The sources of inflammatory mediators in the lung after silica exposure

The expression of 10 genes implicated in regulation of the inflammatory processes in the lung was studied after exposure of alveolar macrophages (AMs) to silica in vitro or in vivo. Exposure of AMs to silica in vitro up-regulated the messenger RNA (mRNA) levels of three genes [interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2)] without a concomitant increase in the protein levels. AMs isolated after intratracheal instillation of silica up-regulated mRNA levels of four additional genes [granulocyte/macrophage-colony stimulating factor (GM-CSF), IL-1beta, IL-10, and inducible nitric oxide synthase]. IL-6, MCP-1, and MIP-2 protein levels were elevated in bronchoalveolar lavage fluid. Fibroblasts under basal culture conditions express much higher levels of IL-6 and GM-CSF compared with AMs. Coculture of AMs and alveolar type II cells, or coculture of AMs and lung fibroblasts, in contact cultures or Transwell chambers, revealed no synergistic effect. Therefore, such interaction does not explain the effects seen in vivo. Identification of the intercellular communication in vivo is still unresolved. However, fibroblasts appear to be an important source of inflammatory mediators in the lung.     

Effect of silica and volcanic ash on the content of lung alveolar and tissue phospholipids

Silica or volcanic ash (VA) was administered to rats via intratracheal instillation and the changes in extracellular (i.e., lavage fluid) and tissue phospholipids, as well as various biochemical parameters, were monitored over a 6-month period. VA produced relatively minor (up to 2.8-fold) increases in lung tissue or lavage fluid phospholipids that were maximal at 1 month postinstillation. These increases were quantitatively similar to the increases in protein and DNA content of lung tissue and lavage fluid induced by VA and, thus, may be attributable to hypercellularity and accumulation of cellular breakdown products in the alveolar lumen. Instillation of silica produced a much greater (up to 11-fold) increase than VA in total phospholipid over time, primarily due to a 14-fold increase in phosphatidylcholine (PC). The accumulation of PC was more pronounced in the lavage fluid during the first month following silica instillation, but thereafter progressed more rapidly in the lung tissue. The relatively small increases (1.3- to 3.5-fold) in other phospholipids induced by silica appeared to be nonspecific, since they did not differ greatly from the increases in lung weight, DNA, and protein. Collectively, these results indicate that intratracheal instillation of silica induces selective accumulation of lung PC, implying enhanced synthesis and secretion of pulmonary surfactant from alveolar epithelial Type II cells into the lumen.     

Pulmonary phospholipidosis in rats respiring air containing diesel particulates

Rats chronically exposed to diesel particulates (dp) or given intratracheally a single dose of dp show increased levels of phospholipids in the lungs and in pulmonary lavage fluid. Pulmonary phospholipidosis is accompanied by increased lecithin levels and by increased palmitate content in lecithin of both lungs and pulmonary lavage fluid. A de novo increase of pulmonary and hepatic phospholipid (PL) formation was detected 5 days after rats were treated with dp. We hypothesize that a dp-stressed lung releases a pulmonary lipogenic factor (PLF), which stimulates hepatic lipogenesis. This was further tested by an in vitro study in which primary cultures of free hepatocytes were incubated with [2-14C]acetate and various molecular weight fractions of a pulmonary homogenate from rats. The results from these studies indicated that in rat lung homogenates a PLF exists of greater than 100,000 Da molecular mass. The results also indicate that respired air containing a dp concentration of greater than 750 micrograms dp/m3 of air would result in a mild phospholipidosis in the lung, whereas a dp dose in respired air of 250 micrograms dp/m3 of air for 2 years did not alter pulmonary PL content in rats.     

