INTRODUCTION

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Keywords: interleukin-1; nitric oxide; inflammation; apoptosis; silicosis

Silicosis is characterized by the accumulation of inflammatory cells, thickening of the alveolar interstitium, hyalinized silicotic nodules, and deposition of collagen (1, 2). Although complicated silicosis and accelerated silicosis cause significant respiratory impairment, the initial form of the disease, or simple silicosis, is relatively benign. The lack of effective therapeutic strategies to treat silicosis can be attributed, in part, to the poor understanding of the pathophysiology of the disease.

Early events in silicosis include the accumulation of alveolar macrophages and lymphocytes and the release of cytokines. Macrophage-derived growth factors such as platelet-derived growth factor (PDGF), insulinlike growth factor-I (IGF-I), and transforming growth factor (TGF) have been shown to be important for interstitial lung diseases induced by inorganic dusts (3). Activated macrophages release proinflammatory and cytotoxic mediators such as hydrogen peroxide, nitric oxide (NO), peroxynitrite, bioactive lipids, interleukin-1 (IL-1), and tumor necrosis factor- (TNF-) (6). The inflammatory cell-derived products, which include reactive oxygen and nitrogen intermediates such as superoxide and NO, may mediate the early inflammatory processes preceding repair and fibrosis (7).

In a murine model of silicosis, both IL-1 and TNF- were associated with mononuclear cell inflammation (8). IL-1 has been implicated in the deposition of collagen (6) and modulation of PDGF activity (9). IL-1 receptor antagonist reduces pulmonary fibrosis elicited by silica or bleomycin in mice (10). TNF- is also required for the development of silica-induced pulmonary fibrosis, because anti-TNF antibody prevents silicosis, and silicosis is absent in TNF- receptor knockout mice (11, 12).

In addition to the induction of the release of proinflammatory cytokines, silica also induces apoptosis of human alveolar macrophages (13, 14). The fibrogenic potential of a particulate correlated with its ability to induce apoptosis in alveolar macrophages, consistent with the concept that the apoptotic potential may be an important factor in initiating an inflammatory response (14). IL-1 enhances the response of glomerular cells to oxidant-initiated apoptosis (15). In insulin-dependent diabetes mellitus, apoptosis in pancreatic beta cells is mediated by IL-1 and requires NO release (16). IL-1-induced apoptosis of lung fibroblasts has also been shown to be mediated by NO (17). NO is a well-documented cytotoxic molecule implicated in the induction of apoptosis of several cell types (18). Recently, the role of NO in the initiation of the inflammatory response after hemorrhagic shock and the regulation of neutrophil migration in zymosan-induced inflammation has been reported (19, 20). Alveolar macrophages have been shown to upregulate both IL-1 and inducible NO synthase (iNOS) gene expression in response to silica (6, 21).

The role of proinflammatory cytokines in lung inflammation and fibrosis has been well-documented, but the underlying mechanisms remain poorly understood. We have developed a murine silicosis model using IL-1 and iNOS knockout mice, and demonstrate an essential role for IL-1 and NO in silica-induced apoptosis and lung inflammation.

	METHODS

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Animal Care and Treatment

IL-1 gene knockout mice (129/SvEv) (22) were kindly provided by Dr. Lex H. T. Van der Ploeg at Merck (Rahway, NJ). Wild-type 129/ SvEv control mice were purchased from Taconic (Germantown, NY). iNOS (Nos2) gene knockout mice (C57BL/6J × 129/J background) and wild-type control mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice used in the experiments were 8 to 12 wk in age. All animals were maintained in a pathogen-free facility, and the care and use of animals were conducted as approved by the NYU Institutional Animal Care and Use Committee.

