Introduction

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Exposure to silica dust induces lung inflammation which may progress to lung fibrosis, a disease known as silicosis. Although the exact mechanisms leading to silicosis have yet to be elucidated, current evidence suggests that the alveolar macrophage plays a critical role in the development of inflammation and fibrosis. Alveolar macrophages isolated from patients with pulmonary fibrosis are activated, and release a variety of fibrogenic factors and cytokines (1, 2, 3). Among these cytokines, tumor necrosis factor (TNF)- emerges as a crucial modulator of the inflammatory and fibrogenic response to silica exposure (4, 5). When macrophages are exposed to silica in vitro, enhanced TNF- production occurs (6, 7). Increase in TNF- production, in turn, activates an inflammatory response cascade by altering the transcriptional regulation of several genes involved in fibrogenesis including other cytokines, fibronectin, collagen, and TNF- itself (2, 7, 8). Treatment of silica-exposed mice with an anti-TNF- antibody significantly reduces collagen deposition in the lung and, conversely, infusion of recombinant TNF- increases collagen deposition in the lungs of mice (4). In addition, overexpression of TNF- in the lung of transgenic mice resulted in alveolar disruption and mild pulmonary fibrosis (5). Thus, silica-induced lung disease appears to be critically dependent on TNF- gene activation by alveolar macrophages.

Several studies have recently suggested that apoptotic mechanisms are involved in the development of silica-induced pathologic changes (12). However, the signal transduction pathways involved in TNF- induction by silica in the alveolar macrophage are presently unclear. For example, in murine peritoneal macrophages, the TNF- gene promoter contains four nuclear factor (NF)-B-binding sequences (16, 17), but it is likely that other regulatory elements such as AP-1, AP-2, CRE, Egr-1, and Sp-1, which have been identified in a region of the human monocyte promoter that is highly homologous to the mouse TNF- promoter, may also be involved (18, 19). This evidence linking NF-B activation to silica exposure in the alveolar macrophage and TNF--induced cytotoxic effects would suggest that the putative role of NF-B activation may involve antiapototic pathways (10, 11, 20).

NF-B has been implicated in proapoptotic as well as antiapoptotic pathways, depending on the cell type and the stimulus (21). Thus, assessment of silica-induced NF-B activation and TNF- production in the macrophage, and their correlation to silica induction of apoptosis, could add to the current understanding of the intracellular signaling pathways underlying silica-induced fibrogenesis.

The susceptibility to silica-induced injury differs among mouse strains, as evidenced by varying degrees of pulmonary fibrosis, ranging from minimal to severe (25, 26). We have recently shown that gene disruption of the TNF receptor was associated with markedly reduced lung fibrosis following silica exposure (27). In addition, silica exposure enhanced NF-B nuclear binding in the mouse lung (28). Based on these studies, we hypothesized that a mechanism underlying the strain susceptibility differences to silica may involve upstream signaling pathways regulating TNF- gene expression. Macrophage apoptosis is an early event in the pathogenesis of silica injury. The study of silica-induced lung injury requires a better understanding of the signaling pathways underlying tolerance and vulnerability of the macrophage response to silica injury. To address these issues, we identified two murine macrophage-derived cell lines that differ in their TNF- and apoptotic responses to silica in vitro. RAW 264.7 macrophages exhibit enhanced TNF- production and NF-B activation in response to silica. In contrast, IC-21 macrophages do not produce TNF- in response to silica and do not induce NF-B. However, both IC-21 and RAW 264.7 macrophages are able to phagocytize silica particles (29), and induce TNF- production and NF-B activation in response to lipopolysaccharide (LPS). We therefore employed this novel paradigm to further examine relationships between silica-induced apoptosis, TNF- production, and NF-B activation.

