Immunological effects of silica and asbestos.

Silicosis patients (SILs) and patients who have been exposed to asbestos develop not only respiratory diseases but also certain immunological disorders. In particular, SIL sometimes complicates autoimmune diseases such as systemic scleroderma, rheumatoid arthritis (known as Caplan syndrome), and systemic lupus erythematoses. In addition, malignant complications such as lung cancer and malignant mesothelioma often occur in patients exposed to asbestos, and may be involved in the reduction of tumor immunity. Although silica-induced disorders of autoimmunity have been explained as adjuvant-type effects of silica, more precise analyses are needed and should reflect the recent progress in immunomolecular findings. A brief summary of our investigations related to the immunological effects of silica/asbestos is presented. Recent advances in immunomolecular studies led to detailed analyses of the immunological effects of asbestos and silica. Both affect immuno-competent cells and these effects may be associated with the pathophysiological development of complications in silicosis and asbestos-exposed patients such as the occurrence of autoimmune disorders and malignant tumors, respectively. In addition, immunological analyses may lead to the development of new clinical tools for the modification of the pathophysiological aspects of diseases such as the regulation of autoimmunity or tumor immunity using cell-mediated therapies, various cytokines, and molecule-targeting therapies. In particular, as the incidence of asbestos-related malignancies is increasing and such malignancies have been a medical and social problem since the summer of 2005 in Japan, efforts should be focused on developing a cure for these diseases to eliminate nationwide anxiety.

The mechanisms of vitamin K2-induced apoptosis of myeloma cells.

BACKGROUND AND OBJECTIVES: Physiologically, vitamin K compounds act as co-factors for g-carboxylation of selected glutamates at the N-terminus of prothrombin and some other coagulation factors. These congeners have some growth inhibitory effects of human neoplastic cells. Furthermore, vitamin K2 (VK2) cause apoptosis of some leukemic cells. In search for a new candidate agent to use in the maintenance treatment of myeloma, we analyzed the growth inhibitory effects and apoptosis-inducing capacity of VK2 in human myeloma cells. DESIGN AND METHODS: The growth of myeloma, lymphoma and non-lymphoid cells cultured with various concentrations of VK2 with or without dexamethasone or allopurinol was assayed. Flow cytometry was used to detect apoptotic cells, activated caspase-3 and -9, the generation of superoxide by hydroethidine, and mitochondrial membrane potential (E centym). In addition, the activation of apoptosis-inducing MAPK, p38 and JNK, release of cytochrome c from mitochondria, and change in the relative Bcl-XL/Xs expression balance were analyzed by Western blotting. RESULTS: Myeloma cells and B-cell lymphoma cells were sensitive to VK2. The growth inhibition was caused by apoptosis and activation of caspase-3. The generation of superoxide, and inhibitory effects of the xanthine oxidase inhibitor allopurinol, were demonstrated in myeloma cells. The phosphorylation of MAPK was increased by VK2 in myeloma cells. In addition, the mitochondrial apoptotic pathway was activated. INTERPRETATION AND CONCLUSIONS: VK2 may be a good candidate for myeloma patients, particularly patients who are not suitable candidates for intensive cytoreductive chemotherapy due to age and/or complications.

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 auto-immunity 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.

TRAIL expression up-regulated by interferon-gamma via phosphorylation of STAT1 induces myeloma cell death.

BACKGROUND: Although the cellular and molecular biological effects of interferon (IFN)-alpha have been well-investigated, the effects of IFN-gamma are less understood. MATERIALS AND METHODS: Eleven human myeloma cell lines with various myeloma-specific chromosomal translocations and overexpression of oncogenes were cultured with 1000 U/ml of IFN-gamma. In the KMS-20 cells, which showed growth inhibition due to IFN-gamma, trail expression, status of the Janus kinase (JAK)/STAT pathway were analyzed. RESULTS: KMS-20 cells showed marked up-regulation of trail, activation of STAT1 and TRAIL hyperproduction induced by IFN-gamma. CONCLUSION: The effects of IFN-gamma on growth inhibition of KMS-20 cells were characterized by activation of the JAK/STAT signalling pathway, particularly STAT1 phosphorylation, enhanced secretion of TRAIL, and auto/paracrine usage of secreted TRAIL to induce apoptotic cell death. From these results, IFN-gamma may be considered one of the drugs to be used in future multidrug chemotherapeutic regimens for myeloma patients.

