with the present invention, as far as the disease is an autoimmune disease. Such autoimmune diseases include, for example, but not limited to, pemphigus vulgaris, myasthenia gravis, autoimmune hemolytic anemia, Basedow's disease, Hashimoto's disease, Goodpasture's syndrome, autoimmune diabetes mellitus, multiple sclerosis, etc.

Animals to be utilized for creating the model animal are preferably non-human mammals. There is no restriction on such non-human mammals, as far as gene-disrupted animals can be created from them. Preferable animals include rodents, e.g., mouse.

The model animals in accordance with the present invention can be created by immunizing antigen gene-deficient non-human mammals with the antigen protein for the autoimmune disease, removing the immune cells thereof, and then transplanting the cells to other non-human mammals having the antigen protein.

Animals having the disputed antigen gene can be created by a method known to those skilled in the art. The antigen gene to be disrupted includes, for example, but not limited to, the DSG3 gene when the autoimmune disease is pemphigus vulgaris; the acetylcholine receptor gene for myasthenia gravis; the TSH receptor gene for Basedow's disease or Hashimoto's disease; the type IV collagen gene for Goodpasture's syndrome; the myelin basic protein gene for multiple sclerosis, etc.

Further, immune cells can be obtained from the thymus, lymph node, spleen, liver, intestinal epithelium, peripheral blood, etc. but are not limited to those from the tissues. The spleen abundantly contains mature immune cells and thus is a preferable organ for the immune cells. It is preferable that the animal (donor) from which immune cells are prepared and the animal (recipient) to which lymphocytes derived from the immune cells are transferred belong to a same species and have a same genetic background thereby preventing the onset of GVHD which may cause tissue destruction in the recipient.

In addition to this, it is preferable that the recipient has immunodeficiency thereby preventing the rejection of lymphocytes derived from immune cells transferred. For example, SCID mouse, nude mouse as well as an animal of which RAG2 gene has been disrupted may be used as the immunodeficient animal. Furthermore, MHC-knockout mouse or common .gamma. chain-knockout mouse can also be used but it is not limited thereto.

The immunization with the antigen protein from the donor, preparation of immune cells from the donor, and transplantation of the immune cells to the recipient can be carried out, for example, by the methods as described in the Examples.

The model animal created in accordance with the present invention can show phenotype of autoimmune disease through the stable production of antibody reacting to the antigen protein for the autoimmune disease or sustained activation of T cell. In the model animal for pemphigus vulgaris, major phenotype includes weight loss and reversible telogen hair loss. Further, among autoimmune diseases other than pemphigus vulgaris, phenotype may include reduced muscle power in myasthenia gravis; anemia in autoimmune hemolytic anemia; hyperthyroidism in Basedow's disease; hypothyroidism in Hashimoto's disease; nephropathy and pulmonary disorders in Goodpasture's syndrome; glucosuria in autoimmune diabetes mellitus; and neuroparalysis in multiple sclerosis.

One can use these model animals for developing therapeutic agents or methods for the diseases, administering to them test compounds of interest for therapeutic effects on autoimmune diseases and observing phenotypes thereof. Particularly the major phenotype includes weight loss and reversible telogen hair loss in pemphigus vulgaris model mouse prepared in accordance with the present Example and the phenotypes last over 6 months, and therefore it is possible to readily and objectively evaluate the effectiveness of each therapeutic agent or method based on the observation without sacrificing the mouse. In addition, these model mice are very useful for clarifying cellular mechanism underlying the production of antibody against the antigen protein.
 

Claim 1 of 5 Claims

1. An assay for identifying a mouse or rat with the ability to produce autoantibodies directed to an autoimmune disease protein antigen, said assay comprising: identifying a donor mouse or rat which is deficient in the production of the autoimmune disease protein antigen; harvesting immune cells from the identified donor mouse or rat; transplanting the harvested immune cells into a recipient mouse or rat respectively, wherein the recipient mouse or rat is immunodeficient or has the same genetic background as the donor mouse or rat, and is not deficient in the production of the autoimmune disease protein antigen; obtaining a biological sample from the recipient mouse or rat; assaying the biological sample from the recipient mouse or rat for production of autoantibodies directed to the autoimmune disease protein antigen; and identifying a recipient mouse or rat which produces autoantibodies directed to the autoimmune disease protein antigen based on said assaying.

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