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 1:1/4 to 1/2. 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 the T cell receptor Vbeta 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 microg/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-Vbeta (TcR-Vbeta) expression. MT-2Rst cells showed excess expression of various TcR-Vbeta, although TcR-Vbeta-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-Vbeta without clonal expansion, whereas SIL patients revealed significant overpresentation of TcR-Vbeta 7.2. These experimental and clinical analyses indicate the superantigenic and dysregulation of autoimmunity-inducing effects of asbestos and silica, respectively.

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.

Activation of human CD4+CD45RA+ T cells by chrysotile asbestos in vitro.

Chrysotile asbestos stimulates T lymphocyte subsets. Cell surface CD4 or CD45RA expression in peripheral blood mononuclear cells (PBMC) was downregulated after incubation with chrysotile asbestos in vitro temporarily. The percentage of CD4+CD45RA+ cells and mean fluorescence intensity in CD4 or CD45RA decreased after incubation with asbestos and returned to the original level after 24 h of incubation, which suggests that chrysotile asbestos activates CD4+CD45RA+ cells. No change was observed in CD29 expression. An increased percentage of IL-2R positive cells and an elevated intracellular Ca++ level were also indicative of the activation of PBMC by chrysotile asbestos.

Crocidolite asbestos suppresses the differentiation of HL-60 cells induced by DMSO.

This study was undertaken to determine if the biological function of inducers for cell differentiation is affected by asbestos fibers, which are sometimes deposited in human tissues. Protein kinase C activity, c-myc protein expression and cell surface CR3 expression were used as the markers of cell differentiation. The function of dimethylsulfoxide (DMSO), an inducer of cell differentiation, was suppressed by the co-culturing of crocidolite asbestos, because DMSO reacted with the hydroxyl radical released after the stimulation with crocidolite and spent itself. Superoxide dismutase (SOD) inhibited the effect of crocidolite, reacting rapidly with .O2- before the secondary release of .OH. Asbestos fibers deposited in tissues may inhibit the function of inducers which stimulate immature cells to differentiate, because such inducers frequently are also radical scavengers.

Human cd4+ cd45ra+ lymphocytes-T can be stimulated by crocidolite, anthophyllite and amosite asbestos invitro.

Crocidolite, anthophyllite and amosite asbestos stimulate T lymphocyte subsets. Cell surface CD4 or CD45RA expression in peripheral blood mononuclear cells (PBMC) was downregulated temporarily after incubation with asbestos in vitro. The percentage of CD4+ CD45RA+ cells significantly decreased 12 h after incubation with asbestos, suggesting activation of the cells. An early increase in the intracellular Ca++ level was also indicative of activation. An elevated Ca++ level was observed after incubation in both PBMC and purified T cell fractions.

Activation-induced cell death in human peripheral blood lymphocytes after stimulation with silicate in vitro.

Silica and related substances such as silicate have been proven to possess "adjuvant effects". We have previously reported a finding of polyclonal human T cell activation induced by silicate as a superantigen in vitro. In this study, we observed activation-induced cell death in human lymphocytes after stimulation with chrysotile, a kind of silicate. Apoptotic cells were detected flow cytometrically using the TUNEL assay, and the maximum appearance of TUNEL positive cells occurred on day 4 of incubation. Simultaneously the manifestation of small-sized cells in the specimens increased implying apoptosis. Fas expression on lymphocytes increased to day 3 of incubation with chrysotile, and then spontaneously decreased on day 4 when remarkable apoptosis could be detected. Based on these results it is conceivable that activation-induced cell death occurred through Fas-Fas ligand interaction in lymphocytes after stimulation with silicate in a concentration with which no acute cytotoxicity has been detected. Whether and how the repeated apoptosis in definite clones of lymphocytes causes the induction of sFas synthesis need clarification.

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.

Inactive cathepsin B-like enzyme in human melanoma culture medium.

An inactive cathepsin B-like enzyme with a molecular mass of 40 kD was found in a human melanoma culture medium. The inactive form of this enzyme was converted into an active form with a molecular mass of 28 kD by pepsin treatment. This activated cathepsin B-like enzyme had almost the same characteristics regarding molecular size, substrate specificity, dependence on chemical reagents, and Km values as intracellular cathepsin B. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by electroblotting with an antiserum against cathepsin B yielded inactive cathepsin B-like enzyme fractions which showed two immunoreactive bands with molecular masses of 40 and 28 kD, respectively. On the other hand, alkali treatment of the inactive cathepsin B-like enzyme fractions released a cysteine proteinase inhibitor with a molecular mass of 12 kD. These data suggest that these inactive cathepsin B-like enzymes in melanoma culture medium are present not only in the precursor form, but that they are also present as enzyme-inhibitor complexes, both of which can be activated enzymatically in vitro.

