Introduction

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Inhalation of silica dust induces a lung disease characterized by interstitial inflammation, fibrosis, and cancer (Green and Vallyathan, 1995; Shi et al., 1998). The recruitment of phagocytic cells to the lung is found, and alveolar macrophages are considered to play an important role in the development of silicosis by releasing a variety of mediators (Fantone et al., 1987; Sibille and Reynolds, 1990; Driscoll et al., 1995). The destruction of lung tissue in silicosis is mediated partly by free radicals. Alveolar macrophages exposed to silica release excessive amounts of free radicals, cytokines, and chemokines that are involved in the onset of inflammation and fibrosis. In addition, silica is suggested to cause tissue damage by the generation of silicon-based free radicals and by the interaction of silica with cell components. The reactive species, including peroxynitrite, induce damage of lipids, proteins, and DNA and oxidation of protein and nonprotein thiols (Blackford et al., 1994; Gatti et al., 1994; Koh et al., 1998).

The activation of protein kinases is initiated during the phagocytosis of silica. The increase in tyrosine phosphorylation and the phosphorylation of p38 mitogen-activated protein kinases are found in alveolar macrophages and lung epithelial cells exposed to silica (Holian et al., 1994; Ding et al., 1999). Inhibitors of PKC suppress the production of reactive oxygen species (Lim et al., 1997) and decrease the cytosolic acidification and depolarization in alveolar macrophages activated by silica (Tarnok et al., 1997). Elevation of intracellular calcium is observed in the silica-activated alveolar macrophages (Chen et al., 1991; Rojanasakul et al., 1993). Alteration of the intracellular calcium homeostasis is suggested to contribute to the silica-induced cell death in macrophages. Although the mechanisms involved in the silica-induced apoptosis in alveolar macrophages are not clearly elucidated, apoptosis has been postulated to initiate the inflammatory response resulting in pulmonary fibrosis (Iyer and Holian, 1997; Lutjohann et al., 1998).

Ambroxol, trans-4[(2-amino-3,5-dibromobenzyl)amino]cyclohexanol HCl, is known to promote bronchial secretion and is used as an expectorant (Disse, 1987). Ambroxol has been shown to improve the clinical course of respiratory distress syndrome, including bronchopulmonary dysplasia (Wauer et al., 1992), and to reduce postoperative pulmonary complication (Fegiz, 1991). Ambroxol shows antioxidant action and anti-inflammatory effect. Ambroxol inhibits the lipid peroxidation of lung tissue induced by hydrogen peroxide (Nowak et al., 1994), protects damage of hyaluronic acid and collagen caused by iron and ascorbate (Koh et al., 1998), and reduces the hypochlorous acid-induced inactivation of ₁-antiproteinase (Cho et al., 1999). Ambroxol inhibits chemotaxis, respiratory burst, and cytokine production in neutrophils, monocytes, and macrophages activated by stimulating agents such as lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) (Stockley et al., 1988; Bianchi et al., 1990; Lee et al., 1999; Park et al., 1999).

The compounds that exert an antioxidant ability may have a therapeutic usefulness in some inflammatory diseases. Antioxidant glutathione is reported to attenuate the cytokine production in murine macrophages after the exposure of silica (Barrett et al., 1999). In this respect, the present study examined the effect of ambroxol on the stimulated respiratory burst and granule enzyme release and on the cell death in rat lung alveolar macrophages exposed to silica. The activities of PKC and PTK and the change in intracellular calcium level were measured to assay the action of ambroxol. The results suggest that ambroxol may decrease the stimulated responses and cell death in rat alveolar macrophages exposed to silica by inhibition of the activation processes, protein kinases, and calcium transport.

