Intratracheal Instillation Of Silica Research Articles

Fermented cordyceps powder alleviates silica-induced pulmonary inflammation and fibrosis in rats by regulating the Th immune response

BackgroundSilicosis is an important occupational disease caused by inhalation of free silica and is characterized by persistent pulmonary inflammation, subsequent fibrosis and lung dysfunction. Until now, there has been no effective treatment for the disease due to the complexity of pathogenesis. Fermented cordyceps powder (FCP) has a similar effect to natural cordyceps in tonifying the lung and kidney. It has started to be used in the adjuvant treatment of silicosis. This work aimed to verify the protective effects of FCP against silicosis, and to explore the related mechanism.MethodsWistar rats were randomly divided into four groups including the saline-instilled group, the silica-exposed group, the silica + FCP (300 mg/kg) group and the silica + FCP (600 mg/kg) group. Silicosis rat models were constructed by intratracheal instillation of silica (50 mg). Rats in the FCP intervention groups received the corresponding dose of FCP daily by intragastric gavage. Rats were sacrificed on days 7, 28 and 56 after treatment, then samples were collected for further analysis.ResultsFCP intervention reduced the infiltration of inflammatory cells and the concentration of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) at days 7, 28, 56, and decreased the expression of collagen, α-smooth muscle actin (α-SMA) and fibronectin (FN) at days 28 and 56 in the lung of silicosis rats. FCP also decreased the immune response of Th1 and Th17 at days 7, 28, 56 and inhibited the enhancement of the Th2 response at day 56.ConclusionsFCP intervention could alleviate silica-induced pulmonary inflammation and fibrosis, the protective effect may be achieved by reducing Th1 and Th17 immune responses and inhibiting the enhancement of the Th2 response.

Yangqing Chenfei formula alleviates silica-induced pulmonary inflammation in rats by inhibiting macrophage M1 polarization

BackgroundYangqing Chenfei formula (YCF) is a traditional Chinese medicine formula for early-stage silicosis. However, the therapeutic mechanism is unclear. The purpose of this study was to determine the mechanism for the effects of YCF on early-stage experimental silicosis.MethodsThe anti-inflammatory and anti-fibrotic effects of YCF were determined in a silicosis rat model, which was established by intratracheal instillation of silica. The anti-inflammatory efficacy and molecular mechanisms of YCF were examined in a lipopolysaccharide (LPS)/interferon (IFN)-γ-induced macrophage inflammation model. Network pharmacology and transcriptomics were integrated to analyze the active components, corresponding targets, and anti-inflammatory mechanisms of YCF, and these mechanisms were validated in vitro.ResultsOral administration of YCF attenuated the pathological changes, reduced inflammatory cell infiltration, inhibited collagen deposition, decreased the levels of inflammatory factors, and reduced the number of M1 macrophages in the lung tissue of rats with silicosis. YCF5, the effective fraction of YCF, significantly attenuated the inflammatory factors induced by LPS and IFN-γ in M1 macrophages. Network pharmacology analysis showed that YCF contained 185 active components and 988 protein targets, which were mainly associated with inflammation-related signaling pathways. Transcriptomic analysis showed that YCF regulated 117 reversal genes mainly associated with the inflammatory response. Integrative analysis of network pharmacology and transcriptomics indicated that YCF suppressed M1 macrophage-mediated inflammation by regulating signaling networks, including the mTOR, mitogen-activated protein kinases (MAPK), PI3K-Akt, NF-κB, and JAK-STAT signaling pathways. In vitro studies confirmed that the active components of YCF significantly decreased the levels of p-mTORC1, p-P38, and p-P65 by suppressing the activation of related-pathways.ConclusionYCF significantly attenuated the inflammatory response in rats with silicosis via the suppression of macrophage M1 polarization by inhibiting a “multicomponent-multitarget-multipathway” network.