矽宁对实验性矽肺磷脂变化的影响

应用高效液相色谱分析法，对实验性矽肺大鼠的支气管肺泡灌洗液、肺泡巨噬细胞及肺组织的磷脂组分进行动态观察，并探讨抗矽肺新药矽宁对磷脂作用的特点。实验结果表明：实验性矽肺支气管肺泡灌洗液中的总蛋白及总磷脂含量伴随矽肺纤维化病变的进展而出现逐步增长的趋势。在这些生物样品的磷脂组分中，以ＰＣ含量增长最为明显，其次为ＰＥ及ＳＰＨ。矽宁治疗组的支气管肺泡灌洗液中磷脂含量明显低于矽肺对照组，且其ＰＥ／ＳＰＨ及ＰＣ／ＳＰＨ组分的比值高于矽肺对照组，但肺泡巨噬细胞中的这种比值都低于矽肺对照组。本实验提示矽宁的疗效作用可能与抑制磷脂合成、改变其各组分含量有一定关系。     

The role of complement in experimental silicosis

The role of the complement system in the pathogenesis of crystal-induced pulmonary inflammation and fibrosis was evaluated using a mouse model of silicosis and congenitally complement-deficient mice. Mice lacking the fifth component of complement (B10.D2/o) were compared to C5-sufficient animals (B10.D2/n) for pulmonary changes following intratracheal instillation of silica crystals. Complement-deficient mice demonstrated a significant reduction compared to complement-sufficient mice in both cell number and protein content of lung lavage fluid throughout the 12 weeks following silica exposure. Lung hydroxyproline content (indicative of collagen deposition) was equivalent for both strains and significantly higher than controls at all time points following silica instillation. Moreover, studies in vitro have shown that silica crystals are capable of activating complement via the alternative pathway. These studies indicate that the complement system may be responsible for some of the pulmonary inflammation, but not fibrosis elicited by silica exposure.     

N-acetylcysteine and deferoxamine reduce pulmonary oxidative stress and inflammation in rats after coal dust exposure

Coal dust inhalation induces oxidative damage and inflammatory infiltration on lung parenchyma. Thus, the aim of this study was to determine whether N-acetylcysteine (NAC) administered alone or in combination with deferoxamine (DFX), significantly reduced the inflammatory infiltration and oxidative damage in the lungs of rats exposed to coal dust. Forty-two male Wistar rats (200-250 g) were exposed to the coal dust (3mg/0.5 mL saline, 3 days/week, for 3 weeks) by intratracheal instillation. The animals were randomly divided into three groups: saline 0.9% (n=8), supplemented with NAC (20mg/kg of body weight/day, intraperitoneal injection (i.p.)) (n=8), and supplemented with NAC (20 mg/kg of body weight/day, i.p.) plus DFX (20 mg/kg of body weight/week) (n=8). Control animals received only saline solution (0.5 mL). Lactate dehydrogenase activity and total cell number were determined in the bronchoalveolar lavage fluid. We determined lipid peroxidation and oxidative protein damage parameters and catalase and superoxide dismutase activities in the lungs of animals. Intratracheal instillation of coal dust in the lungs of rats led to an inflammatory response and induced significant oxidative damage. The administration of NAC alone or in association with DFX reduced the inflammatory response and the oxidative stress parameters in rats exposed to coal dust.     

Effect of particle size of intratracheally instilled crystalline silica on pulmonary inflammation

Crystalline silica, known as a causal substance of silicosis, has been carefully evaluated for its carcinogenicity and fibrogenicity. In this study, we instilled crystalline silica of two different size (S(1.8) :1.80 microm (S.D. 2.0), S(0.7) :0.74 microm (S.D. 1.5)) into the trachea of rats to evaluate the size effects of the particles on pulmonary inflammation. S(1.8) and S(0.7) samples were administered to rats by a single intratracheal instillation (2 mg/ 0.4 ml saline). At three days, 1 wk and 1, 3 and 6 months after the instillation, the blood, bronchoalveolar lavage fluid (BALF), and pulmonary tissues were analyzed. Six images per HE-stained section were digitally captured and examined by the point counting method (PCM). Polymorphonuclear leukocyte (PMN)-in-blood specimens and cytospin specimens from BALF were stained immunohistochemically with BrdU. At six months after the instillation, the effects on inflammatory cells in the pulmonary tissues and BALF tended to be more marked in the rats instilled with S(1.8) than those instilled with S(0.7). Particularly, clear differences were observed in the number of inflammatory cells in BALF. Even if the particles are of the same chemical composition, the results suggest that, their biological effects vary depending on their particle size. Therefore, when such particles are used in workplaces, strict control systems should be established according to the risks present by different sizes of particles.     