IL-1 knockout and iNOS knockout mice and their corresponding wild-type controls were exposed to crystalline silica (Min-U-Sil 5, 1 to 5 µm particle size) from U.S. Silica (Berkeley SPGS, WV) by means of a nose-only inhalation chamber using a Wright dust feeder. The concentration of silica in the inhaled air was 250 mg/m³. The duration of exposure was 5 h/d for 10 d. The silica dust standard, as recommended by the National Institute of Occupational Safety and Health (NIOSH), is 50 µg/m³ for an 8-h day as a time-weighted average. This is to be averaged over a 20-yr working life and approximates 250 mg (50 µg × 250 d × 20 yr) to prevent silicosis. The level of silica exposure given to our mice each day was equal to this lifetime exposure, which was enough to cause lung inflammation. Mice were killed at 1, 4, 6, or 12 wk after exposure and lungs were harvested. Four mice were killed for each timepoint and each exposure experiment was performed two times.

Macrophage Cell Line

Mouse macrophage cell line IC-21 was purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin/streptomycin. All tissue culture reagents were purchased from Cellgro (Herndon, VA).

Histology and Immunohistochemistry

After sacrifice of animals, whole lungs were inflated and fixed in formaldehyde and embedded in paraffin. Lung sections (6 to 8 µM) were stained with hematoxylin and eosin (H & E) and observed at 100 or 200× using an Olympus BH2 microscope. The number of lesions per square centimeter and the range of lesion size were evaluated on 10 random fields from each lung section using image analysis software (Image Pro Plus; Media Cybernetics, Del Mar, CA).

For immunohistochemical staining, paraffin-embedded lung sections were heated at 65° C overnight and processed through xylene, 100%, 90%, 80%, 70%, and 50% ethanol in mentioned order. Sections were then washed in phosphate-buffered saline (PBS) and blocked with PBS-bovine serum albumin (BSA) (4 mg/ml) purchased from Sigma (St. Louis, MO). Macrophages were stained with antibody F4/80 specific for mouse macrophage obtained from Caltag (Burlingame, CA) and detected by fluorescein isothiocyanate (FITC)-labeled goat anti-rat secondary antibody (Sigma). iNOS was stained with a rabbit antibody specific for iNOS/NOS type II from BD Transduction Laboratories (San Diego, CA) and detected by FITC-labeled goat anti-rabbit secondary antibody. Sections were visualized at 100 or 400× using an Olympus BH2 fluorescent microscope.

TUNEL Assays

1 × 10⁶ IC-21 cells or alveolar macrophages were plated on glass coverslips in 6-well tissue culture plates and cultured in RPMI 1640 (supplemented with 10% FBS for IC-21 cells) overnight. Cells were then treated with 100 µg/ml crystalline silica and incubated at 37° C. For studies with inhibitors, cells were pretreated for 1 h with 100 µM NOS inhibitor L-NAME (Sigma) or 10 µg/ml neutralizing anti-IL-1 antibody before addition of silica. At various timepoints after treatment, cells were fixed in 4% formaldehyde and apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay using a Fluorescent Apoptosis Detection System (Promega, Madison, WI). TUNEL staining in lung sections was performed after deparaffinization and permeabilization with Triton-X100. Lung sections or fixed cells were incubated with FITC-labeled dUTP and TdT enzyme. After removal of unincorporated FITC-dUTP, cells or lung sections were stained with 1 µg/ml propidium iodide (Sigma) and mounted for visualization using an Olympus BH2 fluorescent microscope. Apoptotic nuclei stained green or yellow, whereas nuclei of viable cells stained red with propidium iodide.

NO Assays

NO oxide release from macrophages was evaluated by a colorimetric assay using the Cayman Nitrite Nitrate detection kit obtained from Alexis Biochemicals (San Diego, CA). 1 × 10⁶ IC-21 cells were plated in 6-well tissue culture plates and maintained in RPMI 1640. Cells were then treated with 100 µg/ml silica and incubated at 37° C. Silica-induced NO release was also assayed in the presence and absence of 100 µM NOS inhibitor N^G-nitro-L-arginine-methyl ester (L-NAME) (Alexis Biochemicals, San Diego, CA) or 10 µg/ml neutralizing anti-IL-1 antibody from R&D Systems (Minneapolis, MN). Cell supernatants were harvested at various timepoints after treatment and stored at 70° C. For NO detection, supernatants were thawed and used according to instructions provided with the kit. The working principle of the assay is the enzymatic conversion of nitrites to nitrate and the generation of a colored azo chromophore by Griess reaction. Experiments were repeated three times and all samples were assayed in duplicates.