	Experimental Procedures

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Materials. Ambroxol, silica (amorphous, particle size 0.5-10 µm), staurosporine, genistein, N^G-monomethyl-L-arginine (NMMA), L-N⁶-(1-imminoethyl)lysine (L-NIL), ferricytochrome c, phenol red, horseradish peroxidase, NADPH, FAD, nitrate reductase (from Escherichia coli), sulfanilamide, N-(1-naphthyl)ethylene-diamine dihydrochloride, assay kit for acid phosphatase, Micrococcus lysodeikticus, fura-2/AM, 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin, Ficoll-Hypaque solution, Hanks' balanced salt solution (HBSS), and Dulbecco's modified Eagle's medium (DMEM) were purchased from Sigma-Aldrich (St. Louis, MO). PKC assay kit (SpinZyme Format) and PTK assay kit (enzyme-linked immunosorbent assay based) were obtained from Pierce Chemical (Rockford, IL), and ApoAlert CPP32/Caspase-3 assay kit was from CLONTECH (Palo Alto, CA). All other reagents were of analytical grade. Polystyrene 96- and 48-well plates (Costar, Cambridge, MA) were purchased from Corning Glassworks (Corning, NY). Polystyrene tubes (Falcon) used in the preparation of macrophages and the fura-2 loading were obtained from Becton Dickinson (Franklin Lakes, NJ). Silica particle suspensions were sonicated at 55 W for 15 s four times by using a sonifier cell disruptor (Branson sonifier, model W185D, Danbury, CT) before addition to the reaction mixtures.

Isolation of Rat Alveolar Macrophages. Sprague-Dawley rats weighing between 230 and 270 g each were anesthetized by intraperitoneal injection of 40 mg/kg pentobarbital sodium. A tracheal cannula was inserted through an incision in the neck, and 6 ml of cold Ca²⁺, Mg²⁺-free Dulbecco's phosphate-buffered saline (PBS), pH 7.4, was instilled the lung via a syringe attached to the cannula. Instillation of PBS was repeated three times to obtain macrophages. Cell suspension was treated with hypotonic solution for the lysis of erythrocytes. The cell pellets suspended in Ca²⁺, Mg²⁺-free HBSS were placed on a Ficoll-Hypaque gradient and were centrifuged at 400g for 45 min at 4°C. Macrophages were collected from the interphase of the gradient. The cells were washed with HBSS and were suspended in the same solution (Lee et al., 1999).

Measurement of Superoxide Anion Production. The superoxide anion produced was assayed by superoxide dismutase-inhibitable reduction of ferricytochrome c. The reduction of ferricytochrome c in the reaction mixture containing alveolar macrophages continuously increased up to 24 h of incubation as demonstrated in our report (Kim et al., 2001). The reaction mixtures (200 µl) in 96-well microplates contained 3 × 10⁵ macrophages, 75 µM ferricytochrome c, 100 µg/ml silica particle, and HBSS, pH 7.4, and were placed in a 5% CO₂ incubator at 37°C for 6 h. Absorbance was measured using a microplate reader (Spectra MAX 340; Molecular Devices, Sunnyvale, CA). The amount of reduced ferricytochrome c was expressed as nanomolar concentration by using the extinction coefficient of 2.1 × 10⁴ M¹ cm¹ at 550 nm (Park et al., 1999).

Measurement of Hydrogen Peroxide Production. Macrophages (3 × 10⁵ cells/well) were incubated in 200 µl of DMEM containing 0.1 mg/ml phenol red and 0.2 mg/ml horseradish peroxidase for 6 h at 37°C. The reaction was terminated by adding 20 µl of 1 N NaOH, and absorbance was measured at 610 nm (Kim et al., 2001). The concentration of hydrogen peroxide was calculated using hydrogen peroxide solution as the standard. We prepared 2 µM stock after calculating the hydrogen peroxide concentration by absorbance at 230 nm and added 1 to 50 nM/well.

Measurement of Nitrite/Nitrate Production. Nitric oxide production by the nitric-oxide synthase in macrophages was measured by assaying nitric oxide metabolites, nitrite and nitrate (NO_X) (Gilad et al., 1998; Kim et al., 2001). Macrophages (3 × 10⁵ cells/200 µl) were incubated in DMEM containing silica particles for 6 h at 37°C. Nitrate in the culture medium was reduced to nitrite by incubation with nitrate reductase (500 mU/ml), 160 µM NADPH, and 4 µM FAD at room temperature for 2 h. The medium were mixed with an equal amount of Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride, and 2.5% phosphoric acid). Absorbance was measured at 550 nm, and the amount of nitrite produced was determined using sodium nitrite as the standard. The results were expressed as total nitrite equivalents (NO_X).