Expression of heme oxygenase-1 in the lungs of rats exposed to crystalline silica

Oxidative stress is thought to be the pathogenesis of pulmonary fibrosis induced by particles, and heme oxygenase-1 (HO-1) protects lung tissue against oxidative stress. We hypothesized that HO-1 is also associated with oxidative lung injury caused by exposure to particles. The present study was conducted to investigate the time course of HO-1 expression of lungs exposed to crystalline silica in vivo. Male Wistar rats were administered 1 mg or 2 mg of crystalline silica suspended in saline by a single intratracheal instillation and were sacrificed at 3 d, 1 wk, 1 month, 3 months and 6 months of recovery time. The expression of HO-1 was observed by western blot analysis and immunostaining. Protein levels of HO-1 were increased compared to the controls at 3 d, and from 1 month to 6 months following intratracheal instillation of 2 mg of crystalline silica. The levels of HO-1 were increased compared to the controls from 1 month to 6 months following intratracheal instillation of 1 mg of crystalline silica. Many HO-1 positive cells were found particularly in the alveolar macrophages during immunostaining. These findings suggest that HO-1 is related to lung injury arising from exposure to crystalline silica.     

低剂量染石英尘大鼠支气管肺泡冲洗液磷脂谱变化的研究

本文报道了给大鼠气管注入低剂量石英尘后其BALF中总磷脂、DPPC、PI、PS、Sph和LPC含量的动态变化。实验分为对照组和石英组,分别于染尘后7天、15天、60天和120天分组剖检。结果表明,总磷脂、DPPC染尘后7～120天一直呈上升趋势,Sph、PI也明显增加,PS、LPC无明显改变。并对结果进行了讨论。     

Response of mouse lung to carbon deposition during injury and repair.

Increased respiratory disease and daily mortality rates are associated with higher levels of fine particulate air pollutants. We examined the possibility that deposition of even inert particles to previously injured lungs may accentuate pulmonary damage by investigating how the lung handles small carbon particles delivered during acute injury or during fibrotic repair. Mice received 2 mg carbon by intratracheal instillation into lungs already showing acute injury, 3 days after bleomycin (BL), or into lungs with fibrosis, 4 weeks after BL. At 3 days after BL, injury to the type I alveolar epithelium resulted in high protein levels in lavage fluid. Instilling carbon at this time induced a large increase in inflammatory cells, though many particles reached the interstitium, and a high proportion was retained up to 16 weeks later. However, fibrosis in these mice was equal to that found after BL alone. In the mice that received carbon 4 weeks after bleomycin, fibrotic repair had already occurred, and the epithelial surface was restored before particle instillation. After carbon, the subsequent inflammatory reaction cleared most particles, little reached the interstitium, and carbon retained at 16 weeks was not different from that in the carbon-only group. Instilling particles into fibrotic lung did not induce additional fibroblast growth or collagen production. The results indicate that instillation of fine particulates to the alveoli at a time of epithelial injury results in increased translocation to the interstitium. However, deposition of pure carbon into injured lungs does not further stimulate an ongoing fibrotic process, although it alters the patterns of particle deposition and retention in the lung.     

Complement facilitates macrophage phagocytosis of inhaled iron particles but has little effect in mediating silica-induced lung inflammatory and clearance responses.