Statistical Analysis

Statistical analysis was done by performing one-way analysis of variance, statistical significance was determined by Bonferroni's t test using SigmaStat software, and p values were calculated. A p value < 0.05 was considered significant.

	RESULTS

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Role of IL-1-Dependent Nitric Oxide Release in Silica-induced Apoptosis

Silica-induced apoptosis of alveolar macrophages has been proposed to be a key event in the initiation of silicosis because the fibrogenic potential of a particulate parallels its ability to induce apoptosis in macrophages (14). To determine the role of IL-1 and NO in silica-induced apoptosis of macrophages, in vitro studies were performed using the IC-21 mouse macrophage cell line.

First, we treated IC-21 cells with silica in vitro and observed that the levels of nitrate produced were upregulated beginning at 2 h and reached a maximal level after 5 h (Figure 1A). Silica-induced NO release was significantly inhibited by pretreatment of cells with the NOS inhibitor L-NAME (p < 0.001, 3 to 24 h), consistent with activation of the L-arginine-dependent pathway. To ascertain the role of IL-1 in silica-induced NO production, the cells were pretreated with IL-1 neutralizing antibody, which markedly reduced the levels of nitrate production (p values were 0.004 and 0.008 at 3 h and 4 h, respectively; p value < 0.001, 5 to 24 h). After 12 h of exposure to silica, an appreciable number of macrophages were apoptotic, as evident by nuclear condensation (data not shown). By 24 h, almost all cells were apoptotic by TUNEL staining (Figure 1B). Pretreatment with IL-1 neutralizing antibody, which inhibited NO release, significantly abrogated silica-induced apoptosis. The contribution of NO to silica-induced apoptosis was further confirmed using L-NAME, which potently inhibited silica-induced apoptosis (Figure 1B). Similar results, namely, protection from silica-induced apoptosis by pretreatment with L-NAME or IL-1 neutralizing antibody, were obtained using human bronchoalveolar lavage cells that contained more than 95% alveolar macrophages (data not shown).

View larger version (12K): [in this window] [in a new window]   View larger version (29K): [in this window] [in a new window]   Figure 1.   NO release and apoptosis in IC-21 cells after silica exposure in vitro. (A) NO release. NO release in the cell supernatants was quantitated by measuring nitrate concentrations using a colorimetric assay. The means ± SD of three experiments were plotted. Silica induced NO production beginning 2 h after exposure and reached a maximal level after 5 h. Pretreatment with the NOS inhibitor L-NAME (100 µM) markedly inhibited nitrate accumulation and NO release. Nitrate accumulation was also significantly reduced by preincubation of cells with anti-IL-1 neutralizing antibody (10 µg/ml). silica; silica + L-NAME; Silica + anti IL-1. (B) Evaluation of silica-induced apoptosis by TUNEL staining. TUNEL staining indicated that significant numbers of macrophages were apoptotic by 12 h (data not shown) and almost all cells were apoptotic by 24 h. Pretreatment with L-NAME or anti-IL-1 antibody prevented silica-induced apoptosis.

IL-1-Deficient Mice Are Protected from Silica-induced Lung Inflammation

Silicosis is characterized by mononuclear cell inflammation, with macrophage activation and accumulation of mononuclear cells in silicotic lesions and bronchial-associated lymphoid tissue (BALT) (8). To determine the specific role of IL-1 in silicosis, IL-1 knockout (IL-1^/) mice and wild-type control mice (129/SvEv) were exposed to air or silica using an inhalation chamber. After 1 wk of exposure to silica, wild-type mice displayed patches of alveolar inflammation marked by infiltration of mononuclear cells and macrophages localized to subbronchial areas (Figure 2). At 6 wk, the inflammatory lesions enlarged, with increased accumulation of macrophages and lymphocytes. The alveolar septum in the affected areas showed mild disintegration with some septal cell hypertrophy. At 12 wk, wild-type mice displayed large silicotic lesions with densely packed inflammatory cells within BALT structures and complete obliteration of the alveolar septum in the affected areas. In striking contrast, the chronic inflammatory responses to silica observed in the wild-type mice were absent in IL-1^/ mice. The alveolar septum of the latter appeared normal throughout the 12-wk time period, with little or no cellular inflammation in the lungs. The number of lesions per square centimeter of lung ranged from 9 to 20 in wild-type mice compared with 1 in IL-1^/ mice, and the lesions were much larger in the former (Table 1) (p values for 1, 6, and 12 wk are < 0.001). No lung inflammation was observed in wild-type or IL-1^/ mice exposed to air (data not shown).

