| Silicosis is characterized of chronic pulmonary fibrosis which is caused by prolonged inhalation of productive dust containing more than 10% quartz. It is of the most serious occupational disease, because the fibrosis is irreversible and incurable. Thus, it is significant to study the mechanism of silicosis caused by silica for the early diagnosis, treatment and prevention of silicosis.A growing number of studies have shown that oxidative stress and lung inflammatory response play key roles in the development of silicosis induced by silica dust. Recently, in vitro and in vivo experimental studies have demonstrated that high level of IL-1βinduced by silica dust, however, the mechanism of adverse health effects caused by IL-1βhas yet fully understood.To explore the mechanism and influencing factors of inflammatory response induce by different types of silica dust, we collected productive silica dust from a pottery factory and a tungsten mine in Jiangxi Province and a tin mine in Guangxi Province, different sizes of quartz dusts as experimental dusts. We selected differentiated THP-1 macrophages stimulated by PMA as target cells. as target cells to assess and compare the toxic and inflammatory responses induced by different types of dust, then combine with the vital status of dust-exposed workers to evaluate the pathogenic potentials of silica dust. This study was divided into three parts:(1) chemical elements of silica dust from different worksites and their effects on dust-exposed workers'health; (2) the injured and inflammatory responses induced by silica dust on macrophages and their associations with adverse health effects on dust-exposed workers; (3) the roles of IL-1βin inflammatory responses induced by quartz on macrophages.Part 1 Chemical elements of silica dust from different worksites and their effects on health of dust-exposed workersObjective:To explore chemical elememts of silica dust and their effects on dust-exposed workers' health.Methods:The productive slica dust were selcected from above mentioned three worksites. The content of free silica in silica dust were determined by pyrophosphate quality method and the contents of chemical elements were assayed by atomic absorption spectrometry and atomic fluorescence spectrometry. Cohort was established basing on the three worksites. The cohort included all dust-exposed workers who were employed for at least one year during 1960-1974 in the studied workplaces. Vital status of the cohort subjects were followed-up till the end of 2003. The causes of death were analyzed and the SMR was calculated based on the Chinese national mean mortality (1973-2003) from Health Ministry.Results:(1) The contents of free silica in silica dusts from pottery factory and tin mine were similar, determined as 43.39%,43.60%, respectively. the content of free silica in silica dust from tungsten mine is higher than that from pottery factory and tin mine, reaching 57.51%.(2) In the three worksites, the content of aluminum element (10.64%) in silica dust from pottery factory was highest, but the content of other elements were very low. The content of iron element was highest in silica dust from tungsten mine, reaching 3.47%, the content of zinc element was lowest,0.09%. the contents of calcium, zinc and arsenic were highest in tin mine dust, reaching 9.62%,0.33%,0.34%, respectively; but the content of aluminum element (2.80%)was lowest.(3) The number of dust-exposed workers in pottery factory was 907, among which 148 were diagnosed as pneumoconiosis. The incidence of pneumoconiosis was 2.82%. The SMRs from all cause death, pneumoconiosis, malignant tumors and lung cancer were 1.15, 85.26,0.58,0.77, respectively. In tungsten mine, the number of dust-exposed workers was 2350, of which 788 were diagnosed as pneumoconiosis. The incidence of pneumoconiosis was 8.44%. The SMRs from all cause death, pneumoconiosis, malignant tumors and lung cancer were 1.24,101.80,0.67,0.73, respectively. The number of dust-exposed workers in tin mine was 3108, of which 466 were diagnosed as pneumoconiosis. The incidence of pneumoconiosis was 2.42%. The SMRs from all cause death, pneumoconiosis, malignant tumors and lung cancer were 1.07,48.67,1.44,2.43, respectively.It is suggested that the content of silica has played a key role in the incidence of pneumoconiosis. The incidence of pneumoconiosis of dust-exposed workers in pottery factory was low but the cumulative total silia dust was relative high, in that the content of Al element in silica dust was higher than other two mineral mines. In addition, it is possible that there is correlation between the high SMRs of malignant tumors and the content of As element.Part 2 The injured and inflammatory responses induced by silica dusts on macrophages and their associations with adverse health effects on dust-exposed workersSection 1 The optimal model of IL-1βinduced by quartz (DQ12) on macrophagesObjective:To investigate the condition, time-course and dose-response relationship of IL-1βproduction induced by DQ12 on macrophages.Methods:Differentiated THP-1 macrophages stimulated by PMA were used as target cells. We selected three types of DQ12 which were DQ12-PM1(<1μm), DQ12-PM3-5(3-5μm), DQ12-PM5 (5μm), respectively. Differentiated THP-1 macrophages were then treated with DQ12 at concentrations of 900,600,300,150,75,37.5μg/ml for a series of time points (3,6,12,18,24 h). The levels of IL-1βinduced by macrophages were measured.Results:(1) Elevated trends of IL-1βlevel released by macrophages were observed at all concentrations of three types of DQ12 when exposure time reached 3,6,12,18 and 24 h. Especially, the levels of IL-1βinduce by DQ12-PM3-5 and DQ12-PM1 reached a peak at all concentrations when exposure time was at 6 h.