Nicotinamide Mononucleotide Ameliorates Silica-Induced Lung Injury through the Nrf2-Regulated Glutathione Metabolism Pathway in Mice

Nicotinamide mononucleotide (NMN) is a natural antioxidant approved as a nutritional supplement and food ingredient, but its protective role in silicosis characterized by oxidative damage remains unknown. In this study, we generated a silicosis model by intratracheal instillation of silica, and then performed histopathological, biochemical, and transcriptomic analysis to evaluate the role of NMN in silicosis. We found that NMN mitigated lung damage at 7 and 28 days, manifested as a decreasing coefficient of lung weight and histological changes, and alleviated oxidative damage by reducing levels of reactive oxygen species and increasing glutathione. Meanwhile, NMN treatment also reduced the recruitment of inflammatory cells and inflammatory infiltration in lung tissue. Transcriptomic analysis showed that NMN treatment mainly regulated immune response and glutathione metabolism pathways. Additionally, NMN upregulated the expression of antioxidant genes Gstm1, Gstm2, and Mgst1 by promoting the expression and nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf2). Gene interaction analysis showed that Nrf2 interacted with Gstm1 and Mgst1 through Gtsm2. Promisingly, oxidative damage mediated by these genes occurred mainly in fibroblasts. In summary, NMN alleviates silica-induced oxidative stress and lung injury by regulating the endogenous glutathione metabolism pathways. This study reveals that NMN supplementation might be a promising strategy for mitigating oxidative stress and inflammation in silicosis.

Identification of key pathways and genes in the progression of silicosis based on WGCNA

Silicosis, induced by inhaling silica particles in workplaces, is one of the most common occupational diseases. The prognosis of silicosis and its consequent fibrosis is extremely poor due to limited treatment modalities and lack of understanding of the disease mechanisms. In this study, a Wistar rat model for silicosis fibrosis was established by intratracheal instillation of silica (0, 50, 100 and 200 mg/mL, 1 mL) with the evidence of Hematoxylin and Eosin (HE) and Masson staining and the expressions of inflammatory and fibrotic proteins of rats’ lung tissues. RNA of lung tissues of rats exposed to 200 mg/mL silica particles and normal saline for 14 d and 28 d was extracted and sequenced to detect differentially expressed genes (DEGs) and to identify silicosis fibrosis-associated modules and hub genes by Weighted gene co-expression network analysis (WGCNA). Predictions of gene functions and signaling pathways were conducted using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. In this study, it has been demonstrated the promising role of the Hippo signaling pathway in silicosis fibrosis, which will be conducive to elucidating the specific mechanism of pulmonary fibrosis induced by silica and to determining molecular initiating event (MIE) and adverse outcome pathway (AOP) of silicosis fibrosis.

Gas6 or Mer deficiency ameliorates silica-induced autophagosomes accumulation in mice lung

Published evidences have shown that autophagy plays an important role in silica-induced lung inflammation and collagen deposition. Our previous study found that the level of growth arrest-specific protein 6 (Gas6) in bronchoalveolar lavage fluid was increased after silica exposure. However, it is unclear whether Gas6 is involved in the regulation of silica-induced autophagy dysfunction. In this study, we observed an autophagosomes accumulation in wild-type C57BL/6 (WT) mice lung after silica intratracheal instillation and then investigated whether genetic loss of Gas6 (Gas6−/−) could ameliorate it. Our data showed that Gas6−/− mice exhibited a limited autophagosomes accumulation from days 7–84 after silica exposure, revealed by reduced induction and increased degradation of autophagosomes in mice lung tissue. Interestingly, silica particles could elevate the expression of Mer receptor, which was significantly decreased in Gas6−/− mice (P < 0.05). Furthermore, we found that Mer deficiency (Mer−/−) could also reduce the formation of autophagosomes and restore the function of impaired lysosomes in silica-treated mice. Taken together, our results indicate that genetic loss of Gas6 attenuates silica-induced autophagosomes accumulation partly through down-regulating the expression of Mer receptor. Targeting Gas6/Mer-mediated autophagy pathway may provide a novel insight into the prevention and therapy of silica-induced pulmonary fibrosis.