Evaluation of cases with silicosis using the parameters related to Fas-mediated apoptosis.

To establish a new clinical index for immunological abnormalities occurring in silicosis, several clinical parameters related to Fas-mediated apoptosis; i.e., membrane Fas expression on peripheral blood lymphocytes (mFas), serum soluble Fas levels (sFas), serum soluble Fas ligand levels (sFasL), and soluble/membrane Fas mRNA expression ratios (s/mFas ExR) in peripheral blood mononuclear cells (PBMC) were investigated. Fifty-eight silicosis patients with no clinical symptoms of autoimmune diseases were the subjects of this study. Factor analysis was performed using 12 clinical parameters including four parameters related to Fas-mediated apoptosis. Two common factors were identified. Factor 1 which consisted of the following parameters; duration of exposure, symptomatic dyspnea, PO2, PCO2, and A-aDO2, should be designated as the respiratory factor for cases with silicosis. The parameters of factor 2 were serum IgG, sFas with high factor loading, titer of ANA, sFasL, and s/mFas ExR. These parameters of factor 2 are indicative of the immunological disorders occurring in silicosis cases. Some cases exhibited abnormalities in parameters of factor 2 but not factor 1. The factor analysis clearly demonstrated that the parameters related to Fas-mediated apoptosis should be the most beneficial for predicting the pre-clinical status of complicated autoimmune diseases in silicosis.

Man-made mineral fibers induce apoptosis of human peripheral blood mononuclear cells similarly to chrysotile B.

The aim of this study was to investigate whether man-made mineral fibers (MMMF) induce apoptosis of human peripheral blood mononuclear cells (PBMC), as we recently demonstrated for chrysotile B. In vitro cultivation of PBMC with various MMMF as well as chrysotile B clearly produced apoptotic cells. The alteration of the expression for apoptosis related genes at the mRNA level during in vitro cultures of PBMC with various MMMF revealed upregulation of Flice and Apaf-1 genes and down regulation of TNF receptor 1 and Bid genes. These results indicate that MMMF induce apoptosis of PBMC in a similar manner to chrysotile B. However, the process may be mediated not only by the Fas-related apoptotic pathway but also a mitochondrial pathway. Thus, one should be aware that respiratory and immunological abnormalities may occur in workers who are exposed to MMMFs.

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-IbA 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 examination 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.

[Effects of asbestos on the cell cycle of PHA-stimulated human peripheral blood lymphocytes].

It is well known that persons exposed to asbestos display systemic immunological alterations: impaired lymphocyte response to PHA, the appearance of autoantibodies and elevation of the serum immunoglobulin level. The authors intend in this report to determine whether asbestos fibres (crocidolite, chrysotile and amosite) have any effect on the cell cycle of human lymphocytes after PHA stimulation. Peripheral blood mononuclear (PBM) cells were incubated with 10 micrograms/ml PHA for 2 days. After PHA stimulation, a decrease in the percentage of cells in the G0 phase and an increase in that of cells in the G1A, G1B and S phases was observed. When asbestos fibres or titanium dioxide (TiO2) was added to the culture dish at the beginning of the experiment, the progression of the cell cycle was inhibited only by asbestos fibres, in which case the percentage of cells in the G0 phase was significantly increased, while those of cells in the G1B and S phases were significantly decreased. The experiment for determining the critical period for inhibition of blastogenic response revealed that no inhibition was demonstrable when crocidolite fibre was added at 24 hr after PHA stimulation. Although the mechanisms of asbestos-fibre-mediated suppression remain to be clarified, these results showed that asbestos fibres act at an early stage (G0 phase) of the cell cycle and suppress the PHA stimulation of PBM cells.

[Suppressive effect of crocidolite fibers on the differentiation of HL-60 cells induced with DMSO].

