Spontaneous animal models with systemic autoimmune diseases

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University of California at Davis (UCD) 200 strain as an animal model for progressive systemic sclerosis (SSc) - scleroderma

SSc is a human disease that is classified among the autoimmune connective tissue diseases that also comprise systemic lupus erythematosus (SLE), dermatomyositis and polymyositis, rheumatoid arthritis and several others. While there are several well-established, spontaneously occurring animal models available for SLE, models for the other conditions in this group are either nonexistent or of questionable significance. This is also true for SSc for which homologous disease of rats has been proposed as a model, as it is associated with chronic skin lesions with increased thickening and collagenization of the dermis. Significant titers of antinuclear antibodies (ANAs) are, however, lacking and the presence of immune complexes in vascular walls is also not characteristic for SSc. Another model for human scleroderma is the tight-skin (TSK) mouse, a dominant mutant of the inbred B10.D2 (58N/SN) mouse strain. The TSK mouse is characterized by cutaneous and skeletal hyperplasia but - in contrast to human SSc - these animals do not show gastrointestinal involvement, particularly the characteristic vascular pathology. There is also no evidence in these mice of early mononuclear cell infiltration of the skin.

The UCD 200 chicken model, described below, has many more similarities with SSc than all the models described so far, but it has to be emphasized that there are also some dissimilarities, including the development of glomerulonephritis, particularly in older animals, that may, however, be a sequel of secondary infection.

The first (male) chickens showing signs of a genetically determined fibrotic disease resembling SSc were discovered in 1942 by P. Bernier at the Department of Poultry Husbandry, Oregon State University, Corvallis. These birds exhibited comb and dermal swelling accompanied by fibrosis. The affected birds were then selectively bred for over 40 years and a new strain, now denoted UCD 200, was developed where males and females are affected to about the same extent.

200 strain UCD chickens exhibit severe alterations of the comb, neck and extremities. The comb already becomes visibly swollen and red 1-2 weeks after hatching, and at the age of 4 weeks about 90% of all birds develop comb necrosis with subsequent resorbtion ('self-dubbing'). Similar events occur in the digits of 30-40% of the birds, again beginning at 2-3 weeks of age, including polyarthritis of peripheral joints. At 2-4 weeks of age the skin of the neck also becomes involved, but the constant mechanical irritation of the neck in the outlets of the cages to gain access to food and water may also contribute to the lesions at this particular location.

A mononuclear cell infiltration is first observed in the affected skin areas and can be demonstrated in about 70% of line 200 birds at 1 week of age. Mononuclear cell infiltration starts perivascularly and is followed by vessel occlusion, similar to one of the hallmarks of human SSc. This acute inflammatory process reaches a peak at 2 weeks of age and subsides at the age of 5 months, when no more skin inflammation can be shown macroscopically or microscopically. At 5 months of age the esophagus becomes involved with a severe cellular infiltration around vessels and in the lamina propria of mucous glands in about 50% of the animals, increasing to 70% or more at the age of 1 year. Similar changes can be observed in the lamina propria of the small intestine, albeit at a lower frequency of about 30%. Furthermore, mononuclear cell accumulations can be found in the lung, starting at 1 week of age (60%) and decreasing to about 30% at 4-6 weeks, in the heart and kidney (30-40% at 5-6 weeks of age) and the testes, beginning at 4-6 weeks of age.

The early mononuclear cell infiltrates in the skin are composed mainly of T cells expressing the 7/8 receptor. An influx of a/(3 T cell receptor-positive cells is first observed later, i.e. at the age of 20 days. While •y/S-positive T cells occur mainly in the upper layers of the dermis, a/(3-positive cells prevail in the deeper areas. Most of these latter cells express B-L, the chicken MHC class II analog.

The vascular changes arc very characteristic in UCD 200 birds and can be found in most of the organs mentioned above. They are first noted in the comb and the feathered skin at 1 week of age, where the vessels show hyperplasia of the media and intimal thickening. In the internal organs the vascular changes are found later but always precede the macroscopic and clinical symptoms.

The nature of the increased deposition has not yet been studied in great detail by chemical and molecular biological methods but only by immunohistology on frozen sections, using specific antibodies against the different collagen variants. With this approach an increase of type III and type VI collagens can be noted in the skin and the terminal vascular ratification becomes clearly evident when antibodies against basement membrane components, such as type IV collagen or laminin, are applied. The accumulation of connective tissue, as manifested by fibrosis of the lung, severe myocardial muscle fiber destruction, subpericardial fibrosis, thickening of the esophageal wall and effects on other internal organs, leads to the premature death of these birds. In addition, they are prone to secondary infections and show a low fertility rate due to the massive involvement of the gonads, particularly the testes.