Complement-mediated mechanisms are known to play a role in pulmonary inflammation and clearance responses to some types of inhaled particles. The present studies were undertaken to investigate the role of complement in mediating pulmonary inflammation and/or phagocytosis as a function of particle clearance in rats exposed to silica or carbonyl iron (CI) particles. Both particle types were shown to be weak activators of serum complement in vitro. In these studies, normal and complement-depressed (CVF-treated) rats were exposed to aerosols of CI or silica particles for 6 hr at 100 mg/m3. Following exposure, alveolar fluids and cells from sham and dust-exposed animals were recovered by bronchoalveolar lavage (BAL) at several time periods postexposure and measured for a variety of biochemical and cellular indices. In addition, pulmonary macrophages were cultured and studied for morphology and phagocytosis. Our results showed that CI exposure did not produce cellular or biochemical indices of pulmonary inflammation, either in normal or complement-depleted rats. However, fewer phagocytic macrophages were recovered from the lungs of CVF-treated, CI-exposed rats than from normal exposed animals. In contrast, silica inhalation produced a sustained PMN inflammatory response in the lungs of exposed rats, measured up through 1 month postexposure, along with significant increases in BAL fluid levels of LDH, protein, and alkaline phosphatase (P less than 0.05) and deficits in pulmonary macrophage phagocytic functions. Cobra venom factor (CVF) treatment prior to exposure in rats had no significant effect upon the silica-induced parameters, suggesting that complement may not play an important role in the acute pulmonary response to silica. The results indicate that complement may play a role in mediating CI-related macrophage clearance responses but has little effect upon sustained silica-induced pulmonary inflammatory parameters.     

多壁碳纳米管致大鼠急性肺毒性

[目的]探讨多壁碳纳米管（multi-wall carbon nanotubes，MWCNTs）对Wistar大鼠的急性肺毒性作用。[方法]用脱氧核苷酸钠盐（deoxyribonucleic acid sodium salt，DNA钠盐）提高MWCNTs的分散度，设3个浓度组（2、10、20mg／mL）和空白对照组及溶剂对照组，采用气管滴注的方法染毒，一次滴注量为1．5mL／kg体重，每天1次，连续滴注3d，染毒结束后24h，计算肺灌洗液中各类白细胞百分比，测定乳酸脱氢酶（LDH）、碱性磷酸酶（AKP）、灌洗液中总蛋白（TP）等细胞毒性及谷胱苷肽（GSH）、丙二醛（MDA）、超氧化物岐化物（SOD）等细胞氧化损伤指标，并观察肺病理改变。[结果]中、高剂量染毒组肺灌洗液中细胞分类计数中性粒细胞和淋巴细胞较对照组有不同程度的升高，差异有统计学意义；随着染毒剂量的增加，肺泡灌洗液中TP、AKP、LDH和MDA含量随之升高，而GSH和SOD含量随之降低，且各个指标的变化呈一定的剂量-效应关系。肺组织病理显示炎症性病变，主要表现为单核细胞和淋巴细胞浸润，尘细胞聚集，黏膜损伤和血管出血等。[结论]MWCNTs具有致大鼠急性肺毒性的作用。     

Persistent biological reactivity of quartz in the lung: raised protease burden compared with a non-pathogenic mineral dust and microbial particles.

This study assessed the potential harmfulness of particles in the lung by measuring their ability to elicit and maintain an inflammatory response and to damage lung tissue. It compared the inflammogenicity of two nondurable, biological particulates (Corynebacterium parvum and zymosan) with a pathogenic mineral dust (quartz) and a nonpathogenic dust (titanium dioxide) by dosing rats via the intratracheal route and measuring the consequent alveolitis. The magnitude and duration of the inflammatory response were assessed by measuring the total number of leucocytes and the percentage of neutrophils obtained by bronchoalveolar lavage. Two key functional parameters of the lavaged leucocytes--ability to degrade fibronectin and production of plasminogen activator--were also measured. A marked inflammatory response had occurred by one day after instillation, characterised by increases in total leucocyte numbers and percentage of neutrophils in the bronchoalveolar lavages, with all four test materials. In all but the quartz exposed animals, the inflammation subsided rapidly thereafter, approaching control levels by 15 days after injection; in the quartz exposed animals the alveolitis persisted for up to 30 days. All of the inflammogens generated chemotaxins in rat serum in vitro and so, by analogy, might also be expected to generate chemotactic activity in alveolar lining fluid which could contribute to the generation of an inflammatory response. The cellular inflammatory response was accompanied by a concomitant increase in the proteolytic activity of the bronchoalveolar lavage leucocytes but production of plasminogen activator remained unchanged. In vitro exposure to the inflammogens had no effect on the proteolytic activity against fibronectin or on the plasminogen activator activity of bronchoalveolar leucocytes.     