Efferocytosis mediated by TAM receptors maintains lung homeostasis and controls inflammation in a mouse model of silicosis

Abstract Alveolar macrophages (AMs) are key cells for the establishment of pulmonary homeostasis. Efferocytosis is one of the main regulatory mechanisms for maintaining tolerance and homeostasis. The TAM receptor family (Tyro3, Axl and MerTk) mediates efferocytosis and inhibits proinflammatory pathways through Gas6 or Protein S binding to phosphatidylserine on apoptotic cells. Here we investigated how lung macrophages are regulated by TAM receptors in homeostasis and during silicosis. We found that LPS-stimulated AMs have an impaired ability to secrete IL-1b, IL-6, IFN-b, and MCP-1, but produce high levels of TNF-a and lower levels of IL-10, compared to bone marrow derived macrophages (BMDM). During homeostasis, AMs from wild type (WT) mice showed high expression of Axl, MerTk, and Gas6 by RT-PCR. Compared to WT mice, Axl−/− and MerTk−/− mice had more airway cells, less TGF-b and IL-10, and higher levels of IL-17 and KC within BAL. In addition, both KO mice had a higher frequency and number of AMs in the BAL, and secreted high levels of MCP-1, IFN-b, GM-CSF, and IL-10 following LPS stimulation compared to WT mice. Fifteen days after intra-tracheal silica instillation (SiO2; 20 mg), Axl−/− and MerTk−/− mice lost more body weight, and had increased lung weight, and percentage and number of neutrophils in the BAL compared to WT mice. Furthermore, both KO mice had a decreased percentage of AMs, and higher levels of KC, TNF-α and TGF-β but lower levels of IL-10 in the BAL compared to WT mice. Finally, Axl−/− mice had higher levels of IL-1β and IL-6 in the BAL than MerTk−/− mice. Our data suggest that efferocytosis mediated by Axl and MerTk receptors plays an important role in maintaining homeostasis and controlling inflammation in the pulmonary mucosae.

The clinical application value of ultrasound-guided percutaneous lung biopsy in the diagnosis of peripheral lung lesions of silicosis

Objective To determine the clinical application value of percutaneous lung biopsy guided by ultrasound in the diagnosis of peripheral lung lesions of silicosis. Methods Experimental silicosis was produced in rabbits by the intratracheal administration of silica with non-exposure method. Imaging changes were observed in 36 rabbits on 60 days after intratracheal instillation of silica. To contrast with CT results, percutaneous lung biopsy of peripheral lesions was guided by ultrasound. The success rate of sufficient material, the diagnosis rate of coincidence between biopsy and pathology, and the incidence of complications were calculated. The biopsy with sufficient material, biopsy findings coincided with pathological results and no complications were defined as strictly success of the puncture. The baseline data and monitoring index were compared between successful biopsy group and unsuccessful biopsy group. Each rabbit was intravenously administrated by 10 000 U of heparin for the antiocoagulation and sacrificed by fast injection of 10% KCl through jugular vein catheterization. Specimens from lung tissue were collected and stained with hematoxylin-eosin. Pathological changes of lung tissue were observed through an optical microscope. Results Of 36 silicosis rabbits, peripheral lung lesions of silicosis were observed in 30 rabbits. Biopsy procedures were performed with ultrasound guidance in 30 rabbits. The total success rate of biopsy was 70% (21/30). The success rate of sufficient material was 93% (28/30), the diagnosis rate of coincidence between biopsy and pathology 86%(24/28), and the incidence of complications was 10% (3/30) respectively. Compared with failure group, peripheral lesions in successful biopsy group were bigger in size, closer to the chest wall, and lower respiratory rate, the difference was statistically significant (P 0.05). The pathological results showed typical silicosis nodules. Silica-exposed rabbit also showed signs of vascular remodeling with pulmonary artery muscularization, vascular occlusion, and medial thickening. Conclusions Ultrasound-guided percutaneous lung biopsy is an effective and safe method in peripheral lung lesions of silicosis. It can serve as an important technical means to monitor the evolution of silicosis lesions and provide reference for the diagnosis of silicosis. Observation of small vascular changes in silicosis nodules by lung biopsy is helpful for clinical intervention treatment. Key words: Ultrasonography; Silicosis; Vascular remodeling; Biopsy, needle