Induction of CD69 antigen expression in peripheral blood mononuclear cells on exposure to silica, but not by asbestos/chrysotile-A.

While cases of silicosis are often complicated by various autoimmune disorders, patients with asbestosis develop malignant tumors such as lung cancer and malignant mesothelioma. These differences may derive from different biological effects, particularly on immunological cells, of silica and asbestos. To find differences between silica and asbestos, the early activation antigen, CD69, on T cells was examined because dysregulated and continuous activation of T cells may promote the survival of self-recognizing T cells. After cultivation of peripheral blood mononuclear cells with or without silica or chrysotile-A, an asbestos, only silica induced CD69 expression on the lymphocytes. This induction of CD69 expression was mediated by protein kinase C activation. In addition, cell-cell contact mediated by HLA-DR was more important than soluble factors secreted from silica-phagocytosed cells such as IL-1beta, IL-6, and IL-8, even though IL-6 and IL-8 were produced during the culture of PBMCs with silica and chrysotile-A. It should be examined how these activated, CD69-expressing lymphocytes affect other immune systems as well as alter themselves in terms of cytokine production and cell-cell interaction, leading to autoimmune disorders in silicosis patients.

[Dysregulation of Fas and related molecules in silicosis patients].

Silicosis patients not only suffer from respiratory disorders but also from autoimmune diseases. To clarify the mechanisms involved in the occurrence of the dysregulation of autoimmunity found in silicosis patients, we have been focusing on investigation of the Fas and Fas-related molecules in the Fas-mediated apoptotic pathway, since Fas is one of the most important molecules regulating autoimmunity in mainly T cells. Our findings showed that silicosis patients exhibited elevated serum soluble Fas levels, increased relative expression of soluble Fas and DcR 3 genes in peripheral blood mononuclear cells, other highly detectable variant messages of Fas transcripts, relatively decreased expression of several physiological inhibitors (Sentrin, I-Fline, ICAD/DFF45, and survivin), and dominancy of lower membrane Fas expressers in lymphocytes when compared with healthy volunteers. These parameters also constitute immunological factors with serum immunogulobulin G and the titer of anti-nuclear autoantibodies. In addition, anti-caspase 8 autoantibody and anti-Fas autoantibody were also detected in the serum from silicosis patients, and a functional assay showed that anti-Fas antibody stimulated Fas-mediated apoptosis. Taken together, we hypothesize that there are two subpopulations of silicosis lymphocytes. One is a long-term survival fraction including a self-recognizing fraction showing lower membrane Fas and inhibition of Fas/Fas ligand bindings in the extracellular region. The other is a fraction exhibiting apoptosis caused by silica/silicates, recruiting from bone marrow, showing higher membrane Fas and sensitive to anti-Fas autoantibody. Further investigations should be performed to confirm the effects of silica/silicates on the human immune system.

Expression of angiogenic factors including VEGFs and the effects of hypoxia and thalidomide on human myeloma cells.

Angiogenic factors are major causes of tumor progression in hematological malignancies, particularly multiple myeloma, as well as solid tumors. The introduction of thalidomide as an anti-angiogenic agent in myeloma treatment has demonstrated the importance of angiogenic factors in the progression of myeloma. However, the direct effects of angiogenic factors, particularly VEGFs, hypoxia, and thalidomide, on myeloma cells are not been documented. In this study, we demonstrate increased expression and production levels of VEGF in myeloma compared to non-myelomatous hematological lines, resistance to hypoxia and enhancement of VEGF-A production by hypoxia in myeloma, and direct growth inhibition of myeloma cells due to apoptosis and G1 arrest caused by TNFalpha upregulation induced by thalidomide. These findings may encourage the clinical use of anti-angiogenic agents for their cytostatic effects and the prevention of progression.

IL-6 is a key factor in growth inhibition of human myeloma cells induced by pravastatin, an HMG-CoA reductase inhibitor.