	Materials and Methods

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Cell Culture, Silica Exposure, and TNF- Determination

The mouse macrophage-like cell lines RAW 264.7 (derived from BALB/c mouse; TIB 71) and IC-21 (derived from C57BL/6 mouse; TIB 186) were purchased from the American Type Culture Collection (Rockville, MD), and cultured in DMEM and RPMI 1640 medium (Gibco BRL, Rockville, MD) respectively, supplemented with 10% FCS, 1% glutamine, 1% penicillin, and 1% streptomycin. Silica particles < 1 µm were selected by sedimentation according to Stokes' law, acid hydrolyzed, and baked overnight. For stimulation experiments cells were seeded in 6-well plates at 3-5 × 10⁵ cells/cm², grown for 3 d, and exposed to 0.2 or 1 mg/ml silica for 6 or 24 h in duplicate wells. Untreated cells were used as negative controls and cells were exposed to LPS (2 µg/ml; Sigma, St Louis, MO) as positive control. Cell viability was verified by trypan blue exclusion. Following exposure, culture supernatants were collected for measurements of TNF- using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Pierce-Endogen, Rockford, IL). Cells were scraped in 1 ml PBS, collected by centrifugation, and resuspended in adequate lysis buffer for electrophoretic mobility shift assay (EMSA) analysis. For apoptosis detection, a Cell Death Plus ELISA kit was used according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN).

Detection of Apoptosis

3-5 × 10⁵ cells/cm² grown for 3 d in 6 wells and exposed to 0.2 or 1 mg/ml silica for 6 h in duplicate wells floating and adherent cells were pooled and lysed with 500 µl of lysis buffer (1% SDS, 10 mM Tris, pH 7.4) for 30 min. Lysed cells were transferred to 1.5-ml Eppendorf tubes and centrifuged at 1,000 × g for 10 min to separate low molecular weight DNA (oligonucleosome-sized fragments derived from apoptotic cells) from high molecular weight DNA (from viable cells). A 20-µl aliquot of a 1:5 dilution of the supernatant containing oligonucleosomes was used to detect apoptosis using an ELISA kit (Cell Death Detection ELISA Plus; Roche Molecular Biochemicals). Background values (incubation buffer alone) were subtracted, and OD values representing nucleosomal DNA fragments in treated samples were compared with those values obtained from untreated control cells, and expressed as fold increase. Duplicate wells were treated for each experimental condition and ELISA was performed in duplicate for each treated well. For inhibition of NF-B activation, cells were pretreated with 50 µM of the NF-B inhibitor, BAY 11-7082 (Biomol, Plymouth Meeting, PA) for 1 h, then stimulated with LPS or silica.

Electrophoretic Mobility Shift Assay

Pellets were resuspended in 150 µl lysis buffer (20 mM HEPES, 125 nM sodium chloride, 5 mM magnesium chloride, 12% vol/vol glycerol, 5 mM DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin), sonicated on ice for 15 s, and centrifuged at 14,000 RPM at 4°C. Protein concentration was determined using a DC-BioRad protein assay (BioRad, Hercules, CA). For retardation assays NF-B consensus oligonucleotide 5'-AGT TGA GGG GAC TTT CCC AGG C-3' (Santa Cruz, Santa Cruz, CA) was end-labeled with [-³²P] ATP and T4 polynucleotide kinase (Gibco BRL). 5 µg protein of crude nuclear extract was mixed with the labeled probe and buffer (10 mM Hepes, 7.2% vol/vol glycerol, 3 mM magnesium chloride, 3 mM DTT, 3 µl Nonidet, 60 µg BSA, 360 µg spermidine, 1.5 µg poly [dI-dC] in a 20 µl total volume) and incubated to allow binding to the probe. DNA-protein complexes were separated on 6% polyacrylamide gels (Invitrogen, Carlsbad, CA), gels were vacuum-dried, and labeled complexes were detected by autoradiography.

Transient Transfections and Luciferase Assay

RAW 264.7 cells were transfected using lipofectamine reagent (3 µl/µg DNA; Gibco BRL). In a standard experiment, cells (60-80% confluence in 6-well plates) were transfected with 2 µg/well of reporter plasmid/well in OptiMEM I media (Gibco BRL), using cis-reporter plasmids containing the luciferase reporter gene driven by a basic promoter element (TATA box) joined to tandem repeats of NF-B (Stratagene, La Jolla, CA), or by pTNF (1185), pTNF (615), and pTNF (36), three truncated TNF promoters, previously described and kindly provided by Dr. J. S. Economou (30). Following a 6-h incubation at 37°C in a 5% CO₂ incubator, the media was replaced with DMEM media, and cells were stimulated with silica (0.1 and 0.2 mg/ml), or with LPS (2 µg/ml) or 4Phorbol 12 myristate 13acetate (PMA; 20 ng/ml) as positive controls. Untreated transfected cells served as a control. Luciferase gene expression was assayed using a Luciferase Assay System kit (Promega, Madison, WI) and compared with Luciferase expression in transfected untreated control cells.