Therapeutic effects of adipose-tissue-derived mesenchymal stromal cells and their extracellular vesicles in experimental silicosis

BackgroundSilicosis is an occupational disease that affects workers who inhale silica particles, leading to extensive lung fibrosis and ultimately causing respiratory failure. Mesenchymal stromal cells (MSCs) have been shown to exert therapeutic effects in lung diseases and represent an alternative treatment for silicosis. Recently, it has been suggested that similar effects can be achieved by the therapeutic use of extracellular vesicles (EVs) obtained from MSCs. The aim of this study was to investigate the effects of adipose-tissue-derived MSCs (AD-MSCs) or their EVs in a model of silicosis.MethodsSilicosis was induced by intratracheal instillation of silica in C57BL/6 mice. After the onset of disease, animals received saline, AD-MSCs, or EVs, intratracheally.ResultsAt day 30, AD-MSCs and EVs led to a reduction in collagen fiber content, size of granuloma, and in the number of macrophages inside granuloma and in the alveolar septa. In addition, the expression levels of interleukin 1β and transforming growth factor beta in the lungs were decreased. Higher dose of EVs also reduced lung static elastance when compared with the untreated silicosis group.ConclusionsBoth AD-MSCs and EVs, locally delivered, ameliorated fibrosis and inflammation, but dose-enhanced EVs yielded better therapeutic outcomes in this model of silicosis.

Regulation of silicosis formation by lysophosphatidic acid and its receptors

ABSTRACTSilicosis is a serious occupational disease characterized by lung fibrosis that is caused by long-term inhalation of silica-containing fine particles. Lysophosphatidic acid (LPA) and LPA1/3 plays a role in lung fibrosis. Until recently, there has been little research investigating the role of LPA and LPA receptors (LPAR) in silica-induced development of pulmonary fibrosis. In this study, we evaluated the hypothesis that LPA and LPA1/3 may play a role in silicosis pathogenesis using rat silicosis models induced by intratracheal instillation of silica, and randomly divided into control, silica, and VPC-12249 groups. LPA serum and bronchoalveolar lavage fluid (BALF) levels were quantified by ELISA. α-smooth muscle actin (α-SMA), type I and III collagen protein expression was quantified by western blotting (WB), and type I and III collagen mRNAs detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Lung hydroxyproline (HYP) levels were detected using alkaline hydrolysis, with hematoxylin and eosin (H&E) and picrosirius red staining used for pathological examination. In vitro experiments showed that LPA stimulated fibroblasts proliferated in a time and dose-dependent manner and promoted expression of α-SMA, and type I and III collagen. Moreover, LPA serum and BALF levels increased in silica-instilled rats. In vivo and in vitro experiments revealed that α-SMA expression and collagen deposition reduced significantly after VPC-12249 treatment, and histopathological results show VPC-12249 alleviates silicosis progression. In conclusion, our findings suggest that LPA promotes the proliferation, transformation, and collagen synthesis of fibroblasts, and that LPA-LPA1/3 are involved in the development of silicosis and may serve as novel therapeutic targets for treatment.

Pranlukast, a Cysteinyl Leukotriene Type 1 Receptor Antagonist, Attenuates the Progression but Not the Onset of Silica-Induced Pulmonary Fibrosis in Mice

