Fuminori Hyodoh

Over‐expression of the decoy receptor 3 (DcR3) gene in peripheral blood mononuclear cells (PBMC) derived from silicosis patients

Dysregulation of apoptosis, particularly in the Fas/Fas ligand (FasL) pathway, is considered to be involved in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Recently, a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds FasL and inhibits FasL‐induced apoptosis, has been identified. Silicosis is clinically characterized not only by respiratory disorders but by immunological abnormalities. We have found that serum soluble Fas (sFas) levels are elevated in silicosis patients and that sFas message is dominantly expressed in PBMC derived from these patients. This study examined DcR3 gene expression in PBMC derived from patients with silicosis, SLE, or progressive systemic sclerosis (PSS), and compared it with that in healthy volunteers (HV). The relative expression level of the DcR3 gene was examined in PBMC derived from 37 patients with silicosis without clinical symptoms of autoimmune disease, nine patients with SLE, 12 patients with PSS, and 28 HV using the semiquantitative multiplex‐reverse transcriptase‐polymerase chain reaction (MP‐RT‐PCR). The correlation between the relative expression level of the DcR3 gene and multiple clinical parameters for respiratory disorders and immunological abnormalities in individuals with silicosis was analysed. The DcR3 gene was significantly over‐expressed in cases of silicosis or SLE when compared with HV. In addition, the DcR3 relative expression level was positively correlated with the serum sFas level in silicosis patients. It is unclear, however, whether over‐expression of the DcR3 gene in silicosis is caused by chronic silica exposure, merely accompanies the alteration in Fas‐related molecules, or precedes the clinical onset of autoimmune abnormalities. It will be necessary to study these patients further, establish an in vitro model of human T cells exposed recurrently to silica compounds, and resolve whether the increase in DcR3 mRNA expression is a cause or consequence of disease.

Alterations of fas and fas-related molecules in patients with silicosis

Persons with silicosis have not only respiratory disorders but also autoimmune diseases. To clarify the mechanisms involved in the dysregulation of autoimmunity found in patients with silicosis, we have been focusing on Fas and Fas-related molecules in the Fas-mediated apoptotic pathway, because Fas is one of the most important molecules regulating autoimmunity involving T cells. Our findings showed that patients with silicosis exhibited elevated serum soluble Fas levels, an increased relative expression of the soluble fas and dcr3 genes in peripheral blood mononuclear cells, high levels of other variant messages of the fas transcript, relatively decreased expression of genes encoding several physiological inhibitors (such as survivin and toso), and dominancy of lower-membrane Fas expressers in lymphocytes, which transcribe soluble fas dominantly, compared with soluble fas transcription in healthy donors. These findings are consistent with known features regarding immunological factors, such as serum immunogulobulin G levels and the titer of anti-nuclear autoantibodies in silicosis. In addition, anti-caspase 8 autoantibody and anti-Fas autoantibody were detected in serum specimens from patients with silicosis, and a functional assay showed that anti-Fas antibody stimulated Fas-mediated apoptosis. We hypothesize that there are two subpopulations of silicosis lymphocytes. One is a long-term surviving fraction that includes self-recognizing clones showing lower levels of membrane Fas and inhibition of Fas/Fas ligand binding in extracellular spaces. The other subpopulation exhibits apoptosis caused by silica and silicates, is recruited from bone marrow, shows higher levels of membrane Fas, and is sensitive to anti-Fas autoantibody. Further investigation should be performed to confirm the effects of silica and silicates on the human immune system.

Reduced function of CD4+25+ regulatory T cell fraction in silicosis patients.

The quality and quantity of CD4+25+ regulatory T cells (Treg) in silicosis patients (SIL) were examined and compared with results from healthy donors (HD) because SIL often develop autoimmune diseases along with pulmonary disorders. Peripheral blood mononuclear cells from 57 SIL and 50 HD were analyzed for Treg. Treg frequency and clinical parameters were subjected to a factor analysis. Treg and CD4+25- T cells (Tneg) from five HD and five SIL, sorted by flow-cytometer, were used for functional assays of Treg, the expression pattern of Treg specific genes (FoxP3, GITR and CTLA-4) and activation-related genes (CD122 and CD123;. Although the actual frequency of Treg did not differ between SIL and HD, the age-corrected level was reduced in SIL. The factor analysis showed that Treg frequency was positively associated with the serum level of IL-2. The inhibitory effect of Treg on Tneg activation was decreased when the Treg:Tneg ratio was 1:¼ to ½. In addition, Treg dominancy of FoxP3 and CTLA-4 expression and Tneg dominancy of CD132 expression found in HD were lost in SIL. These results indicated that the Treg fraction in SIL may be substituted with chronically activated T cells due to recurrent exposure to silica, resulting in a reduction in the frequency and function of Treg. Since the reduction of Treg may precede the clinical manifestation, as silicosis may be a pre-clinical status for autoimmune diseases, control of Treg function using cell and/or gene therapy may be a good way to manage autoimmune disease.