Although recent developments in initial chemotherapeutic regimens and stem cell transplantation have achieved improvements of initial remission for myeloma patients, relapse and recurrence are still major problems. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been developed for treating hyperlipidemia. Recently, there have been several reports concerning the effects of statins on cancer cells including liver, colon, leukemia, malignant B, stomach, and breast cells. In this study, the in vitro effects of pravastatin on human myeloma cells and the factors closely related to its growth inhibitory effects were examined. Although concentrations were higher than those used clinically, 4 out of 10 myeloma lines showed growth inhibition by pravastatin. The study of factors related to the inhibition indicated IL-6 is important. Indeed, rhIL-6 abolished pravastatin-induced growth inhibition in KMS-21BM cells which did not express IL-6. Statins may be useful in maintenance therapy for myeloma after the screening of IL-6 status.

Expression of HER family receptors and effects of anti-HER2-antibody on human myeloma cell lines.

We have recently studied expression of estrogen receptors and the growth inhibitory effects of antiestrogens on human myeloma cells. In myeloma chemotherapy, Antiestrogens in combination with other chemotherapeutic agents, may have applications in which melphalan/predonisolone still remains the standard treatment. In this study, we examined expression of HER family molecules in myeloma cells to clarify the possible usage of anti-HER2-monoclonal antibody in the treatment of myeloma. Although the mRNA levels of HER family genes analyzed by RT-PCR were significantly lower in myeloma cells than breast cancer cells, some cell lines expressed a certain amount of HER2 and HER4 proteins. In addition, an anti-HER2 monoclonal antibody, rhumAbHER2, caused significant growth inhibition in six out of eight myeloma cell lines studied and these inhibitory effects were similar to those in the breast cancer cells studied previously. The rhumAbHER2 induced up-regulation of p21 family CDK-Is (cyclin dependent kinase inhibitors) and down-regulation of VEGF genes. Moreover, combination treatment with antiestrogen had an additive growth inhibitory effect. Such analyses may provide for use of rhumAbHER2 in myeloma treatment for the future.

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.

Effects of an HMG-CoA reductase inhibitor, simvastatin, on human myeloma cells.

To look for new candidates for agents to use in maintenance therapy for myeloma patients, the growth inhibitory effects of a 3-hydroxy-3-mehtylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin), simvastatin, was analyzed using human myeloma cell lines. Several investigations have indicated growth reduction in certain lineages of cancer cells including one report on myeloma, and inhibitory effects of statins on GTPases and involving MAP-kinases. Most (12 out of 13) myeloma lines examined showed growth inhibition when cultured with various concentrations (1-30 microM) of simvastatin in a dose-dependent manner. Simvastatin in combination with other biological response modifiers such as ATRA or DEX had additional effects on growth. In addition, anti-oxides prevented the simvastatin-induced growth inhibition and apoptosis. Furthermore, myeloma cells treated with simvastatin clearly showed inactivation of various MAP-kinase pathways such as ERK1/2, MEK1/2, JNK, and p38. Based on these findings, statins may be suitable for clinical usage in maintenance therapy for myeloma patients.

Effects of genetic and nutritional factors on bone mineral density in young adults.

To estimate the genetic and dietary factors influencing bone mineral density (BMD) in young adults, a total of 53 healthy volunteers (HV) (age 20.89+/-1.34), from whom informed consent was obtained, answered a questionnaire on dietary factors and had DNA from peripheral blood mononuclear cells analyzed for single nucleotide polymorphisms (SNPs) for vitamin (Vit) D receptor (VDR), estrogen receptor alpha (ERalpha), interleukin 1 receptor antagonist (IL1RA), and apolipoprotein E (ApoE) genes. Daily intakes of Vit C, fiber, soybean and related foods, and green and yellow vegetables showed a correlation with % BMD. In addition, Vit B2 as well as Vit C, and vegetables were identified as important factors for BMD by Stepwise regression analysis. Among the SNPs analyzed, the B+ type of the VDR gene tended to be associated with a lower BMD, and pp type of the ER gene digested by the PvuII enzyme in females indicated a significantly lower BMD than that in males. In addition, these SNPs were also identified by factor analysis to be associated with BMD. These results suggested that a complex array of genetic factors, such as two or more SNPs or SNPs and gender, may be important to BMD.