Background: Although cysteinyl leukotrienes (CysLTs) have been implicated in the etiology of acute inflammatory diseases, recent studies have suggested that they also directly stimulate fibroblasts. However, their precise role in the pathogenesis of pulmonary fibrosis is unclear. Methods: In this study, we evaluated the effect of both short- and long-term treatment with pranlukast, a CysLT type 1 (CysLT₁) receptor antagonist, on silica-induced pulmonary fibrosis in mice, which is characterized by persistent progression of fibrosis in the chronic phase. Pranlukast (30 mg/kg/day) was administered orally to mice for 2 or 10 weeks after intratracheal silica instillation. Results: Pranlukast treatment for 10 weeks significantly attenuated the progression of pulmonary fibrosis, and decreased the content of CysLTs and LTB₄, which were markedly increased in the bronchoalveolar lavage fluid (BALF) and lung tissues of silica-instilled mice in the chronic phase. However, pranlukast treatment for 2 weeks neither affected the acute inflammatory response induced by silica instillation nor inhibited the onset of fibrosis. The expression of TGF-β1 and TNF-α was not affected by pranlukast treatment for either 2 or 10 weeks. Conclusions: Pranlukast attenuates the progression of pulmonary fibrosis in the chronic phase but has no effect on the acute inflammatory response or on the onset of pulmonary fibrosis. The antifibrotic effect of pranlukast may be exhibited by antagonizing the direct profibrotic effect of CysLTs, without affecting the expression of other profibrotic cytokines such as TGF-β1 and TNF-α, and also by decreasing the production of CysLTs and LTB₄.

Murine pulmonary inflammation model: a comparative study of anesthesia and instillation methods

Various techniques have been utilized historically to generate acute pulmonary inflammation in the murine system. Crystalline silica exposure results in acute inflammation followed by pulmonary fibrosis. Methods of exposure are varied in their techniques, as well as types of anesthesia. Therefore, the current study sought to compare the effects of two major anesthesia (isoflurane and ketamine) and three routes of instillation, intranasal (IN), intratracheal (IT), and trans-oral (TO), on markers of inflammation. Mice were anesthetized with isoflurane or ketamine and instilled IN with silica or phosphate-buffered saline (PBS). Mice were sacrificed and lavaged after 3 days. To assess inflammation, alveolar cells were assessed by cytospin and lavage fluid was analyzed for inflammatory cytokines and total protein. While all parameters were increased in silica-exposed groups, regardless of anesthesia type, there were significant increases in neutrophils and total protein in mice anesthetized with ketamine, compared to isoflurane. In comparing instillation techniques, mice were anesthetized with isoflurane and instilled IN, IT, or TO with silica. Increases were observed in all parameters, except tumor necrosis factor-α, following IT silica instillation as compared to the IN and TO instillation groups. In addition, fluorescent microsphere uptake by alveolar macrophages supported the notion that all methods of instillation were uniform, but IT had significantly greater dispersion. Taken together, these data show that each method of exposure tested generated significant inflammation among the silica groups, and any differences in parameters or techniques should be taken into consideration when developing an animal model to study pulmonary diseases.

Exposure In Vivo to Silica or Lipopolysaccharide Produces Transient or Sustained Upregulation, Respectively, of PYPAF7 and MEFV Genes in Bronchoalveolar Lavage Cells in Rats

A family of proteins containing PAAD [for PYRIN, AIM (absent in melanoma), apoptosis-associated protein speck-like protein containing a caspase recruitment domain, and death domain] domain was found to be involved in modulating inflammatory responses, by its ability to regulate nuclear factor (NF)-κB and procaspase-1 activation. In this study, intratracheal instillation of silica in rats was found to produce transient upregulation of mRNA levels of the PAAD family of proteins, PYPAF7 (PYRIN containing Apaf1-like protein; Apaf stands for apoptosis activating factor) and MEFV (for Mediterranean fever), in bronchoalveolar lavage (BAL) cells. The levels were markedly elevated at 4 h, returning to basal levels by 24 h. In contrast, intratracheal instillation of LPS produced a sustained upregulation of the two genes in BAL cells. In vitro exposure of BAL cells to silica or lipopolysaccharide (LPS) produced no changes in the expression of these genes, indicating that silica or LPS exposure in vivo induces some factors that are responsible for the upregulation of PYPAF7 and MEFV. The mRNA levels of these two genes in peripheral blood monocytes and PMN following LPS exposure did not change, indicating that AM and peripheral blood cells show similar response to LPS exposure in vitro. This study provides the basis for a physiological model to study the effects of these two genes in modulating the inflammatory response after particle exposure.