View larger version (101K): [in this window] [in a new window]   Figure 2.   Histologic evaluation of lung tissues from mice exposed to silica. H & E staining of paraffin-embedded lung sections. Wild-type mice (IL-1+/+) (top panel ) display the early inflammatory response and the evolution of silicotic lesions. Inflammatory cell aggregations were seen subbronchially and were mostly macrophages. At 6 wk, the silicotic lesions became larger and lymphocytes started to appear (arrows). By 12 wk, the silicotic lesions continued to enlarge and consisted predominantly of lymphocytes. In contrast, silica-induced inflammation and silicotic lesions were absent in the lung tissues of IL-1 knockout mice (IL-1/) (bottom panel ) at all timepoints. B = bronchiole, V = blood vessel.

Requirement for IL-1 for Accumulation of Macrophages and Induction of iNOS and Apoptosis

Exposure to crystalline silica causes lung inflammation characterized by accumulation of mononuclear cells and formation of silicotic lesions. These were shown to be predominantly macrophages using the F4/80 monoclonal antibody (Figure 3A). Because silica has been shown to induce NO production and upregulate iNOS messenger RNA (mRNA) expression in monocytic cells (21), and because NO can induce apoptosis of macrophages (23), we examined the expression of iNOS in macrophages. Lung sections of silica-exposed wild-type and IL-1^/ mice were stained with antibody specific for iNOS. iNOS-positive staining was localized to aggregations of alveolar macrophages in the lungs of wild-type mice and was more evident at early timepoints (1 wk postexposure) (Figure 3B). In contrast, IL-1^/ mice showed few to no iNOS-positive cells.

View larger version (27K): [in this window] [in a new window]   Figure 3.   Immunodetection of macrophages and iNOS expression in lung sections of mice exposed to silica. (A) Paraffin-embedded lung sections of silica-exposed wild-type mice (1 wk) showed distinct accumulation of macrophages (left) as detected by the F4/80 antibody specific for mouse macrophages. Consistent with the absence of inflammation, macrophage staining was not observed in the lungs of silica- exposed IL-1/ mice (right). (B) Immunodetection of iNOS expression in paraffin-embedded lung sections of wild-type (left) and IL-1/ mice (right) 1 wk after exposure to silica. Wild-type mice displayed positive iNOS staining in lung sections in response to silica inhalation, whereas lung sections from IL-1/ mice showed little or no staining for iNOS.

The lung sections of silica-exposed wild-type and IL-1^/ mice were examined for the presence of apoptotic cells by TUNEL staining. As shown in Figure 4, apoptotic cells were present within the silicotic lesions in the lungs of wild-type mice at all timepoints observed. Consistent with the absence of iNOS expression (Figure 3B) and lung inflammation in the lung sections of IL-1^/ mice, little or no apoptosis was observed in the lung sections of the latter, supporting the role of apoptosis as an early event in the initiation of lung inflammation in silicosis.

