Tamayo Hatayama

Dysregulation of the immune system caused by silica and asbestos

Silica and asbestos cause pneumoconioses known as silicosis and asbestosis, respectively, that are each characterized by progressive pulmonary fibrosis. While local effects of inhaled silica particles alter the function of alveolar macrophages and sequential cellular and molecular biological events, general systemic immunological effects may also evolve. One well-known health outcome associated with silica exposure/silicosis is an increase in the incidence of autoimmune disorders. In addition, while exposure to silica—in the crystalline form—has also been seen to be associated with the development of lung cancers, it remains unclear as to whether or not silicosis is a necessary condition for the elevation of silica-associated lung cancer risks. Since asbestos is a mineral silicate, it would be expected to also possess generalized immunotoxicological effects similar to those associated with silica particles. However, asbestos-exposed patients are far better known than silicotic patients for development of malignant diseases such as lung cancer and mesothelioma, and less so for the development of autoimmune disorders. With both asbestos and crystalline silica, one important dysregulatory outcome that needs to be considered is an alteration in tumor immunity that allows for silica- or asbestos- (or asbestos-associated agent)-induced tumors to survive and thrive in situ. In this review, the immunotoxicological effects of both silica and asbestos are presented and contrasted in terms of their abilities to induce immune system dysregulation that then are manifest by the onset of autoimmunity or by alterations in host-tumor immunity.

Asbestos-Induced Cellular and Molecular Alteration of Immunocompetent Cells and Their Relationship with Chronic Inflammation and Carcinogenesis

Asbestos causes lung fibrosis known as asbestosis as well as cancers such as malignant mesothelioma and lung cancer. Asbestos is a mineral silicate containing iron, magnesium, and calcium with a core of SiO2. The immunological effect of silica, SiO2, involves the dysregulation of autoimmunity because of the complications of autoimmune diseases found in silicosis. Asbestos can therefore cause alteration of immunocompetent cells to result in a decline of tumor immunity. Additionally, due to its physical characteristics, asbestos fibers remain in the lung, regional lymph nodes, and the pleural cavity, particularly at the opening sites of lymphatic vessels. Asbestos can induce chronic inflammation in these areas due to the production of reactive oxygen/nitrogen species. As a consequence, immunocompetent cells can have their cellular and molecular features altered by chronic and recurrent encounters with asbestos fibers, and there may be modification by the surrounding inflammation, all of which eventually lead to decreased tumor immunity. In this paper, the brief results of our investigation regarding reduction of tumor immunity of immunocompetent cells exposed to asbestos in vitro are discussed, as are our findings concerned with an investigation of chronic inflammation and analyses of peripheral blood samples derived from patients with pleural plaque and mesothelioma that have been exposed to asbestos.

Intramolecular epitope spreading among anti-caspase-8 autoantibodies in patients with silicosis, systemic sclerosis and systemic lupus erythematosus, as well as in healthy individuals

Dysregulation of apoptosis through the Fas‐Fas ligand pathway is relevant in autoimmune disease onset. We recently reported elevated serum levels of sFas in patients with silicosis, systemic sclerosis (SSC) and systemic lupus erythematosus (SLE), and proposed a block of apoptosis in the pathogenesis. The disturbance of apoptosis in lymphocytes including autoreactive clones could induce autoantibody production. Since autoantibodies directed against unknown antigens are present in the sera of these patients, the sera samples were examined for the presence of autoantibodies directed to caspase‐8.

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.