Activation of telomerase by silica in rat lung

By measuring the activity of telomerase in a silica-instilled rat lung, the study found a significant increase in telomerase activity compared to that of the control. Pneumoconiosis displays the characteristics of fibroblast-proliferation and accumulation of collagen, which finally causes the pathologic changes of irreversible and progressive fibrosis of the lung. On the basis of the hypothesis that cellular proliferation may trigger telomerase-activity, the experiment was carried out with telomerase-activation in silicosis. Silica-instilled rat lungs showed increased activity of telomerase, which was measured by TRAP (telomeric repeat amplification protocol) assay, at the time of the 1st, 5th and 8th week after intratracheal instillation of silica in vivo. However, no activity was shown in silica-co-cultured fibroblast in vitro. By summarizing these results, the activity of telomerase is thought to be a very sensitive marker for the evaluation of pathogenicity, showing cellular immortalization in an experimental silicosis model.

USE OF TETRANDRINE TO DIFFERENTIATE BETWEEN MECHANISMS INVOLVED IN SILICA- VERSUS BLEOMYCIN-INDUCED FIBROSIS

Animals exposed to silica or bleomycin ( BLM) develop pulmonary fibrosis. Tetrandrine (TT) has been shown to inhibit stimulant-induced macrophage respiratory burst and effectively reduce silica-induced lung injury. The present study employed TT as a probe to assess the differences in mechanisms involved in silica- and BLM-induced pulmonary responses. Rats received a single intratracheal instillation of silica (40 mg/ rat, sacrificed 4 wk postexposure) or BLM (1 mg/kg or ~0.25 mg/rat, sacrificed up to 2 wk postexposure). TT was administered orally at 18 mg/kg, 3 times/wk for desired time periods beginning 5 d before silica or BLM exposure. Both the silica and BLM exposures resulted in a significant increase in lung weight, total protein, lactate dehydrogenase (LDH), and phospholipids (PL) content in the acellular fluid from the first lavage, and hydroxyproline content in the lung tissue. Alveolar macrophages (AM) isolated from rats exposed to silica or BLM exhibited significant increases in secretion of interleukin-1 (IL-1), tumor necrosis factor (TNF-alpha) , and transforming growth factor (TGF-beta). TT treatment significantly lowered the silica- or BLM-induced increase in lung weight, while marginally reducing the release of IL-1 and TNF-alpha by AM. TT, however, markedly inhibited the silica-induced increase in the acellular protein, LDH and PL, hydroxyproline content, and the production of TGF- by AM but had no marked effect on these same parameters in BLM-exposed rats. Histological examination of rats exposed to BLM for 14 d showed pulmonary inflammation and fibrosis. TT treatment had only a small effect on limiting the extent of these lesions and did not significantly affect their severity. In summary, data indicate that many inflammatory and fibrotic effects of in vivo silica exposure are substantially attenuated by TT, whereas the stimulation by BLM is only marginally affected by this drug. Since TT acts to attenuate AM-mediated reactions, these results suggest that AM may play a pivotal role in silica-induced fibrotic development and may be less involved in the pathogenesis of BLM-induced fibrosis.

L-arginine uptake and metabolism by lung macrophages and neutrophils following intratracheal instillation of silica in vivo.