Detection of alternatively spliced variant messages of Fas gene and mutational screening of Fas and Fas ligand coding regions in peripheral blood mononuclear cells derived from silicosis patients

Silicosis is clinically characterized not only by respiratory disorders but by immunological abnormalities such as the appearance of autoantibodies and complications of autoimmune diseases. Dysregulation of apoptosis, particularly in the Fas/Fas ligand (FasL) pathway, has been considered to play a role in the pathogenesis of autoimmune diseases. It has been found that serum soluble Fas (sFas) levels are elevated in silicosis patients (SIL) and the sFas message is dominantly expressed in peripheral blood mononuclear cells (PBMC) derived from these individuals. In the present study, one tried to detect alternatively spliced variant messages including typical sFas message and found four that were highly and frequently expressed, and which possess a signal peptide domain, but not transmembrane and signal transducing domains, in PBMC derived from SIL. Functional mutations were not detected in Fas and FasL genes in silicosis PBMC. Still, alternative spliced variants of the Fas gene including typical sFas message appear to play an important role in the immunological dysregulation in SIL.

Different Distribution of HLA Class II Alleles in Anti-Topoisomerase I Autoantibody Responders between Silicosis and Systemic Sclerosis Patients, with a Common Distinct Amino Acid Sequence in the HLA-DQB1 Domain

Autoantibodies against DNA topoisomerase I (anti-topo I) have been reported to be specific to systemic sclerosis (SSc), however, anti-topo I was detected in patients with silicone breast implants, SLE without features of SSc, and rheumatic diseases. We detected anti-topo I positive silicosis patients without any symptoms of autoimmune diseases. The correlation between anti-topo I autoantibody responses and HLA class II has been established. HLA-DRB1*1502; DQB1*0601 has been reported to be the most frequent anti-topo I associated haplotype among Japanese SSc patients. In this study, haplotype HLA-DR15; DQ6 was detected in all 4 anti-topo I positive Asian Japanese SSc patients randomly selected. Furthermore, HLA-DQB1*0402 was identified in 3 of 4 anti-topo I positive silicosis patients. These findings coincide with the results of a previous study, in which all 4 Japanese patients with anti-topo I had the DQB1*04 alleles, whereas no studies among Caucasian-Americans, African-Americans and Choctaw Indians found the involvement of DQB1*04. We investigated common features among various DQB 1 alleles. HLA-DQB I with a distinct characteristic is clearly involved in the anti-topo I response irrespective of ethnic groups, the main disease, or silica exposure. A common positioning of distinct amino acids, (i.e. positions 14, 30, 57 and 77 of the DQbeta1 domain are methionine, tyrosine, aspartic acid and threonine, respectively,) seems to be associated with anti-topo I response. The above-mentioned amino acid sequence is detected in alleles *0301, *0303, *0306, *0401, *0402, *0601 and *0602.

Detection, epitope-mapping and function of anti-Fas autoantibody in patients with silicosis

Dysregulation of apoptosis through the Fas–Fas ligand pathway is associated with the onset of autoimmune disease. Since autoantibodies directed against unknown antigens are present in the sera of these patients, sera samples were examined for the presence of autoantibodies directed against the Fas molecule. Using Western blotting and a ProteinChip analysis, autoantibodies against Fas were detected in patients with silicosis, systemic lupus erythematosus (SLE) and systemic sclerosis (SSc), and weakly detected in healthy individuals. Using epitope mapping employing 12‐amino‐acid polypeptides with the SPOTs system, a minimum of four epitopes and a maximum of 10 epitopes were found. Several amino acid residues involved in binding FasL, such as C66, R87, L90, E93 and H126, were presented within the epitopes. Serum containing a large amount of anti‐Fas autoantibody from silicosis patients inhibited the growth of a Fas‐expressing human cell line, but did not inhibit the growth of a low Fas‐expresser nor a Fas‐expresser in which the Fas gene had been silenced by small interference RNA. All epitopes in the intracellular region of Fas were located in the death domain. The possible roles of anti‐Fas autoantibody detected in healthy volunteers and patients with silicosis or autoimmune diseases are discussed here.

Candida albicans aspartic proteinase cleaves and inactivates human epidermal cysteine proteinase inhibitor, cystatin A.