As in the human disease, no gross alteration at the genomic level of collagen genes has been found by means of restriction fragment length polymorphism (RFLP) analysis, thus suggesting that the UCD 200 model is an appropriate counterpart for studying the altered collagen metabolism in SSc. Investigation of the putative role of endogenous viruses (eu) for the development of SSc led to the discovery of ev23, which is found only in UCD 200 chickens. So far, the role of ev23 in the development of avian scleroderma is unclear.

Similar to human patients with SSc, sera from UCD 200 chickens contain ANAs in high frequency. At 1 month of age over 70% of the birds show ANAs with various staining patterns, including speckled centromeric and nucleolar fluorescence that are also characteristic of human SSc. These antibodies belong mainly to the IgY isotype, homologous to mam malian IgG, and can also be demonstrated on mammalian substrates, such as HEp-2 cells.

Although the UCD 200 chickens have now been kept for over 40 years, there are only a very few reports in the literature addressing the possible pathogenetic mechanisms leading to the SSc-like disease in this strain. The group of Gershwin and colleagues has shown that thymectomy on the day of hatching significantly reduces - but not completely abolishes - the comb/neck lesions, when assessed at the age of 8 weeks. Furthermore, the severity of the lesions in general was significantly reduced by this manipulation.

In vitro, UCD 200 peripheral blood T lymphocytes show a significantly decreased mitogen-induced proliferation associated with a decreased capacity to produce IL-2 and to express IL-2 receptors compared with normal White Leghorn chickens. In contrast to the deficient in vitro IL-2 production, the sera of UCD 200 chickens contain significantly higher levels of IL-2 bioactivity.

UCD 200 chickens also show a disturbed immuno-endocrine feedback, characterized by a prolonged secretion of corticosterone after application of certain cytokines. Although the peak serum concentrations of the glucocorticoids are equal to that of controls, UCD 200 have to secrete twice as much ACTH to achieve this corticosterone serum level, owing to an apparent hyporesponsiveness of the adrenal gland to this secretagogue. The altered cyto-kine-induced glucocorticoid secretion is found in early as well as in chronic, sclerotic stages of the disease.

Recent studies by Sgonc and coworkers brought forward an interesting aspect on the pathogenesis of SSc: simultaneous in situ analysis of frozen skin sections for apoptosis and immunofluorescence staining for cell markers show that endothelial cells are clearly the first cells to undergo apoptosis in the skin of UCD 200 chickens, a process that seems to be induced by antibody-dependent cytotoxicity. Endothelial cell apoptosis is found before any other macroscopical or microscopical alterations are visible. Analysis of early lesion skin biopsies from human SSc patients shows a similar course of events.

The UCD 200 chickens are homozygous for a variation of the MHC haplotype B17. In addition they have been typed for some of the other blood groups and found to carry Al, C2, D3, E7 and P2. They are homozygous for the immunoglobulin 7S Ig (homologous to mammalian IgG) allotype 1.7. Since UCD 200 chickens are still segregating at the B locus and some other major blood group loci, attempts have been made in recent years to develop a subline that matches one of the related inbred normal lines

(line 058) in order to allow for mutual cell transfer experiments. Such a subline, denoted line 206, carrying the B15 haplotype has now been established.

In summary, the UCD 200 line of chickens seems to provide the best model for human SSc available so far. It mimics most of the clinical, histopatho-logical and immunological hallmarks of SSc, albeit in an accelerated fashion. With respect to more derailed further studies, particularly the development of new therapeutic strategies, this accelerated course of the disease is an experimental advantage.