Nitric oxide (NO) has been associated with lung inflammation following exposure to silica. L-arginine can be converted to NO and L-citrulline by nitric oxide synthase (NOS), or into urea and L-ornithine by arginase. We tested the hypothesis that after instillation of silica into rat lungs in vivo, lung inflammatory cells increase L-arginine metabolism by both NOS and arginase, which is associated with an increase in L-arginine uptake. We isolated lung inflammatory cells 3 d after silica or saline (control) exposure. The uptake of [3H]L-arginine at 24 h by cells from silica-exposed lungs (73.9 +/- 4.8%) was significantly greater than uptake by control cells (24.7 +/- 2.2%; P < 0.05) and was a saturable process. The greater [3H]L-arginine uptake by cells from silica-exposed lungs was associated with greater NO and urea production than by control cells. The uptake of [3H]L-arginine by cells from control or silica-exposed lungs was blocked in a dose-dependent manner by L-ornithine (an inhibitor of L-arginine transport) and by Nomega-nitro-L-arginine methyl ester (L-NAME) (an NOS inhibitor), but not by L-valine (an arginase inhibitor). The production of NO by cells from silica-exposed lungs was completely blocked by L-NAME. The addition of L-arginine to media resulted in dose-dependent production of NO and urea. The results show that lung inflammatory cells increase L-arginine uptake and metabolism by both NOS and arginase following in vivo silica exposure. The increase in L-arginine uptake may represent a mechanism to maintain an intracellular supply of this amino acid. NO can react to generate peroxynitrite, a potential mediator of lung injury following silica exposure.

Silica induced Expression of IL-1beta, IL-6, TNF-beta, TGF-alpha, in the Experimental Murine Lung Fibrosis

Background: Silica-induced lung diseases is characterized by the accumulation of inflammatory cells at early stage and fibrosis in pulmonary parenchyma and interstitium at late stage. As a consequence of inflammation, silicosis is accompanied with the expansion of interstitial collagen and the formation of fibrotic nodule. In this process, several kinds of lung cells produce cytokines which can amplify and modulate pulmonary fibrosis. The alveolar macrophage is a potent source of proflammatory cytokines and growth factor. But in the process of silicotic inflammation and fibrosis, there are many changes of the kinetics in cytokine network. And the sources of cytokines in each phase are not well known. Method: 2.5 mg of silica was instillated into the lung of C57BL/6J mice. After intratracheal instillation of silica, the lungs were removed for imunohistochemical stain at 1, 2, 7 day, 2, 4, 8, 12 week, respectively. We investigated the expression of IL-1, IL-6, TNF- and TGF- in lung tissue. Results: 1) The expression of IL-6 increased from 1 day after exposure to 8 weeks in vascular endothelium. Also peribronchial area were stained for IL-6 from 7 days and reached the peak level for 4 weeks. 2) The IL-1 was expressed weakly at the alveolar and peribronchial area through 12 weeks. 3) The TNF- expressed strongly at alveolar and bronchial epithelia during early stage and maintained for 12 weeks. 4) TGF- was expressed strongly at bronchial epithelia and peribronchial area after 1 week and the strongest at 8 weeks. Conclusion: The results above suggests IL-6, TNF- appear to be a early inflammatory response in silica induced lung fibrosis and TGF- play a major role in the maintenance and modulation of fibrosis in lung tissue. And the regulation of TNF- production will be a key role in modultion of silica-induced fibrosis.

Role of interleukin-10 in the lung response to silica in mice.

There is evidence that, following exposure to crystalline silica, the release of several proinflammatory cytokines contributes to the induction of unbalanced inflammatory reaction leading to lung fibrosis. We have examined the potential contribution of interleukin-10 (IL-10), an anti-inflammatory cytokine, in the development of silicosis. In a mouse model of inflammatory lung reaction induced by intratracheal instillation of silica (0.5 mg and 5 mg DQ12/mouse), the levels of IL-10 protein (determined by ELISA) both in cells obtained after bronchoalveolar lavage (BAL) and in lung tissue homogenates were significantly increased when compared with controls. After in vitro lipopolysaccharide (LPS) stimulation (1 microg/ml), BAL cells obtained from silica-treated animals produced significantly more IL-10 protein and mRNA than cells obtained from control animals. To examine the role of IL-10 in the lung reaction induced by silica, IL-10-deficient animals were instilled with 5 mg of silica. Twenty-four hours after treatment, the amplitude of the inflammatory response (lactate dehydrogenase [LDH], protein and number of inflammatory cells in BAL) was significantly greater in IL-10-deficient animals than in the wild type. In contrast, the fibrotic response, evaluated by measuring lung hydroxyproline content and by histopathologic analysis 30 days after silica, was significantly less important in IL-10-deficient than in wild-type mice. Together, these data suggest that increased IL-10 synthesis induced by silica can limit the amplitude of the inflammatory reaction, but also contributes to amplify the lung fibrotic response.