It is known that the cysteine proteinase inhibitor, cystatin, has a defence function against exogenous pathogens. Human epidermal cysteine proteinase inhibitor, cystatin A, which is a member of the cystatin family, is localized in the upper epidermal layer. In this study, the relationship between cystatin A and Candida aspartic proteinase (CAP), a putative Candida virulence factor, was studied. CAP activity was not affected by human epidermal cystatin A, while 90% of cystatin A activity was lost after incubation with CAP for 12 h at 37 degrees C. Human epidermal cystatin A was cleaved into small peptides by CAP, and the released peptides had no cystatin activity. These results suggest that CAP may induce an imbalance between cysteine proteinase and its inhibitor in cutaneous Candida infectious lesions through the degradation and inactivation of epidermal cystatin A.

Inhibitory effects of anti-oxidants on apoptosis of a human polyclonal T-cell line, MT-2, induced by an asbestos, chrysotile-A.

To clarify the effects of silica and silicates on cellular features of lymphocytes, a human T‐lymphotropic virus type‐1‐immortalized polyclonal T‐cell line, MT‐2, was exposed to various concentrations of chrysotile‐A, an asbestos classified as silicate. MT‐2 cells underwent apoptosis in a dose‐ and time‐dependent manner. The mitochondrial apoptotic pathway was activated during chrysotile‐A‐induced apoptosis of MT‐2 cells, because of the phosphorylation of JNK and p38, increase of BAX and release of cytochrome‐c from mitochondria to cytoplasma. In addition, anti‐oxidants such as hydroxyl‐radical excluders and capturers of superoxide and inhibitors of superoxide production effectively reduced the size of the apoptotic fraction in MT‐2 cells cultured with chrysotile‐A. These results indicate that the activation of reactive oxygen species may play a central role in asbestos‐induced T‐cell apoptosis, and anti‐oxidants may help to prevent complications of pneumoconiosis.

Reduced Expression of the Inhibitory Genes for Fas-Mediated Apoptosis in Silicosis Patients.

Reduced Expression of the Inhibitory Genes for Fas‐Mediated Apoptosis in Silicosis Patients: Takemi Otsuki, et al. Department of Hygiene, Kawasaki Medical School—Silicosis cases are characterized not only by respiratory disorders but by various immunological abnormalities, but the cellular biological effects of silica compounds on human lymphocytes have not been well investigated. Our previous studies revealed high serum soluble Fas (sFas) levels in silicosis patients without any clinical symptoms of autoimmune disease and the dominant expression of sFas mRNA in peripheral blood mononuclear cells (PBMC) derived from these patients. These observations indicate that dysregulation of the Fas gene may play an important role in the pathogenesis of the immunological abnormalities found in silicosis patients. Recently several molecules with inhibitory or regulatory effects on Fas‐mediated apoptosis have been identified and their cellular biological roles investigated. We examined the mRNA expression of inhibitory genes (TOSO, sentrin, and cFLIP), regulatory genes (CPAN and DFF45), and caspases (caspase‐1, ‐3, and ‐8) in PBMC derived from silicosis patients. The results showed a reduced expression of sentrin, cFLIP and DFF45 and an overexpression of caspase‐1. Nevertheless, considering the remarkable dominant expression of sFas in silicosis, the dysregulation of inhibitory genes and caspase‐1 may be evidence of a Fas‐mediated apoptotic pathway activated via the remaining membrane Fas molecules in the PBMC of silicosis patients.

Expression of the T Cell Receptor Vβ Repertoire in a Human T Cell Resistant to Asbestos-Induced Apoptosis and Peripheral Blood T Cells from Patients with Silica and Asbestos-Related Diseases

To explore the effects of asbestos and silica on the human immune system, an experimental model of low-dose and long-term exposure was established using a human HTLV-1-immortalized polyclonal T cell line, MT-2 (MT-2Org). MT-2 cells were continuously exposed to asbestos at a concentration (10 μg/ml) which does not induce complete cell death during short-term exposure. After acquiring resistance to CB-induced apoptosis (designated MT-2Rst), an immunological comparison was made between the MT-2Org and MT-2Rst lines in terms of T cell receptor-Vβ (TcR-Vβ) expression. MT-2Rst cells showed excess expression of various TcR-Vβ, although TcR-Vβ-overpresenting cells were characterized as undergoing apoptosis due to first contact with CB. Patients with asbestos-related diseases (ARD), such as asbestosis and malignant mesothelioma, were compared with silicosis (SIL) patients as a disease control and with healthy donors (HD). SIL and ARD not only differed in their causative materials, silica and asbestos as mineral silicates, but also in terms of complications; autoimmune disorders in SIL and tumors in ARD. ARD patients showed a restricted overpresentation of TcR-Vβ without clonal expansion, whereas SIL patients revealed significant overpresentation of TcR-Vβ 7.2. These experimental and clinical analyses indicate the superantigenic and dysregulation of autoimmunity-inducing effects of asbestos and silica, respectively.