Persistent biological reactivity of quartz in the lung: raised protease burden compared with a non-pathogenic mineral dust and microbial particles

This study assessed the potential harmfulness of particles in the lung by measuring their ability to elicit and maintain an inflammatory response and to damage lung tissue. It compared the inflammogenicity of two nondurable, biological particulates (Corynebacterium parvum and zymosan) with a pathogenic mineral dust (quartz) and a nonpathogenic dust (titanium dioxide) by dosing rats via the intratracheal route and measuring the consequent alveolitis. The magnitude and duration of the inflammatory response were assessed by measuring the total number of leucocytes and the percentage of neutrophils obtained by bronchoalveolar lavage. Two key functional parameters of the lavaged leucocytes--ability to degrade fibronectin and production of plasminogen activator--were also measured. A marked inflammatory response had occurred by one day after instillation, characterised by increases in total leucocyte numbers and percentage of neutrophils in the bronchoalveolar lavages, with all four test materials. In all but the quartz exposed animals, the inflammation subsided rapidly thereafter, approaching control levels by 15 days after injection; in the quartz exposed animals the alveolitis persisted for up to 30 days. All of the inflammogens generated chemotaxins in rat serum in vitro and so, by analogy, might also be expected to generate chemotactic activity in alveolar lining fluid which could contribute to the generation of an inflammatory response. The cellular inflammatory response was accompanied by a concomitant increase in the proteolytic activity of the bronchoalveolar lavage leucocytes but production of plasminogen activator remained unchanged. In vitro exposure to the inflammogens had no effect on the proteolytic activity against fibronectin or on the plasminogen activator activity of bronchoalveolar leucocytes.     

Pulmonary toxicity of components of textile paint linked to the Ardystil syndrome: intratracheal administration in hamsters.

OBJECTIVES: It was hypothesised from an epidemiological investigation that a formula change from Acramin FWR (a polyurea) to Acramin FWN (a polyamide-amine) had led to severe pulmonary disease in textile printing sprayers in SPAIN AND ALGERIA. To verify this, the pulmonary toxicity of the components of the paint systems involved was assessed in experimental animals. METHODS: Individual components and relevant mixtures, diluted in phosphate buttered saline, were given by intratracheal instillation of 2 ml/kg to hamsters. Pulmonary toxicity was assessed on days 3, 7, 14, 28, and 92 after a single intratracheal instillation, by histology and by measuring wet and dry lung weight, protein concentration, the activities of lactate dehydrogenase, alkaline phosphatase, beta-N-acetyl-glucosaminidase, and gamma-glutamyltransferase, inflammatory cell number and distribution in bronchoalveolar lavage fluid (BALF), and hydroxyproline content in dried lung tissue. RESULTS: Based on the doses that killed 50% of the animals (LD50s), the various components were found to be 10 to 1250 times more toxic when given intratracheally than when given orally (according to reported oral LD50s in rats). Acramin FWN, Acramin FWR, Acrafix FHN, or their mixtures caused lung damage. Protein concentration, enzyme activities, total cell number, and percentage of polymorphonuclear neutrophils were increased in BALF during the first week after intratracheal instillation. Lung weights remained high for at least a month. Histology showed inflammatory cell infiltration and subsequent fibrosis with collagen deposition. This finding was confirmed by an increased hydroxyproline content in dried lung tissue. Acramoll W did not show toxic effects. CONCLUSIONS: The study suggests that there is no major difference, in hamsters, between the acute intratracheal toxicity of Acramin FWR and that of Acramin FWN. Consequently, there is no simple toxicological explanation for the epidemiological hypothesis. However, the pulmonary toxicity of these non-irritant polymeric compounds is surprisingly high. The Ardystil disaster and these results should serve as a strong warning that conventional toxicity testing of chemicals does not necessarily protect workers against respiratory toxicity.     