(2) A positive dose-response relationship was observed between three types of DQ12 and the increased level of IL-1βinduced by macrophages at concentrations ranged from 75μg/ml to 300μg/ml. The release of IL-1βreached maximum level at concentration of 300μg/ml. Thus, we seleceted concentrations of 37.5,75,150 and 300μg/ml to do the following experiments.Section 2 The effects of sizes on the injured and inflammatory response induced by dust on macrophagesObjective:To explore the injured and inflammatory responses induced by different micron particle sizes of silica dust and evaluate the role of particle size in the pathway of inflammatory and fibrotic responses.Methods:Differentiated THP-1 macrophages stimulated by PMA were used as target cells. We selected two different sizes of quartz (DQ12-PM1,1μm; DQ12-PM3-5,3-5μm). Differentiated THP-1 macrophages were then treated with above four types of dust at concentrations of 300,150,75,37.5μg/ml for 6 h. Untreated cells were used as negative control. Cell viability, the release of reactive oxygen species (ROS) and the levels of IL-1β, TNF-α, IL-6, TGF-β1, IL-18 and IL-33 were determined to assess the inflammatory responses of silica dust.Results:The cell viability induced by these two types of silica dust on macrophages decreased in a dose-dependent manner. And dose-response relationships were observed between different sizes of dusts and the levles of ROS and inflammatory cytokines. The level of inflammatory cytokines such as IL-1β, TNF-α, IL-6 and IL-33 induced by DQ12-PM1 dust was slightly higher than DQ12-PM3-5 dust at concentrations of 37.5,75,150μg/ml, but which was lower than DQ12-PM3-5 at concentration of 300μg/ml.Section 3 The injured and inflammatory responses induced by productive silica dust on macrophages and their associations with adverse health effects on dust-exposed workersObjective:To assess the inflammatory responses induced by productive dust on macrophages and explore theire asoocations with adverse health effects on dust-exposed workers.Methods:Differentiated THP-1 macrophages were then treated with respirable silica dusts from above mentioned three worksites at concentrations of 300,150,75,37.5μg/ml for 6h. Standard quartz was used as positive control, and untreated cells as negative control. Other methods were same as the section 2 of part 2.Results:The cell viability decreased when the concentrations of silica dust samples increased. Clear dose-response relationships were observed between all silica dust samples from the three worksites and the release of IL-1β, TNF-α, IL-6, TGF-β1, IL-18 and IL-33. The silica dust from pottery factory resulted in the strongest decrease of cell viability, followed by tin mine and the weakest of tungsten mine. The levels of infalmmatory cytokines (IL-1β, TNF-αand IL-6) induced by silica dust from tungsten and tin mines were higher than that from pottery factory. The levels of IL-1βand TNF-αinduced by silica dust from tungsten and tin mines were higher than standard quartz at concentrations of 37.5and 75μg/ml, but the highest level of IL-1βwas induced by standard quartz at concentration of 300μg/ml. The levels of TNF-αinduced by silica dust from tungsten and tin mines were higher than standard quartz at all concentrations. However, silica dust from tungsten and tin mines were incapable of inducing the release of TGF-β1 and IL-18/IL-33, respectively.The levels of proinflammatory cytokines (IL-1β, TNF-αand IL-6) induced by silica dust from tungsten and tin mines on macrophages were higher than that from pottery factory whereaa lower level of TGF-β1. It is consistent with the results of the higher risk of silicosis in dust-exposed workers from tungsten and tin mines and lower risk in pottery factory workers. In conclusion, to some extent, it is suggested that IL-1βcould predict the fibrotic potency of silica dust.Part 3 The roles of IL-1βin inflammatory responses induced by quartz on macrophages.Objective:To investigate the roles of IL-1βin the pathogenesis of silicosis caused by silica.Methods:Differentiated THP-1 macrophages stimulated by PMA were used as target cells. Differentiated THP-1 macrophages were exposed to recombinant IL-1βat concentrations of 1600,800,400,200, 100pg/ml for 6h or 24h. In addition, differentiated THP-1 macrophages were pretreated with 50ng/ml of LPS for 6h, then which were exposed to quartz at concentrations of 300,150,75,37.5μg/ml for another 6h. Differentiated THP-1 macrophages were treated with both 2.5μg/ml IL-1βmonoclonal antibody and different sizes of quartz according to concentrations of 300,150,75,37.5μg/ml for 6h or 24h. the levels of IL-1β, TNF-αadn IL-6 in cell culture supernatant were determined to assess inflammatory responses.Results:(1) Clear dose-response relationships were observed between recombinant IL-1βand the release of TNF-αand IL-6 by macrophages. And the levels of TNF-αand IL-6 increased with the prolonged time course. However, recombinant IL-1βis incapable of eliciting IL-1βrelease from macrophages, and the level of recombinant IL-1βgradually decreased when the time extended from 6h to 24h.(2) Dose-response relationships were observed between quartz and the release of IL-1β, TNF-αand IL-6 by LPS pretreated macrophages at 6 h. The level of IL-1βreleased by LPS pretreated macrophages were significantly higher than that from non-LPS macrophages when cells were treated with silica dust.(3) The level of IL-1βinduced by quartz on macrophages was significantly reduced by IL-1βmonoclonal antibody at concentrations of 150,300μg/ml, which was lower than that form macrophages without IL-1βmonoclonal antibody. The blocking role of IL-1βmonoclonal antibody on the smaller size of quartz (DQ12-PM1) was more effective than the larger size of quartz (DQ-PM3-5). IL-1βmonoclonal antibody almost completely blocked the release of TNF-αand IL-6 induced by two sizes of quartz.
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