Data Analysis

Experiments were replicated three to five times to ensure reproducibility. Within each culture trial, each condition was performed in duplicate, and numbers generated by each condition were averaged and treated as individual results. For gel data analyses, the bands corresponding to each transcription factor of interest were scanned and the dynamic range of the grayscale was examined by pixel histogram to prevent saturation. Numeric data were subjected to two-way ANOVA followed by Newman-Keuls post hoc tests for multiple comparisons or summary t tests as appropriate. A P value < 0.05 was considered to be statistically significant.

	Results

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Induction of Apoptosis in RAW 264.7 and IC-21 Macrophages

Silica stimulation (0.2 or 1 mg/ml) for 6 h resulted in increased apoptosis in both the RAW 264.7 and IC-21 macrophages. However, the magnitude of the apoptotic response was significantly higher in RAW 264.7 compared with IC-21 cells (Figure 1; P < 0.05). In contrast, LPS treatment did not induce apoptosis in any of the two cell lines (Figure 1). Inhibition of NF-B by pretreatment with 50 µM BAY-11-7082, an inhibitor of IB phosphorylation, resulted in a significant increase of apoptosis in RAW 264.7 untreated cells, as well as in LPS- and silica-treated cells (Figure 1). However, there was no additional increase in apoptosis after silica or LPS treatment in RAW 264.7 macrophage pretreated with BAY 11-7082, suggesting that inhibition of NF-B stimulates peak levels of apoptosis that cannot be enhanced by silica proapoptotic stimulation (Figure 1). In contrast, addition of BAY 11- 7082 to IC-21 cells inhibited silica-induced apoptotic response (Figure 1). These data suggest that RAW 264.7 macrophages are more sensitive to silica injury than IC-21. In addition, the reduced apoptotic response to silica in IC-21 cells pretreated with the NF-B inhibitor indicates that in these cells, baseline levels of NF-B activity are required for the induction of an apoptotic response to silica. This observation contrasts with NF-B activation of antiapoptotic pathways in RAW 264.7 cells.

View larger version (16K): [in this window] [in a new window]   Figure 1.   Apoptotic response of RAW 264.7 macrophages (right panel; open bars) and IC-21 macrophages (left panel; black bars) exposed to LPS (2 µg/ml) or silica (Si; 0.2 or 1 mg/ml), with or without pretreatment with the NF-B inhibitor BAY 11-7082 (50 µM). Results are expressed as fold increase from control (untreated cells).

NF-B Activation in RAW 264.7 and IC-21 Macrophages

To investigate whether differences in NF-B activation could account for the differences in induction of apoptosis by silica in the two cell lines, we examined NF-B activation after silica or LPS stimulation in both cell lines. EMSA showed that NF-B DNA binding activity increased in RAW 264.7 macrophages at 6 h exposure to silica (0.2 or 1 mg/ml) or LPS (2 µg/ml), and this increase was attenuated by pretreatment with the NF-B antagonist, BAY-11-7082 (Figure 2).

View larger version (70K): [in this window] [in a new window]   Figure 2.   Electromobility shift assay illustrating activation of NF-B in IC-21 macrophages stimulated with LPS or silica (Si; 0.2 or 1 mg/ml; right panel), and in RAW 264.7 stimulated with LPS in presence or in absence of BAY 11-7082 (Bay; left panel). A lane with labeled probe but no nuclear extract (probe) and a lane with an excess unlabeled probe (excess) served as controls for binding specificity.

