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The alpha-fodrin N-terminal portion (AFN) autoantigen mediates in vivo immunoregulation of autoimmune responses in primary Sjögren's syndrome (SS). We further examined this process and found that cleavage products of AFN were frequently detected in the salivary gland duct cells of SS patients. In in vitro studies using human salivary gland HSY cells, anti-Fas-induced apoptosis resulted in specific cleavage of alpha-fodrin into the 120-kd fragment, in association of alpha-fodrin with mu-calpain, and activation of caspase 3. Significant proliferative responses against AlphaFN autoantigen were observed in the peripheral blood mononuclear cells (PBMCs) from SS patients with higher pathological score (grade 4) and with short duration from onset (within 5 years). In vivo roles of AFN peptides were investigated using PBMCs from patients with SS, systemic lupus erythematosus, and rheumatoid arthritis. Significant proliferative T-cell responses of PBMCs to AFN peptide were detected in SS but not in systemic lupus erythematosus or rheumatoid arthritis. AFN peptide induced Th1-immune responses and accelerated down-regulation of Fas-mediated T-cell apoptosis in SS. Our data further elucidate the in vivo role of AFN autoantigen on the development of SS and suggest that the AFN autoantigen is a novel participant in peripheral tolerance.

Objective: A wide spectrum of extraglandular manifestations may occur in patients with Sjogren's syndrome (SS), but the mechanisms responsible for in vivo progression are still obscure. We undertook this study to evaluate the age-related changes during the development of extraglandular autoimmune lesions, including arthritis, in the murine model of primary SS, and to evaluate the possible relationship between age-related disturbance of activation-induced cell death and the in vivo kinetics against autoantigens., Methods: A total of 126 NFS/sld mice were investigated at ages 2, 4, 6, 10, 12, 18, 20, and 24 months. Cytokine production was tested using culture supernatants from anti-CD3 monoclonal antibody-stimulated T cells. Anti-single-stranded DNA (anti-ssDNA) antibodies, Ig isotypes (IgG1, IgG2a), rheumatoid factor (RF), and anti-type II collagen (anti-CII) antibodies were detected by enzyme-linked immunosorbent assay. Proliferative T cell responses against each of 3 recombinant alpha-fodrin proteins and against CII were analyzed., Results: Autoimmune arthritis developed in SS model mice until age 24 months. Significant elevations in serum levels of RF, anti-ssDNA antibodies, and anti-CII antibodies were found in aging SS model mice. A high titer of serum autoantibodies against alpha-fodrin fragments (containing different epitopes that were originally identified in primary SS model mice) was frequently detected in young and aged SS model mice. Moreover, we found that alpha-fodrin autoantigen induced Th1 immune responses and accelerated disturbance of Fas-mediated T cell apoptosis in aged SS model mice., Conclusion: These results indicate that age-related disturbance of activation-induced cell death via bystander T cell activation may play a crucial role in the development of autoimmune arthritis in a murine model of SS.

Objective: To determine whether eyedrop administration of an anti-CD4 monoclonal antibody (mAb) is effective in the treatment of Sjögren's syndrome (SS) using a mouse model of the disease., Methods: The anti-CD4 mAb was administered daily into the eyes of mice with SS from ages 4 to 8 weeks or ages 10 to 12 weeks. During treatment, tear volume was monitored and after final treatment, histologic features of the lacrimal and salivary glands, the phenotypes and function of T cells, and serum titers of anti-alpha-fodrin antibody were examined., Results: Eyedrop administration of anti-CD4 mAb before the onset of SS prevented the autoimmune pathology seen in the lacrimal glands but not that in the salivary glands. Furthermore, eyedrop administration of anti-CD4 mAb after the development of SS inhibited mononuclear cell infiltration and the destruction of parenchyma only in the lacrimal glands. Eyedrop administration of anti-CD4 mAb suppressed the local activation of CD4+ T cells rather than deleting CD4+ T cells, which reduced the expansion of pathologic CD4+ T cells against alpha-fodrin., Conclusion: These results demonstrate the remarkable efficacy of anti-CD4 mAb eyedrops in the treatment of SS eye symptoms, which illustrates a new antibody-based therapeutic strategy for patients with eye problems caused by SS as well as other diseases.