Expression of protein gene product 9·5 (PGP9·5)/ubiquitin-C-terminal hydrolase 1 (UCHL-1) in human myeloma cells

The neuron cytoplasmic protein gene product 9·5 (PGP9·5)/ubiquitin‐C‐terminal hydrolase 1 (UCHL‐1) protein is a thiol protease that recognizes and hydrolyzes a peptide bond at the C‐terminal of ubiquitin, and is involved in the processing of ubiquitin precursors and ubiquinated proteins. Although this molecule is known as a specific tissue marker for the neuroendocrine system, many reports have indicated that PGP9·5 is a marker for certain tumour types, such as cancer of the lung, colon, and pancreas. The expression of PGP9·5 in myeloma cells was examined. PGP9·5 seemed to be expressed specifically in myeloma cells as compared with other haematological malignant cells. In addition, in myeloma cells subjected to growth‐factor starvation, the upregulation of PGP9·5 was observed in association with that of p27Kip1, a cyclin‐dependent‐kinase inhibitor, although the upregulation caused by irradiation was milder. In contrast, the hypoxic culture of myeloma cells induced down‐regulation of PGP9·5. These results suggested that PGP9·5 may be a good marker for myeloma among haematological malignancies. In addition, it may indicate certain cellular features of myeloma cells, such as sensitivity to proteasome inhibitors.

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.

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.

Effects of all-trans retinoic acid (ATRA) on human myeloma cells.

All-trans retinoic acid (ATRA) is a natural oxidative metabolite of Vitamin A (retinol) and is known to be a regulator of cell proliferation differentiation, especially in various malignant cells. The cyto-differentiating action of ATRA has led to its usage in the treatment of several malignancies, particularly acute promyelocytic leukemia (APL). There have been many reports regarding the cell biological effects of ATRA on human myeloma cells and a few clinical trials. Most of these reports have revealed growth inhibition by ATRA mediated by down-regulation of the IL-6/IL-6R auto/paracrine loop, and upregulation of p21/Cip1. Here, we review previous reports and introduce experimental results obtained using various myeloma cell lines established in our laboratory.

Soluble Interleukin-2 Receptor as an Indicator of Immunological Disturbance Found in Silicosis Patients

Silicosis patients (SILs) possess not only respiratory disorders but also alterations in autoimmunity. To determine an early indicator of immunological disturbance in SILs, the role of serum-soluble interleukin (IL)-2 receptor (sIL-2R) was analyzed. Of ten SILs, immunological clinical parameters such as immunoglobulin (Ig) G, complements, the titer of autoantibodies including anti-nuclear antibodies (ANA), anti-Scl-70 antibody (Ab) and anti-centromere (CM) Ab, and experimental indicators such as serum-soluble Fas, serum IL-2, CD25+ cells in CD4+ or CD8+ fractions, and sIL-2R were divided from respiratory parameters such as % vital capacity (%VC), percentage of forced expiratory volume in 1 second (FEV1.0%) and v25/Ht (liter/second/m(body height) by a correlation assay. Additionally, a stepwise regression test showed that sIL-2R was correlated with Ig G, ANA and anti-CM Ab. Furthermore, factor analysis revealed that sIL-2R contributed to the subpopulation of SILs with poorer immunological status in the absence of alterations in respiratory status. By defining healthy donors as 1, SILs as 2 and patients with systemic sclerosis as 3 for immunopathological progression status as metric variables, sIL2R and ANA showed a strong positive correlation. This suggests that sIL-2R is a good clinical indicator of immunological disturbance found in SILs without clinical manifestations of any disturbance in autoimmunity. Further analysis using a large-scale number of patients should be performed to confirm these findings.

Secretory IgA in saliva and academic stress.

Several reports have proposed that the concentration of secretory immunoglobulin A (S-IgA) in saliva is an indicator of psychological stress. With this in mind, we decided to examine it in 10 second year medical student volunteers at Kawasaki Medical School course between May 4 and July 13, 2000 and discussed the relationship between S-IgA and the stress from academic examinations. Saliva was collected three times (on rising, at forenoon, and at bedtime) every Thursday. During this period, sporadic academic examinations were held twice and term-end examinations occurred during the last two weeks. Results showed the concentration of S-IgA significantly higher at the on rising time-point than at the other two time-points. There was also a tendency for the S-IgA level in saliva to be higher on the day before academic examinations and during them and lower on the days between these examinations. In addition, daily variations in the S-IgA concentration sometimes seemed to be disturbed by other academic stress. Therefore, it may be possible to use this measurement to monitor psychological stress in students and workers.