Hyaluronic acid in bronchoalveolar lavage in rats exposed to quartz.

Hyaluronic acid, a connective tissue component, in bronchoalveolar lavage fluid (BAL) is correlated with decreased lung volumes in sarcoidosis. To investigate whether hyaluronic acid could be a marker of fibrosis in another interstitial lung disease, silicosis, the level of the substance in BAL fluid from rats exposed to crystalline silica (n = 3), amorphous silica (n = 3), and in one sham injected rat was measured. There was an increase in the total number of alveolar cells recovered in the rats exposed to crystalline silica and also a pronounced increase in the proportions of neutrophils and lymphocytes. In addition, the concentration of hyaluronic acid was high in this group of rats, and electron microscopic investigation of the lungs showed fibrosis. Thus hyaluronate in BAL fluid in rats exposed to crystalline silica seems to be a possible marker of fibrotic changes.     

Expression of clara cell secretory protein in the lungs of rats exposed to crystalline silica in vivo

It has been theorized that Clara cell secretion protein (CCSP) plays a critical role in regulating the acute inflammatory response in the lung. We hypothesized that CCSP is also related to lung injury induced by occupational dust. The present study was conducted to investigate the time course of the expression of CCSP in lungs exposed to crystalline silica in vivo. Male Wistar rats were administered 1 mg or 2 mg of silica suspended in saline by a single intratracheal instillation and were sacrificed at 3 d, 1 wk, 1 month, 3 months and 6 months of recovery time. The expression of CCSP was observed by RT-PCR and western blot analysis. Exposure to 2 mg of silica decreased in levels of CCSP mRNA at 3 d, 1 wk, 1 month and 6 months following intratracheal instillation. The protein level of CCSP in silica-exposed rats was decreased at 3 d, 7 d and 1 month after a single instillation of 2 mg. The decreases in CCSP at the acute phase in this experiment suggest that CCSP may regulate the acute injury of the lung exposed to silica.     

Hyaluronic acid in bronchoalveolar lavage in rats exposed to quartz

Hyaluronic acid, a connective tissue component, in bronchoalveolar lavage fluid (BAL) is correlated with decreased lung volumes in sarcoidosis. To investigate whether hyaluronic acid could be a marker of fibrosis in another interstitial lung disease, silicosis, the level of the substance in BAL fluid from rats exposed to crystalline silica (n = 3), amorphous silica (n = 3), and in one sham injected rat was measured. There was an increase in the total number of alveolar cells recovered in the rats exposed to crystalline silica and also a pronounced increase in the proportions of neutrophils and lymphocytes. In addition, the concentration of hyaluronic acid was high in this group of rats, and electron microscopic investigation of the lungs showed fibrosis. Thus hyaluronate in BAL fluid in rats exposed to crystalline silica seems to be a possible marker of fibrotic changes.     

大鼠慢性支气管炎模型在颗粒物PM_(2.5)毒性研究中的应用

目的研究颗粒物PM2.5对慢性支气管炎大鼠与正常大鼠的急性毒效应,以及这种毒效应在模型组和正常组之间的差异。方法采用气管滴注的方法,对慢性支气管炎模型组和正常组连续染毒3天,在最后一次染毒24小时后处死动物,分析肺灌洗液中白蛋白(ALB)、乳酸脱氢酶(LDH)、碱性磷酸酶(AKP)、丙二醛(MDA)和谷胱甘肽(GSH)的含量。结果颗粒物PM2.5对模型组和正常组动物均产生急性毒作用,且存在剂量反应关系;模型组肺灌洗液中ALB、LDH、AKP和MDA四项指标在高剂量组均明显高于正常组(P<0.05),而GSH低于正常组(P<0.05)。结论颗粒物PM2.5可以导致大鼠急性肺损伤,并且慢性支气管炎模型组比正常组对颗粒物PM更加易感。