Measurement of Acid Phosphatase Release. Macrophages (3 × 10⁵) were incubated in 200 µl of DMEM containing silica particles for 18 h at 37°C. Acid phosphatase released was measured as a hydrolysis of p-nitrophenyl phosphate by using diagnostic kit for the enzyme. Absorbance was measured at 405 nm. One unit was defined as the amount of enzyme activity that liberates 1 µM p-nitrophenol. The activity of acid phosphatase was estimated from the standard curve by using a p-nitrophenol standard reagent and was expressed as munit/3 × 10⁵ cells.

Measurement of Lysozyme Release. The release of lysozyme from macrophages was measured using M. lysodeikticus suspension. Macrophages (3 × 10⁵) were suspended in 200 µl of DMEM containing silica particles and were incubated for 18 h at 37°C. Forty microliters of Micrococcus suspension (50 mg of M. lysodeikticus in 5 ml of 0.65 M potassium phosphate buffer, pH 6.2) was added to the mixtures (100 µl). After a 30-min incubation at 37°C, adding 50 µl of 1 N acetic acid and stopped the incubation. Absorbance was measured at 450 nm (Burt et al., 1994).

Measurement of PKC and PTK Activities. Macrophages (10⁶ cells) were incubated in 500 µl of DMEM with or without silica particles and ambroxol at 37°C. The activities of PKC and PTK were determined by the experimental procedures described in the assay protocols of Pierce Chemical. In the assay of PKC activity, the supernatant obtained by a centrifugation of solute macrophages was incubated with PKC substrate for 30 min at 30°C. Activity of PKC in macrophages was assayed using PKC purified from the rat brain as the standard. One unit of the enzyme activity was defined as the amount that transfers 1 nM phosphate to histone H1 per minute at 30°C. The activity of PKC was expressed as munits. In the assay of PTK activity, the phosphorylation reaction in the reaction mixture was performed for 30 min at 30°C. PTK activity was assayed by using tyrosine kinase biotinylated phosphopeptide as the standard. One unit of the enzyme activity was defined as 0.5 nM phosphate incorporated into peptide substrate per minute. The activity of PTK was expressed as units. Absorbances in the assay mixtures for PKC and PTK were measured at 570 and 450 nm, respectively. We compared the data obtained to the standard curves presented in the protocols.

Measurement of Cytosolic Free Calcium. Fura-2 loading into alveolar macrophages was performed in polystyrene conical tubes, and fluorescence change in the assay mixture was measured as described in Luscinskas et al. (1990). Alveolar macrophages were loaded with 2 mM fura-2/AM to 1 µM/10⁷ cells in the reaction mixtures containing HBSS buffer without Ca²⁺ and Mg²⁺ (HBSS-CMF) and 20 mM HEPES-Tris, pH 7.4, for 10 min at 37°C. The suspension was then diluted 5-fold with HBSS-CMF containing 0.5% bovine serum albumin and further incubated for 15 min at 37°C. The suspension was centrifuged at 200g for 10 min, and macrophages were resuspended in HBSS-CMF containing 0.1% bovine serum albumin. Macrophages were finally suspended in bovine serum albumin-free HBSS-CMF at 5 × 10⁷ cells/ml. We observed the nonaggregation state of macrophages and then counted cell number by using a hemocyanometer in a microscope. Loaded macrophages (8 × 10⁶) were suspended in 1 ml of HBSS containing 1.23 mM Ca²⁺ and 1 mM Mg²⁺, and the reaction was started by addition of silica. Fluorescence changes were measured at an excitation wavelength of 340 nm and an emission wavelength of 505 nm by using a luminescence spectrophotometer (Aminco-Bowman Series 2; Aminco-Bowman, Rochester, NY).

Measurement of Cell Viability with an MTT Assay. The cytotoxic effect of silica and other compounds on alveolar macrophages and the protective effect of ambroxol were measured using the MTT assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases. Macrophages (3 × 10⁵ cells/200 µl) were treated with silica, ambroxol, or inhibitors for 6 h in a 96-well plate. The medium was incubated with 10 µl of 10 mg/ml MTT solution for 2 h at 37°C. The medium was removed and 100 µl of dimethyl sulfoxide was added to each well to dissolve the formazan. Absorbance was measured at 570 nm, and cell viability was expressed as a percentage of the control value (Kim et al., 2001).