Induction of apoptosis in human leukemia cells by naturally fermented sugar cane vinegar (kibizu) of Amami Ohshima Island.

Naturally fermented vinegar such as Kibizu (sugar cane vinegar in Amami Ohshima, Japan), Kurozu (black rice vinegar in Kagoshima, Japan), Kouzu (black rice vinegar in China) and red wine vinegar in Italy had potent radical-scavenging activity analyzed by DPPH method. For the elucidation of food factor for cancer prevention contained in naturally fermented vinegar, the induction of apoptosis in human leukemia cell HL-60 was investigated with sugar cane vinegar Kibizu. Fraction eluted by 40% methanol from Amberlite XAD 2 chromatography of sugar cane vinegar showed potent radical scavenging activity. The fraction also showed the activity repressing growth of typical human leukemia cells such as HL-60, THP-1, Molt-4, U-937, Jurkat, Raji and K-562. On the other hand, the fraction did not have any growth inhibition activity against human fetal lung cell TIG-1. The most potent radical-scavenging activity and the growth repression activity of the leukemia cell were observed in the same chromatographic fraction of methanol 40%. From cell sorting FACS analyses, electron microscopic observations and cytochemical staining of chromatin and nuclear segments in human leukemia cell HL-60 treated with the active fraction, it was concluded that apoptosis was induced in the leukemia cell by the fraction of sugar cane vinegar and resulted in the repression of growth of the human leukemia cells. Chromatographic fraction of sugar cane juice eluted by 20% methanol showed potent activities of radical-scavenging and growth repression of HL-60. These results led us the consideration that active components in sugar cane juice could be converted to more lipophilic compounds with activity to induce apoptosis in HL-60 by microbial fermentation with yeast and acetic acid bacteria.

Involvement of IL-10 and Bcl-2 in resistance against an asbestos-induced apoptosis of T cells.

To analyze the possibility that immunological alteration in asbestos-related diseases (ARDs) such as asbestosis (ASB) and malignant mesothelioma (MM) may affect the progression of cancers, a human adult T cell leukemia virus-immortalized T cell line (MT-2Org) was continuously exposed to 10 mug/ml of chrysotile-B (CB), an asbestos. After at least 8 months of exposure, the rate of apoptosis in the cells became very low and the resultant subline was designated MT-2Rst. The MT-2Rst cells were characterized by (i) enhanced expression of bcl-2, with regain of apoptosis-sensitivity by reduction of bcl-2 by siRNA, (ii) excess IL-10 secretion and expression, and (iii) activation of STAT3 that was inhibited by PP2, a specific inhibitor of Src family kinases. These results suggested that the contact between cells and asbestos may affect the human immune system and trigger a cascade of biological events such as activation of Src family kinases, enhancement of IL-10 expression, STAT3 activation and Bcl-2 overexpression. This speculation was partially confirmed by the detection of elevated bcl-2 expression levels in CD4 + peripheral blood T cells from patients with MM compared with those from patients with ASB or healthy donors. Further studies will be required to verify the role of T cells with enhanced bcl-2 expression in tumor progression induced by asbestos exposure.

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.

Detection of anti-topoisomerase I autoantibody in patients with silicosis.

OBJECTIVES: The aim of this study was to detect anti-topoisomerase I (anti-topo I) autoantibodies, which are known to be limited in systemic sclerosis patients, in silicosis patients with no clinical symptoms of autoimmune disease. METHODS: Serum anti-topo I autoantibodies were detected using ELISA. Differences in clinical parameters between patients with and without anti-topo I autoantibodies were analyzed. RESULTS: Seven of 69 patients had anti-topo I autoantibodies. These 7 patients showed elevated PaCO(2) values (P=0.0212), and inverse correlations between serum soluble Fas levels and PaCO(2) values were found. CONCLUSION: Anti-topo I autoantibodies were detected in 10.1% of silicosis patients without any clinical symptoms of autoimmune disease. The findings here suggest that the genesis of anti-topo I autoantibodies might be related to pulmonary involvement or lung fibrosis associated with progression of silicosis.