View larger version (93K): [in this window] [in a new window]   Figure 4.   Evaluation of apoptosis in vivo in lung sections of mice exposed to silica. TUNEL staining of lung sections showed a significant number of apoptotic cells in wild-type mice (top panel ). Apoptosis was greater at 1 and 6 wk and diminished by 12 wk after exposure. IL-1/ mice (bottom panel ) did not display any significant number of apoptotic cells as suggested by the lack of TUNEL staining.

iNOS-Deficient Mice Are Protected from Silica-induced Apoptosis and Pulmonary Inflammation

To ascertain the role of NO in lung inflammation, iNOS^/ mice (C57BL/6 × 129/J) and wild-type control mice were exposed to silica. At 1 wk, silica induced extensive lung inflammation in wild-type mice (Figure 5). After resolution of the initial inflammation, silicotic lesions began to develop after 4 wk and progressed to 12 wk. Similar to findings in the 129/ SvEv mouse strain (Figure 2), silicotic lesions at 6 wk contained predominantly macrophages, whereas lesions at 12 wk were larger and constituted mainly of lymphocytes within BALT structures. In striking contrast, silica failed to induce lung inflammation in the lungs of iNOS^/ mice at 1 wk and at all other timepoints. As noted in Table 2, fewer lesions were seen in the lungs of iNOS^/ mice, and those observed were markedly reduced in size (p values for 1, 6, and 12 wk are < 0.001). To correlate the incidence of apoptosis with inflammatory lesions induced by silica, lung sections were examined after 4 and 6 wk of exposure. TUNEL staining demonstrated apoptosis in the silicotic lesions in the lungs of silica-exposed wild-type mice (Figure 6). In contrast, the lungs of silica-exposed iNOS^/ mice appeared normal, with few apoptotic cells.

View larger version (105K): [in this window] [in a new window]   Figure 5.   Resistance to silica-induced inflammation in iNOS/ mice. Histologic evaluation was done on lung sections from mice exposed to silica. H & E staining of paraffin-embedded sections demonstrated extensive inflammation in wild-type mice at 1 wk after exposure to silica. In striking contrast, no inflammation was observed in iNOS/ mice. By 6 wk, silicotic lesions, constituted largely of macrophages, began to develop in the wild-type mice after resolution of the early inflammatory responses and progressed to 12 wk with an increase in lesion size and accumulation of lymphocytes. Lungs of silica-exposed iNOS/ mice showed significantly fewer lesions and those observed were markedly reduced in size, even at the late 12-wk timepoint.

View larger version (82K): [in this window] [in a new window]   Figure 6.   iNOS/ mice were protected against silica-induced apoptosis. Evaluation of apoptosis in paraffin-embedded lung sections from mice exposed to silica. TUNEL staining of lung sections showed significantly more apoptotic cells in wild-type mice as compared with iNOS/ mice.

	DISCUSSION

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The early events responsible for the initiation and progression of silicosis in humans are poorly understood because the disease typically presents at an advanced stage. Animal models of silicosis have provided valuable insights into the initiating event of the disease, particularly, the early inflammatory phase. Increased understanding of the latter is essential for the development of effective means for early intervention to prevent disease progression to the advanced stage, for which there is no treatment. In this report, we investigated the role of IL-1 and NO in silica-induced apoptosis in vitro in macrophages after silica exposure and in vivo in murine silicosis. We demonstrated that silica-induced NO production and apoptosis can be blocked by anti-IL-1 antibody and the NOS inhibitor L-NAME. In vivo, wild-type mice exposed to silica developed pulmonary inflammation, whereas IL-1-deficient and iNOS-deficient mice were more resistant to silica-induced inflammation. The silicotic lesions developed in wild-type mice were larger and more numerous than those developed in IL-1^/ and iNOS^/ mice. Furthermore, IL-1^/ and iNOS^/ mice were also more resistant to silica-induced apoptosis in the lungs, suggesting that there is an association between apoptosis and inflammation, mediated by IL-1 and NO production.

The mechanisms by which cytokine networks regulate the development of inflammatory lesions in silicosis are not well- understood. Because fibrogenic particulates such as silica caused apoptosis of alveolar macrophages, it was proposed that this apoptotic potential may be a critical factor in initiating an inflammatory response (14, 21, 24). Ingestion of silica by alveolar macrophages leads to cell death and release of intracellular silica that is taken up by other macrophages. This recurrent cycle of macrophage phagocytosis and cell death perpetuates the inflammatory process (2). The fibrogenic effect of silica may be due to the release of inflammatory cytokines and growth factors by alveolar macrophages activated by silica (3). IL-1 is regarded as a principal mediator of inflammation and is an attractive target for therapeutic intervention in the treatment of inflammatory diseases.