Immunocytochemical characterization of lung macrophage surface phenotypes and expression of cytokines in acute experimental silicosis in mice.

The expression of the surface phenotypical profile and the cytokines TNF-alpha and IL-1beta from murine lung macrophages was studied in parenchymal lung tissue and bronchoalveolar fluid of mice, over a 2-week period, following a single intratracheal instillation of silica. The acute inflammatory reaction, confirmed by a significant augmentation of four times the control values of the number of macrophages recovered by lavage from experimental animals, was followed by organized granulomas in the interstitium. The immunohistochemical analysis of lung tissue sections after silica instillation demonstrated the increased alveolar and interstitial tissue expression of all surface antigens and cytokines studied, mainly Mac-1, F4/80 antigens, TNF-alpha and IL-1beta, which were occasionally observed in normal uninjected and saline-treated mice. These findings show that, after silica instillation, the expression of surface phenotypical markers of lung macrophages increased, and this change was concomitantly associated with an increased expression of the cytokines TNF-alpha and IL-1beta. These changes support the conclusion that an influx of the newly recruited and activated macrophage population, with a different phenotype, is induced by treatment during inflammation. The populational changes involve difference in functional activity and enhance TNF-alpha and IL-1beta expression. These cytokines, produced in the silicosis-induced inflammatory process, are associated with the development of fibrosis and may contribute to disease severity.

Silica-induced apoptosis in alveolar and granulomatous cells in vivo.

Silica is a toxicant that can stimulate cells to produce various cellular products such as free radicals, cytokines, and growth factors. Silica and its induced substances may induce apoptosis to regulate the evolution of silica-induced inflammation and fibrosis. To examine this hypothesis, groups of Wistar male rats were intratracheally instilled with different doses of Min-U-Sil 5 silica (Silica, Berkeley Springs, WV). Ten days after the instillation, we obtained cells by bronchoalveolar lavage and placed them on slides by cytospin preparation. The slides were stained with Diff-Quik (Lab Aids, Sydney, NSW, Australia) and examined under oil immersion. A substantial number of cells with apoptotic features were identified in all silica-instilled rats and the apoptosis was confirmed by agarose gel electrophoresis. The number of apoptotic cells was clearly related to silica dosage. Engulfment of apoptotic cells by macrophages was also noted. Neutrophil influx in silica-instilled rats could be saturated with the increase of silica dosage and the number of macrophages in different dose groups changed in parallel with the proportion of apoptotic cells. Fifty-six days after instillation, morphologically apoptotic cells could be identified in granulomatous cells of lung tissue from silica-instilled rats. We conclude that intratracheal instillation of silica could induce apoptosis in both alveolar and granulomatous cells, and the apoptotic change and subsequent engulfment by macrophages might play a role in the evolution of silica-induced effects.

Inhaled Silica Dust Increases Nitric Oxide and Cytokine Production Associated with Collagen Synthesis and Fibrosis in Rats

Abstract The association of silica with pulmonary inflammation and fibrosis is well documented. However, the mediators involved in the pathogenesis of silica-induced pulmonary fibrosis are not completely defined. In parallel studies, which will be summarized in this presentation, intratracheal instillation of silica resulted in nitric oxide production and pulmonary inflammation within 24 hours. The objective of the current study was to determine the involvement of nitric oxide and cytokines in the pathogenesis of inflammation and fibrosis associated with an occupationally-relevant inhaled exposure to silica. Male Fischer rats were exposed by inhalation to 2 mg/m3 of freshly-generated silica for 2 weeks, 2 months, and 6 months at a rate of 5 days/week, 8 hour per day. Cellular differentials, chemiluminescence, and mRNA levels of inducible nitric oxide synthase (iNOS), transforming growth factor β (TGFβ), and platelet-derived growth factor (PDGF) were all measured in cells obtained by bronchoalveolar lavage...