To further confirm that silica-induced activation of NF-B results in stimulation of gene transcription, we examined silica- and LPS-induced activation of luciferase transcription driven by a NF-B-dependent promoter, using PMA stimulation as a positive control. Silica-induced NF-B- driven luciferase expression in RAW 264.7 cells was comparable to that stimulated by LPS (2 µg/ml) or PMA (20 ng/ml; Figure 3). Unlike RAW 264.7 cells, electromobility shift assays did not show increased nuclear binding of NF-B in silica-treated IC-21 macrophages at 6 h. Longer exposures of IC-21 cells to silica up to 24 h also failed to induce NF-B binding to DNA in these cells (Figure 2). In contrast, LPS elicited significant increases in NF-B DNA binding (Figure 2). These findings suggest that RAW 264.7 macrophages, but not IC-21 macrophages, activate NF-B-dependent transcription in response to silica stimulation. However, both cell types can induce NF-B when treated with LPS, indicating that the disparity in NF-B activation between the two cell lines is specific to silica stimulation.

View larger version (23K): [in this window] [in a new window]   Figure 3.   Luciferase Transcription Assay of NF-B reporter constructs performed on transfected RAW 264.7 cells stimulated for 6 h with PMA, silica (Si 0.1; Si 0.2), and LPS as described in MATERIALS AND METHODS. Results are expressed as fold increase of NF-B-driven luciferase transcription when compared with unstimulated cells.

Induction of TNF- Secretion in RAW 264.7 and IC-21 Macrophages

TNF- induction and NF-B activation have previously been implicated in silica injury (4, 10, 27, 28). To examine whether TNF- induction is related to silica stimulation of apoptosis in RAW 264.7 and IC-21 macrophages, we stimulated both of these cell lines for 6 or 24 h, and determined TNF- protein expression with a TNF- ELISA. In the presence of silica doses sufficient to induce apoptosis in these cells, stimulation of RAW 264.7 macrophages resulted in increased TNF- release, with maximal TNF- levels occurring at 6 h (Figure 4). Interestingly, 24 h exposure to silica using the higher silica concentration (1 mg/ml) did not induce a TNF- response as robust as that observed at 6 h, possibly because of extensive cell death.

View larger version (31K): [in this window] [in a new window]   Figure 4.   TNF- levels in the supernatant of RAW 264.7 cells (upper left panel, open bars), and IC-21 cells (upper right panel, black bars), unstimulated (C), or stimulated with 0.2 or 1 mg/ml silica (Si 0.2, Si 1) for 6 or 24 h. Experiments with RAW 264.7 cells were performed in presence or in absence of BAY 11-7082 (B). Data was obtained by ELISA determination and expressed as pg/ml. TNF- levels in the supernatant of RAW 264.7 cells (lower left panel, open bars), and IC-21 cells (lower right panel, black bars), unstimulated (C), or stimulated with LPS. Experiments with RAW 264.7 cells were performed in presence or in absence of BAY 11-7082 (B). Data was obtained by ELISA and expressed as pg/ml.

Pre-treatment of RAW 264.7 cells with 50 µM BAY 11-7082 inhibited silica-induced TNF- secretion at 6 or 24 h (Figure 4; P < 0.05). In contrast, no TNF- release occurred at either 6 or 24 h of silica stimulation in IC-21 macrophages (Figure 4). However, treatment with LPS (2 µg/ml) stimulated TNF- secretion in both cell lines, albeit with a smaller response in IC-21 compared with RAW 264.7 cells (Figure 4). In this setting, NF-B inhibition of RAW 264.7 cells resulted in no induction of TNF- release following LPS exposure (Figure 4). Thus, abolition of silica- or LPS-stimulated TNF- release by NF-B inhibition in RAW 264.7 cells suggests that NF-B activation is critical to the induction of TNF-. In addition, the absence of TNF- release in silica-stimulated IC-21 cells along with absent NF-B activation further reinforces such a putative role of NF-B.

Silica Induction of TNF- Promoter Activity in RAW 264.7 Cells

To examine the role of silica in the activation of the TNF- promoter, we further examined whether silica stimulation of RAW 264.7 cells induces the transcription of a luciferase gene driven by a TNF- promoter. In addition, we studied whether modifications of this promoter will affect silica-induced transcriptional activation of the TNF- promoter-driven gene. RAW 264.7 cells were transfected with a luciferase gene driven by three truncated TNF- promoters, as previously described by Rhoades and coworkers (30): (i) pTNF (-1185) is a virtually complete TNF- promoter, containing four NF-B-binding sites as well as the AP-1-, Sp1/Egr-1-, Ets-, CRE-, and AP-2-binding sites; (ii) pTNF (615) contains three NF-B-binding sites, as well as all the additional transcription factors' binding sites; (iii) pTNF (36) served as a negative control, and is a truncated TNF- promoter containing only the AP-2- binding site.