IL-1 has been shown to play a significant role in zymosan-mediated inflammation (25) and acute-phase response in localized tissue damage induced by turpentine (22). The likely mechanism for the loss of response to turpentine observed in IL-1^/ mice is the lack of induction of IL-6, a final mediator for the induction of acute-phase response (26). In contrast, in patients with beryllium disease, the increased macrophage expression of TNF- and IL-6 supported the role of TNF- and IL-6 in the granulomatous inflammatory response (27). We propose that the mechanism of resistance of IL-1^/ mice to silica-induced inflammation may be due to the lack of induction of NO, an inducer of apoptosis in macrophages (23). Recently, silica has been shown to induce the expression of Fas ligand, a well-characterized apoptosis inducer, in lung macrophages and promoted Fas ligand-dependent macrophage apoptosis (28). Administration of neutralizing anti-Fas ligand antibody blocked the induction of pulmonary silicosis (28). In addition, Miwa and coworkers (29) had previously demonstrated that Fas ligand induced IL-1 release and inflammation, thus challenging the dogma that apoptosis does not induce inflammation. Because silica induced both Fas ligand expression and IL-1 production, it is conceivable that Fas ligand may be upstream of IL-1-NO-mediated apoptosis in silica-induced lung inflammation.

Upon phagocytosis of silica, alveolar macrophages are activated and secrete IL-1, which induces iNOS and NO production. The requirement of IL-1 for NO release was demonstrated by pretreatment with anti-IL-1 neutralizing antibody. Because apoptosis could be inhibited by L-NAME or IL-1 antibody, IL-1-dependent NO production is required for silica-induced apoptosis. NO has been shown to induce apoptotic cell death of macrophages through activation of c-Jun N-terminal kinase (JNK), p38 kinase, and CPP-32 protease (caspase-3) pathways (30). Because the kinetics of NO production correlated with JNK activation, the JNK pathway may be involved in silica-induced apoptosis. The involvement of p38 kinase or other mitogen-activated protein kinase (MAPK) remains to be determined.

NO and its metabolites have been implicated in the pathogenesis of tissue damage associated with acute and chronic inflammation. NOS activity is elevated in inflammatory lung disease in humans, including asthma, cystic fibrosis, and obliterative bronchiolitis after lung transplantation (31). In the development of autoimmune diabetes, activated islet macrophages have been suggested to participate in the initiation of beta cell damage by releasing IL-1, and inducing NO production and apoptosis in beta cells (16, 32). We propose a similar requirement for IL-1-dependent NO production in our mouse model of silicosis as in autoimmune diabetes and suggest a role for macrophage apoptosis in the pathogenesis of silica-induced lung injury. Using IL-1^/ mice, we have demonstrated that IL-1 activity is crucial to the process of silica-induced apoptosis and lung inflammation. Hence the mechanism of resistance of IL-1^/ mice to silicosis may be due to the lack of induction of NO and apoptosis. The role of NO is further confirmed using iNOS^/ mice, which demonstrated similar resistance to silica-induced apoptosis and lung inflammation. NO production by alveolar macrophages has been implicated in the pathogenesis of bleomycin-induced fibrosis (33), and increased production of the potent oxidant peroxynitrite has been reported in patients with idiopathic pulmonary fibrosis (34).

Further understanding of the molecular mechanisms underlying the inflammatory processes that may be important for the progression to fibrotic diseases is important for the development of novel therapeutic approaches for the treatment of silicosis by inhibiting not only IL-1, but also NO production and apoptosis. Imidazoline compounds have been considered for the treatment of type 2 diabetes because these compounds can inhibit IL-1-induced beta cell apoptosis, possibly by inhibition of the expression of iNOS, a key element in pancreatic beta cell apoptosis (35). If imidazoline compounds can suppress IL-1-induced apoptosis and NO production by macrophages after silica treatment both in vitro and in vivo in murine silicosis, these compounds can be explored as potential therapeutic agents for the treatment of silicosis.