Measurement of Apoptosis by Using a Caspase-3 Activity Assay. Macrophages (2 × 10⁶ cells/ml) were treated with silica in the presence or absence of ambroxol for 6 h at 37°C. Effect of ambroxol on the apoptosis in silica-treated macrophages was determined as described in ApoAlert CPP32/Caspase-3 assay kit user's manual. The supernatant obtained by a centrifugation of solute macrophages was added to the reaction mixture containing dithiothreitol and caspase-3 substrate, DEVD-p-nitroanilide, and incubated for 1 h at 37°C. Absorbance of the chromophore p-nitroanilide produced was measured at 405 nm. The standard curves were obtained from absorbances in the p-nitroanilide standard reagent diluted with cell lysis buffer (up to 20 nM). One unit of the enzyme was defined as 1 nM p-nitroanilide produced.

Data Analysis. Data are expressed as mean ± S.E.M. The data were analyzed by one-way analysis of variance. The analysis of variance justified post hoc comparisons between the different groups and was conducted using the Duncan's test for multiple comparisons. A probability of P < 0.05 was considered to be statistically significant. The traces in Fig. 7 are representative of five replicates in two separate experiments.

	Results

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Effect of Ambroxol on Superoxide and Hydrogen Peroxide Production in Silica-Activated Alveolar Macrophages. Alveolar macrophages exposed to silica are found to release reactive oxygen species. Intact macrophages liberated 1.36 ± 0.10 nM superoxide anion/3 × 10⁵ cells and 1.07 ± 0.03 nM hydrogen peroxide/3 × 10⁵ cells (n = 6) for 6 h of incubation, respectively. Figure 1 shows that when rat alveolar macrophages were incubated with silica, the production of superoxide and hydrogen peroxide was increased in a concentration-dependent manner. Macrophages treated with 100 µg/ml silica for 6 h released 3.26 nM superoxide anion and 2.81 nM hydrogen peroxide, respectively. Ambroxol has been shown to attenuate the responses of phagocytic cells stimulated by various stimulating agents, including LPS. From this finding, we examined the effect of ambroxol on macrophage responses stimulated by silica. Ambroxol attenuated the silica-stimulated superoxide and hydrogen peroxide production in a concentration-dependent manner, and at 100 µM, the production of reactive oxygen species was inhibited by 43 to 68%. Ambroxol (100 µM) decreased the production of reactive oxygen species in macrophages not exposed to silica (Fig. 2).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effect of ambroxol on production of reactive oxygen species in alveolar macrophages activated by silica. Macrophages (3 × 105 cells) were treated either with variable amounts of silica (A) or with 100 µg/ml silica in the presence of variable concentrations of ambroxol (B) for 6 h. The amount of superoxide and hydrogen peroxide produced was measured at the detection wavelengths. Data represent mean ± S.E.M. of five replicate values in two separate experiments. Statistically different from intact macrophages or silica alone (P < 0.05).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effect of protein kinase inhibitors on silica-induced oxidant production. Alveolar macrophages (3 × 105 cells) were incubated with 100 µM ambroxol (AMB), 100 nM staurosporine (STA), 10 µM genistein (GEN), 5 mM EGTA, and 100 µM trifluoperazine (TFP) in the presence or absence of 100 µg/ml silica for 6 h. Data represent mean ± S.E.M. of five and six replicate values in two separate experiments. Statistically different from control or silica alone (P < 0.05).