Silica stimulation (0.1 and 0.2 mg/ml) of RAW 264.7 cells induced a 2-fold increase in pTNF (1185) or pTNF (615) promoter transcriptional activity (Figure 5). Similarly, LPS (2 mg/ml) and PMA (20 ng/ml) stimulations resulted in 6- and 3-fold increases, respectively, in luciferase expression driven by pTNF (1185) promoter. There were no significant differences between silica-, PMA-, and LPS-induced luciferase expression when driven by the pTNF (1185) and the pTNF (615) promoters. However, neither LPS, silica, nor PMA induced luciferase expression when luciferase transcription was driven by the pTNF (36) negative control. These findings indicate that silica induces TNF- gene transcription. In addition, binding to the promoter of the TNF- gene by multiple transcription factors is essential to TNF- induction by LPS, silica, or PMA in RAW 264.7 macrophages. However, the fourth distal NF-B-binding site is not essential for activation of TNF- gene transcription.

View larger version (33K): [in this window] [in a new window]   Figure 5.   Luciferase Transcription Assay of TNF- reporter constructs, pTNF (1185) (striped bars), pTNF (615) (open bars), and pTNF (36) (filled bars), as described in MATERIALS AND METHODS. Assays were performed in RAW 264.7 cells stimulated with LPS, PMA, silica (Si) 0.1 or 0.2 mg/ml for 6 h, and results are expressed as fold TNF promoter-driven transcription when compared with unstimulated cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Silica exposure induces the development of granulomatous inflammation and fibrosis in the lung. Initial recruitment of macrophages is followed by an extensive inflammatory response characterized by the release of cytokines, NF-B activation and induction of apoptosis (8, 15, 26, 31- 33). In a recently published study of in vivo silica induction of apoptosis in mice lung sections, peak apoptosis was detected at 1 and 6 wk of silica exposure, when silicotic lesions start to form, diminishing by 12 wk after silica exposure, when large silicotic lesions are already present (34). Previously published observations reported that apoptotic macrophages were present in rat lung 10 d after intratracheal silica instillation, and that 56 d later, apoptosis was detected in granulomatous cells of lung tissue (12). In contrast, a study of histopathologic changes in rat lymph nodes from 2 to 52 wk after silica exposure, reported granuloma-like structure and macrophage recruitment without signs of apoptosis (35). These observations suggest that macrophage apoptosis is an early event in lung pathogenesis of silicosis. In contrast, induction of apoptosis and subsequent removal of apoptotic cells by recruited macrophages, in response to acute pulmonary inflammation as an inflammation clearing process in the lung, may suggest that the apoptotic process contributes to the repair process (36). Silica-induced alveolar macrophage apoptosis is mediated via a class A scavenger receptor. A unique function of this receptor, apart from its ability to bind silica, is the recognition and clearance of negatively-charged inhaled particles as well as the mediation and regulation of macrophage apoptosis (32, 37) In summary, current in vivo time-course studies indicate a critical role for macrophage apoptosis in the development of lung silicosis, but the signaling pathways involved in this pathogenesis have not yet been fully characterized. The apoptotic response occurring at an early stage in the development of silicosis may represent an attempt to remove injured cells and clear inflammation, resulting in tissue remodeling and preservation of lung function. Following this initial response, subsequent development of silicosis may occur as macrophage apoptosis increases, affecting the removal of injured cells and tissue remodeling. To study the pathways implicated in silica-induced macrophage apoptosis, we identified two different cell lines of murine macrophages that differ in their sensitivity to silica, and established relationships between silica induction of TNF-, NF-B activation, and apoptosis in the more sensitive RAW 264.7 cell line, compared with the more resistant IC-21 cell line. Silica stimulation of RAW 264.7 macrophages led to TNF- release and increased NF-B DNA binding. Inhibition of NF-B inhibited TNF- release and induced a significant increase in silica-induced apoptosis in RAW 264.7 cells, suggesting that NF-B activation is essential to TNF- induction, and plays an antiapoptotic role in these cells. In contrast, silica-exposed IC-21 macrophages did not release TNF-, and did not induce NF-B DNA binding above baseline binding levels. Moreover, in contrast with RAW 264.7 cells, inhibition of this baseline NF-B activity in IC-21 cells prevented silica induction of apoptosis. The discrepancy in the cellular response between these two cell lines appears to be specific to silica, because LPS stimulation induced TNF- release and NF-B DNA binding in both cell lines.