The aims of this study were to evaluate the in vivo effects of estrogen deficiency in MRL/lpr mice as a model for rheumatoid arthritis and to analyze the possible relationship between immune dysregulation and receptor activator of nuclear factor-kappaB ligand (RANKL)-mediated osteoclastogenesis. Experimental studies were performed in ovariectomized (Ovx)-MRL/lpr, Ovx-MRL+/+, sham-operated-MRL/lpr, and sham-operated-MRL+/+ mice. Severe autoimmune arthritis developed in younger Ovx-MRL/lpr mice until 24 wk of age, whereas these lesions were entirely recovered by pharmacological levels of estrogen administration. A significant elevation in serum rheumatoid factor, anti-double-stranded DNA, and anti-type II collagen was found in Ovx-MRL/lpr mice and recovered in mice that underwent estrogen administration. A high proportion of CD4(+) T cells bearing RANKL was found, and an enhanced expression of RANKL mRNA and an impaired osteoprotegerin mRNA was detected in the synovium. An increase in both osteoclast formation and bone resorption pits was found. These results indicate that estrogen deficiency may play a crucial role in acceleration of autoimmune arthritis associated with RANKL-mediated osteoclastogenesis in a murine model for rheumatoid arthritis.

Objective: The pathologic mechanisms responsible for organ-specific tissue damage in primary Sjögren's syndrome (SS) remain unclear, but it has been suggested that the pathology is mediated by autoreactive CD4+ T cells infiltrating the salivary and lacrimal glands. This study was undertaken to investigate whether alpha-fodrin autoantigen-specific autoreactive CD4+ T cells are capable of inducing autoimmune lesions., Methods: A total of 45 synthetic alpha-fodrin peptides designed to be 20 amino acid residues in length were generated. To establish an autoreactive T cell line, limiting dilution analysis (LDA) was performed on lymph node cells (LNCs) in the presence of alpha-fodrin peptides. The effects of adoptive transfer of autoreactive CD4+ T cells into normal syngeneic recipients were investigated., Results: Autoreactive CD4+ T cell lines that recognize synthetic alpha-fodrin peptide, which produced Th1 cytokines and showed cytotoxic activities, were established in a murine model for SS. T cell receptor V(beta) usage and third complementarity-determining region (CDR3) sequences indicated that in some cases V(beta)6-CDR3 genes matched between the tissue-infiltrating T cells and the autoreactive T cell lines. Adoptive transfer of the autoreactive CD4+ T cells into normal syngeneic recipients induced autoimmune lesions quite similar to those of SS., Conclusion: Our data help to elucidate the pathogenic mechanisms responsible for tissue destruction in autoimmune exocrinopathy and indicate that autoreactive CD4+ T cells play a pivotal role in the development of murine SS.

Background and Purpose: Moyamoya disease (MMD) is a rare entity that results in progressive occlusion of the arteries of the circle of Willis, but the pathogenesis of MMD is unknown., Methods: MMD sera (n=32) were tested for anti-endothelial cell antibodies by enzyme-linked immunoassays and flow cytometric analysis. Apoptosis was induced in human umbilical vein endothelial cells by tumor necrosis factor-alpha., Results: We found that a high proportion of MMD sera had anti-endothelial cell antibodies with apoptotic stimuli. Prominent reactivities of MMD sera (72%) with recombinant human alpha-fodrin were observed., Conclusions: Our study demonstrates that MMD sera contain a high incidence of anti-alpha-fodrin autoantibodies, providing new insight into the mechanisms of occlusion of MMD arteries.