Kuppuswamy N. Kasturi

Spontaneous Occurrence of Anti-Fibrillin-1 Autoantibodies in Tight-Skin Mice

Tight-skin (TSK) mouse represents an experimental for systemic sclerosis, displaying cutaneous hyperplasia, connective tissue alterations in the internal organs and developing autoantibodies against several scleroderma target autoantigens. TSK mouse syndrome is associated with a mutation in fibrillin-1 (Fbn-1), the major component of 10 nm microfibrils. Here, we have investigated whether TSK mouse develops autoimmunity to Fbn-1 similar to scleroderma target autoantigens. Our results show that anti-Fbn-1 IgG autoantibodies are present in high titer in many TSK mice. Specificity of these antibodies was confirmed by competitive inhibition assays and Western blotting analysis using recombinant human Fbn-1 protein. TSK mouse autoantibodies recognize a conserved epitope present in the C region of Fbn-1. These results indicate the presence of Fbn-1 specific T and B cells in TSK mouse repertoire.

Self Reactive Repertoire of Tight Skin Mouse: Immunochemical and Molecular Characterization of Anti-Topoisomerase I Autoantibodies

Tight skin (TSK) mice develop cutaneous hyperplasia accompanied by histopathological alterations of skin and collagen metabolism similar to those described in human scleroderma. Diffuse scleroderma, the most severe form of progressive systemic sclerosis, is associated with the production of autoantibodies specific for Scleroderma 70 antigen (topoisomerase I). Our studies show that there is an increase in the level of serum anti-topoisomerase I (topo I) autoantibodies in aged TSK mice. The monoclonal antibodies isolated from TSK mice bind to epitopes which interact with autoantibodies from scleroderma patients. A significant number of TSK monoclonal anti-topo I antibodies and serum immunoglobulin (Ig) from aged TSK mice bear a cross reactive idiotype (Id) recognized by a syngeneic monoclonal anti-Id antibody obtained from a 2 month-old TSK mouse. Analysis of V gene usage by monoclonal anti-topo I antibodies showed that the majority of these antibodies are encoded by VH genes derived from VHJ558 family pairing with VK genes from various families in a stochastic manner.

Self-reactive repertoire of tight skin (TSK/+) mouse: Immunochemical and molecular characterization of anti-cellular autoantibodies

The tight skin (TSK/+) mouse has been proposed as an experimental model for progressive systemic sclerosis because of the biochemical alterations in collagen synthesis and pathological similarities to the human disease. Here, we report the analysis of tight skin mice sera for the presence of anti-cytoplasmic and anti-nuclear autoantibodies and determination of the frequency of hybridomas producing anti-cellular autoantibodies. The binding specificity of TSK mAbs to nuclear and cytoplasmic antigens such as keratin, actin, vimentin, and mitochondria was determined. Of 71 monoclonal antibodies that we have studied, only 3 appear to bind to foreign as well as self-antigens, indicating that the majority of these antibodies do not belong to the class of natural autoantibodies. Our results also showed that the frequency of hybridomas producing anti-nuclear and anti-cytoplasmic antibodies was higher in TSK mice than in C57BL/6 pa/pa, the control mouse strain, used in these studies. The results of the analysis of V gene usage showed that the majority of anti-cytoplasmic and anti-nuclear antibodies are encoded by genes from a restricted number of VH and VK genes families. In the sera of TSK mice we have detected the presence autoantibodies specific for cytoplasmic antigens in addition to anti-nuclear and anti-topoisomerase I antibodies which are characteristic of scleroderma. Since the presence of anti-cytoplasmic antibodies has not been described in scleroderma, the significance of their production in tight skin mice is not clear. However, the presence of such autoantibodies in the animal model provides a basis for investigation of this type of antibodies in human disease.

Tight-skin mouse autoantibody repertoire: analysis of VH and VK gene usage.

In the previous studies we have shown that tight-skin (TSK) mouse is an experimental model for systemic sclerosis. This mutant mouse develops autoantibodies specific for scleroderma target antigens. To determine whether the expansion of autoantibody repertoire in TSK mouse occurs by selective expansion of certain variable region gene families, and whether CD5+ B cells contribute significantly to the production of autoantibodies, we have analyzed a panel of 60 hybridomas producing autoantibodies specific for scleroderma target autoantigens. Northern analysis of RNAs from these hybridomas showed that 70% were expressing genes from VHJ558 family while genes from 36-09 and J606 families were not at all represented. In contrast, in the cDNA libraries derived from splenic B cells, the expression of VHJ558 and 36-09 gene families were at an expected frequency corresponding to their genomic complexity (44% and 11.6%, respectively). These results demonstrate that there is a strong bias toward the use of J558 genes in TSK mouse autoantibody repertoire. On the other hand the expression of VK gene families was mostly random and corresponded to their frequency in splenic C kappa cDNA library. The pairing of VH:VK genes was stochastic. Analysis of the expression of J segments, however, revealed that JH2 and JK2 were predominantly used in the autoantibodies. Analysis of the expression CD5 mRNA in these hybridomas indicate that CD5+ B cells do not contribute significantly to the autoimmunity in TSK mice. These findings suggest that the expansion of peripheral autoreactive B cells in TSK mouse is determined by their immunoglobulin variable region rather than the genetic properties linked to a particular B cell subset.

Role of Immunocompetent Cells in Skin Sclerosis and Autoimmunity in Tight Skin Mice

Scleroderma is a connective tissue disease which affects the skin and internal organs. It is a complex and clinically heterogenous disease with three major clinical forms ranging from limited skin involvement (morphea) to diffuse skin sclerosis and severe internal organ involvement (progressive skin sclerosis, SSc) and CREST syndrome in which skin sclerosis is associated with calcinosis, Raynaud phenomenon, esophageal densitometry and telangiectasia. Although, the exact mechanisms involved in the pathogenesis of SSc are not known, the following three major alterations might play a role in the pathogenesis: a) excessive deposition of collagen within the skin and internal organs. b) vascular lesions of capillaries and small arteries. c) autoimmune manifestations. (LeRoy, 1989) The autoimmune manifestations in scleroderma are characterized by the presence of autoantibodies as well as by abnormalities of cellular immune response.

Biased use of certain Vk gene families by autoantibodies

RNAs obtained from 79 hybridomas producing autoantibodies were hybridized with eight vk gene probes: vk1, vk4, Vk8, Vk10, vkl9, Vk21, Vk22, and Vk24. Our results demonstrated that five of these families are used more frequently: vk1, 14 of 79; Vk4, 11 of 79; V,8, 7 of 79; V,lO, 10 of 79; and Vk19, 10 of 79. Overall, 52 of the 79 hybridomas studied (66%) used these five families (FIG. 1). In a control experiment of the use of V, gene families in a panel of 30 hybridomas obtained by fusion of unstimulated lymphocytes from C57BL mice, we observed a more random distribution. Consistent with these initial results, Southern analysis showed important differences in the autoimmune strains when probed with V, gene probes (FIGS. 2 and 3). There was no RFLP difference with Vk4, Vk8. Vk21, and Vk22. The tight skin (tsk)