HL-60 cells were derived from a patient with myelocytic leukemia, and are known to be in the promyelocytic stage and to differentiate into myelocytes or granulocytes after induction with several materials, e.g., DMSO, retinoic acid, and interferons. The authors intended in this report to determine whether asbestos fibers have any effect on the differentiation processes of HL-60 cells induced with DMSO. The cells were induced to differentiate by incubation with 1.25% DMSO for 4 days. A decrease in the percentage of c-myc-protein-positive cells and an increase in the number of C3bi receptor (CD11b) positive cells were observed after differentiation. When crocidolite (50 micrograms/ml) was added to the culture dishes at the beginning of the experiments, the differentiation was inhibited. An increase in the percentage of c-myc-protein-positive cells and a decrease in that of C3bi-receptor-positive cells were observed compared with the cells induced with DMSO alone. It has been reported that DMSO activates phospholipid- and Ca2(+)-dependent protein kinase and induces the differentiation of HL-60 cells. The mechanisms of inhibition by crocidolite fibers of the effects of DMSO remain to be clarified, but the strength of activation of phospholipid- and Ca2(+)-dependent protein kinase may play an important role in the following induction of cell differentiation.

[Effect of crocidolite fibers on the cathepsin B-like enzyme activity in HL-60 cells].

In a previous study, we found that crocidolite (an asbestos fiber) had an inhibitory effect on the differentiation process of HL-60 cells induced by dimethyl sulfoxide (DMSO). Here we describe the cathepsin B-like enzyme HL-60 cells and the changes of its activity during cell differentiation with or without crocidolite treatment. The cathepsin B-like enzyme in HL-60 cell extracts had almost the same characteristics and the already known cathepsin B as for the pH optima and the effects of proteinase inhibitors. The cathepsin B-like enzyme activity increased according to the cell differentiation induced by DMSO, however, its activity was depressed by crocidolite treatment.

[Effect of shampoo on human hair cystatin extraction].

It is known that cystatins have not only regulatory functions in cellular protein catabolism, but also other physiological functions against viral and/or bacterial infection. Recently we have fond cystatin in the human hair shaft. The activity of human hair cystatin in a water extract of pieces of it reached a plateau after 3-hour incubation. The activity of cystatin was compared among three different lengths of hair. The shorter the hair was cut, the more hair cystatin was extracted. Cystatin was more easily extracted by adding a water-soluble detergent. On the basis of these results, the loss of human hair cystatin from the hair shaft seemed to be affected by the condition of the hair surface and by the detergents in hair shampoo.

[The analysis of peripheral blood lymphocytes of patients with silicosis and effects of silica in vitro].

It is well known that patients with silicosis are frequently associated with hyperglobulinemia, RA or PSS. It is also suggested that silica can drive and activated the immune system. In this study, we intended to measure the percentage of CD4+CD45RA+ cells in the peripheral blood of patients with silicosis and to investigate whether silica can actually activate human lymphocytes in vitro or not. Peripheral blood from 45 patients with silicosis was stained with OKT4 and 2H4 monoclonal antibodies, and measured by FACS analysis at 1st day and also checked similarly after 6 days-incubation without patients' sera. The intracellular Ca++ level was also checked after incubation with the normal human lymphocytes with silica by FACS analysis, using Fluo3-AM. CD4+CD45RA+ T cells decreased significantly in number. One to 5 minutes after the incubation with silica, intracellular Ca++ level increased markedly, which means the activation of lymphocytes in vitro. These results revealed that silica can activate human lymphocytes in vitro, but the role in vivo remains to be clarified.

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 retinoic acid on the differentiation of THP-1 cell lines containing aneuploid or diploid chromosomes.

The effects of retinoic acid on the differentiation of human monocytic leukemia cell lines containing aneuploid (THP-1-Cs5) or diploid chromosomes (THP-1-R) were studied and compared. The induction of cell adhesion to a substratum, phagocytosis of sheep red blood cells (SRBC) or IgG-coated SRBC, pinocytosis of dextran sulfate, and NBT dye reduction by the cells were examined. The occurrence of these processes was much greater in RA-treated THP-1-Cs5 cells than in RA-treated THP-1-R cells. Of all these functional activities, the most remarkable differences between the two cell types were seen for cell adhesion and phagocytosis of SRBC. Morphological changes in RA-treated THP-1-Cs5 cells were observed by light and electron microscopy. RA-treated THP-1-Cs5 cells had a moderately-developed Golgi apparatus, and abundant lysosomes, mitochondria and lipid droplets in the cytoplasm. Among various retinoids examined, RA was the strongest inducer of the differentiation of the THP-1-Cs5 cells into mature cells. These findings suggest that THP-1-Cs5 cells which contain aneuploid chromosomes are more efficiently functionally differentiated by RA than are THP-1-R cells.