Murine strains with SLE-like autoimmunity

Several models of spontaneous SLE in mice have been extensively studied, namely New Zealand Black [NZB], (NZB x New Zealand White [NZW])F1, (NZB x SWR)F1, BXSB, and MRL mice, which exhibit two forms of disease, a late-life variety appearing during the second year of life (male [NZB x WJF1, female BXSB, MRL/Mp-+/+ of both sexes, NZB mice of both sexes) and an acute form beginning a few weeks or months after birth (female [NZB x W|F1, male BXSB, MRL/Mp mice of both sexes homozygous for the lymphoproliferation \lpr\ gene, female and male [NZB x SWR]F1 mice). Accelerating factors are female sex hormones in (NZB x W)F1, a gene located on the Y chromosome (Yaa = Y chromosome-lined autoimmunity accelerator) of BXSB mice, the Ipr gene in MRL mice, or genes from phenotypically normal SWR or minimally affected NZW mice in crosses with the NZB strain. All these models are characterized by hypergammaglobulinemia, anti-DNA and other ANAs, antiretroviral gp70 antibodies, circulating immune complexes, glomerulonephritis, and a low percentage of coronary arthritis. MKL/Mp-lpr/lpr mice also develop necrotizing polyarteritis and a rheumatoid arthritis (RA)-like disease accompanied by rheumatoid factors in serum. The NZB strain develops autoimmune hemolytic anemia.

Experiments addressing the genetics and the cellular or molecular mechanisms of SLE in these murine strains revealed that murine lupus is a polygenetic and multifactorial process, involving a combination of various genetically determined defects, which at least in part are different in each of the five models mentioned above. Studies of bone marrow irradiation chimeras indicate that murine lupus is caused by abnormalities at the hematopoietic stem cell level. The relative roles of T and B cells in the initiation and perpetuation of autoaggression are still a matter of debate. However, it appears clear that B cell hyperreactivity of lupus-prone mice is not due to defects intrinsic to immunoglobulin genes, but rather depends on T helper cells and/or polyclonal acti vation signals. Autoantibodies are of poly- or oligo-clonal origin, the nephritogenic ones belonging to the IgG isotype and being cationic in charge. On the cellular level, dependent of the strain investigated and the age of donors, conflicting results have been obtained as far as the production of cytokines (e.g. IL-2), the response to them (e.g. IL-2 and IL-5), the cooperation between T and B cells, and the distribution of lymphocyte subsets are concerned. Both the Ipr and the phenotypically similar, but nonallelic, gld (generalized lymphoproliferative disease) genes result in a massive accumulation of abnormal Thy-1+ CD5+ CD4" CD8" CD3+ TCR-a|3+ T cells, which anomalously express B cell markers (B220, PCI) and appear to be of polyclonal origin, the V(38.2 and V(38.3 T cell receptor genes being over-represented. Homozygosity in the recessive Ipr or the lpr°s mutations affects the expression (Ipr) or signal-transducing capability [Ipr**) of the CD95/Fas/Ap-1 surface receptor that initiates apoptosis-inducing pathways upon interactions with its ligand FasL, the molecule affected by the gld mutation. Since Fas is involved in the activation-induced cell death of T lymphocytes it is assumed that peripheral deletion is at least partially compromised in Ipr/lpr and gld/gld mice. Moreover, it is known that Fas expressed on anergic B cells can receive apoptosis-inducing signals via interaction with FasL on T cells. Therefore, the Ipr or gld mutation may compromise both T and B cell tolerance.

A dominant NZW locus contributing to severe SLE in (NZB x W)F1 maps within the H-2 region. This may be either due to the unique sequence of the NZW I-Ep chain gene or to reduce production of the MHC class III product tumor necrosis factor (TNF). This latter finding could be pathogenetically relevant since treatment with TNF postpones the development of nephritis. Heterozygosity for the I-A|3 haplo-types of NZB and SWR has also been shown to correlate with the development of glomerulonephritis in crosses of the NZB strain with SWR mice. In other strains, no strong correlation with a particular H-2 haplotype was observed. Heterozygosity for the T cell receptor strongly correlates with nephritis in (NZB x SWR)F1 x NZB backcrosses, thus suggesting that the deletion of about half the T cell receptor V(3 genes which is found in healthy SWR parents might contribute to the development of SLE. In contrast, the NZW T cell receptor 3 chain locus, the C(31, Dpi and J(31 regions of which have been deleted, probably does not contribute to the SLE of (NZB x W) hybrids.

In summary, the etiology of murine lupus appears to be rather complex and awaits further elucidation. Multiple loci accounting for different disease mani festations are currently under molecular study using a combination of classical genetic approaches (phenotyping of intercrosses or backcrosses) and microsatellite-based chromosomal maps. Interestingly, some of the loci determining lupus of (NZB x NZW)F1 mice map close to loci determining glomerulonephritis in MRL//pr mice or diabetes in NOD mice, suggesting the existence of shared genetic mechanisms for some autoimmune disorders.

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