Activation of NF-B can be induced by a wide variety of signals and may act as either a promoter or attenuator of apoptosis (22, 38). NF-B activation by proapoptotic stimuli led to the speculation that this transcription factor is implicated in the induction of programmed cell death. In contrast, another large body of literature has implicated NF-B in survival pathways (22, 23, 39, 40). More specifically, TNF receptors can induce apoptosis via a NF-B activating pathway, and NF-B activation induces antiapoptotic genes to downregulate the ability of TNF- to induce apoptosis (20, 23, 39). Recent evidence suggests that NF-B activation inhibits apoptosis in mouse embryonic fibroblasts via modulation of JNK activity as well as induction of antiapoptotic proteins XIAP, c-IAP-1, c-FLIP, Bcl-_xL, and Bcl-2 (41). Similar observations were reported in a fibrosarcoma cell line with NF-B induction of TRAF1 and TRAF2, in turn activating c-IAP1 and cIAP2 inhibitors of caspase 8 activation in the TNF- signaling cascade (42). In agreement with these reports, our study demonstrated that inhibition of NF-B in RAW 264.7 cells resulted in an increased apoptotic response, even in the absence of a proapoptotic stimulus. In contrast, NF-B inhibition in IC-21 cells affecting baseline levels of NF-B activation decreased silica-induced apoptotic response in these cells. These data suggest that NF-B activity is required to prevent cell targeting into programmed cell death in silica-sensitive RAW 264.7 cells. However, a baseline NF-B activity may be required for silica induction of apoptosis, as apparent in IC-21 cells. In addition, in RAW 264.7 cells, the more robust silica response may induce proapoptotic pathways as well as further activation of NF-B to protect the cells from both TNF-- and silica-induced injury.

NF-B inhibition resulted in the inhibition of silica- as well as of LPS-induced TNF- release, suggesting that in addition to its antiapoptotic role, NF-B activation is also involved in the induction of TNF-. Luciferase reporter gene experiments performed in this study using RAW 264.7 cells confirmed that similar to LPS and PMA, silica was able to induce the TNF- promoter via a NF-B-dependent pathway, and that activation of three out of the four NF-B-binding sites in the TNF- promoter is sufficient for the activation of TNF- gene transcription in RAW 264.7 murine macrophages. Thus, in RAW 264.7 macrophages, NF-B can either become proapoptotic by inducing TNF- transcription, or antiapoptotic, possibly dependent on the magnitude of NF-B activation. Conversely, TNF- may both increase silica-induced apoptosis and stimulate antiapoptotic signaling pathways by inducing NF-B to protect the cells from more extensive apoptosis. We therefore propose that the balance between NF-B activity and TNF- expression will decide cell fate, such that strategies that either induce or inhibit NF-B activation may lead to similar antiapoptotic effects, depending on the background levels of NF-B activity and TNF- expression.

Silica exposure of IC-21 macrophages failed to induce TNF- release or NF-B activation. Thus, silica induction of apoptosis in IC-21 cells does not appear to be mediated by a TNF--dependent pathway, and does not recruit NF-B. The relatively reduced apoptosis levels induced by silica in IC-21 cells could be accounted for by the absence of a TNF- response. If so, this observation would concur with published in vivo findings, whereby decreased pulmonary injury occurred in silica-exposed TNF-receptor KO mice (27). The possibility exists, however, that factors acting upstream of TNF- may explain IC-21 response characteristics to silica. For example, the proapoptotic Fas/Fas Ligand (FasL) signaling pathway has been implicated in silica induction of apoptosis in alveolar macrophages (15). When FasL binds to its membrane receptor, which belongs to the TNF-receptor family, it will induce apoptosis by activating a caspase-dependent cascade (reviewed in Ref. 43). Indeed, silica stimulation has been shown to fail to induce TNF- and pulmonary fibrosis in FasL-deficient mice (15). Thus, the relatively low silica induction of apoptosis in IC-21 cells suggests that these cells may have reduced FasL expression and/or that alternate apoptotic pathways are activated following silica exposure. Alternatively, recently published data using IC-21 cells implicates another proinflammatory cytokine, interleukin (IL)-1, as well as nitric oxide synthase in the development of apoptosis and lung inflammation, and demonstrates that silica-induced apoptosis may be inhibited with a nitric oxide synthase inhibitor, or with an antibody to IL-1 (34). In addition, these authors detected minimal apoptotic lesions in the lungs of iNOS^/ and IL-1^/ mice, consistent with a role for IL-1 and nitric oxide synthase in the in vivo induction of apoptosis by silica (34). NF-B DNA-binding sites are present in both IL-1 and iNOS promoters and inhibition of baseline levels of NF-B activation in IC-21 cells may affect this proinflammatory pathway (44).

There is an increasing body of evidence that LPS can induce apoptosis. For example, increased apoptotic cell death occurred after LPS in endothelial cells (47, 48), and in hepatocytes (49). A substantial component of LPS proapoptotic role has been attributed to TNF- and NF-B induction (11, 23, 50). However, the TNF-/NF-B response cannot explain the disparities in cellular apoptosis following LPS stimulation. In the present study, although both LPS and silica were able to stimulate TNF- secretion and NF-B activation in RAW 264.7 macrophages, only silica induced apoptosis in this cell line. These observations suggest that TNF- and NF-B may only contribute to silica-induced apoptosis in RAW 264.7 macrophages, and one or more additional factors induced by silica exposure are essential to proapoptotic effect. In support of this observation, IC-21 had a diminished silica-induced apoptotic response and no TNF-/NF-B induction. Concurrent with those studies supporting a proapoptotic role for LPS, a substantial number of studies suggest that LPS may also induce antiapoptotic pathways to counterbalance the proapoptotic effects of TNF-. The higher levels of TNF- protein and of NF-B nuclear activity induced by LPS in RAW 264.7 cells could have induced an antiapoptotic response to the initial proapoptotic effects of TNF- and NF-B responses. Thus, the early proapoptotic response would be superseded by a later antiapoptotic response, both responses involving TNF- protein and NF-B nuclear activity. In support of this hypothesis, antiapoptotic gene activation and induction of cellular inhibitors of apoptosis by LPS have previously been reported in myeloid cells, human monocytes, and in J774.1 and U937 human macrophages, as well as in HeLa cells (23, 51). Furthermore, LPS has been shown to degrade IB- and IB-, leading to persistent NF-B activation. This is in contrast with short-lasting NF-B activation induced by proapoptotic stimuli, in which IB- degradation is minimal (38, 52). In addition, several studies reported in macrophages the induction of iNOS by LPS, leading to NO production, which in turn inhibits IL-1 production at the transcription level via an NF-B-mediated pathway, and may inhibit cytokine-dependent proinflammatory and proapoptotic responses (46, 55). Thus, the divergent apoptotic response to LPS appears to preferentially involve antiapoptotic pathways in the case of RAW 264.7 and IC-21 macrophage cell lines. Such responses may underlie a protective mechanism for macrophages whose physiologic role is to secrete proinflammatory mediators stimulating cell death during inflammatory events, providing these cells with the ability to trigger anticellular defense mechanisms without inducing their own death.

In summary, silica induces apoptosis in IC-21 cells independently from TNF- and NF-B activation, and may induce alternative pro- and antiapoptotic pathways. In contrast, silica substantially enhances apoptosis in RAW 264.7 cells via TNF- and NF-B-dependent pathways. In addition, LPS failure to induce apoptosis in both RAW 264.7 or IC-21 cells, despite potent TNF- responses in both cell types, further suggests that TNF- signaling is not sufficient to induce apoptosis, but may be synergistic with other silica-induced pathways to promote apoptosis in RAW 264.7 cells. This novel cellular approach will allow the elaboration of new strategies to study molecular mechanisms of cellular vulnerability and tolerance to silica injury. Our current findings further stress the critical importance of concomitant interactions among the various elements mediating the macrophage apoptotic cascade. These observations further imply that therapeutic strategies for silicosis should not target one signaling molecule, but rather a signaling pathway.