Inhibition of Silica-Induced Nitric Oxide Production in Macrophages by Ambroxol. We examined the effect of ambroxol on nitric oxide production in macrophages exposed to silica. In the present study, nitric oxide produced was assayed by measuring nitric oxide metabolites (NO_X). Intact alveolar macrophages liberated 2.10 ± 0.08 µM NO_X/3 × 10⁵ cells (n = 5) for 6 h of incubation. As shown in Fig. 3, the effect of silica on nitric oxide production was different from that on the production of reactive oxygen species. Silica up to 50 µg/ml enhanced NO_X production in macrophages, whereas beyond this concentration, the production was declined. Therefore, the effect of ambroxol on nitric oxide production was observed at the concentration of silica, 50 µg/ml. The NO_X production in macrophages treated with or without silica was significantly decreased by variable concentrations of ambroxol and by 500 µM NMMA, an inhibitor of nitric-oxide synthase, and L-NIL (50 and 100 µM), a selective inhibitor of inducible nitric-oxide synthase (Table 1). Staurosporine (100 nM) and 10 µM genistein decreased the silica-induced production of nitric oxide, whereas the effect was not seen in macrophages without treatment of silica.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Nitric oxide production in alveolar macrophages activated by silica. Macrophages (3 × 105 cells) were treated with variable amounts of silica for 6 h. Values represent mean ± S.E.M. of three replicate values in one experiments. Statistically different from intact macrophages (P < 0.05).

Effect of Ambroxol on Granule Enzyme Release from Macrophages Exposed to Silica. The secretion of granule enzymes from alveolar macrophages was assayed by measuring the release of acid phosphatase and lysozyme. Alveolar macrophages exposed to silica discharged acid phosphatase and lysozyme. The present study examined the effect of ambroxol on the silica-induced release of granule enzymes. Figure 4 shows that ambroxol inhibited the release of acid phosphatase and lysozyme from macrophages activated by silica in a concentration-dependent manner, and at 100 µM, 63 to 80% inhibition was observed. We examined regulatory roles of protein kinases and calcium in the degranulation process in silica-activated macrophages. The silica-induced release of acid phosphatase and lysozyme was decreased by addition of 100 nM staurosporine, 10 µM genistein, 5 mM EGTA, and 100 µM trifluoperazine (Fig. 5). Unlike free radical production, the granule enzyme release from the nontreated macrophages was not affected by additions of ambroxol, EGTA, and trifluoperazine.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of ambroxol on silica-induced granule enzyme release. Alveolar macrophages (3 × 105 cells) were incubated with 100 µg/ml silica in the presence of variable concentrations of ambroxol for 18 h. Acid phosphatase (A) and lysozyme (B) released by silica were measured. Data represent mean ± S.E.M. of five replicate values in two separate experiments. Statistically different from silica alone (P < 0.05).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effect of protein kinase inhibitors on silica-induced granule enzyme release. Alveolar macrophages (3 × 105 cells) were incubated with 100 µM ambroxol (AMB), 100 nM staurosporine (STA), 10 µM genistein (GEN), 5 mM EGTA, and 100 µM trifluoperazine (TFP) in the presence or absence of 100 µg/ml silica for 18 h. Data represent mean ± S.E.M. of five to seven replicate values in two separate experiments. Statistically different from control or silica alone (P < 0.05).

Effect of Ambroxol on PKC and PTK Activities. The previous findings suggested the involvement of protein kinases in the regulation of macrophage responses stimulated by silica. Therefore, we examined the changes in protein kinase activities in macrophages exposed to silica. When macrophages were treated with silica, the increase in PKC activity was found. As shown in Fig. 6, the peak activity of PKC was observed at 5 min of exposure time, and then the activity declined and was maintained up to 1 h of incubation. The increased activity of PTK also was found in the silica-activated macrophages. Dissimilar to PKC, the activity of PTK was gradually increased with increasing exposure times up to 1 h. The activation of protein kinases in macrophages exposed to silica was attenuated by addition of specific kinase inhibitors, staurosporine and genistein. The present study examined the effect of ambroxol on the activated protein kinases. Table 2 shows that ambroxol decreased the silica-induced increases in PKC and PTK activities in a concentration-dependent manner.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Silica-induced increase in protein kinase activities. Alveolar macrophages (106 cells/500 µl) were incubated with 100 µg/ml silica for the indicated times. Protein kinase activities were determined using the enzyme assay kits. Values represent mean ± S.E.M. of four and six replicate values in two separate experiments (munit in PKC, and unit in PTK). Statistically different from intact macrophages (P < 0.05).

Effect of Ambroxol on Intracellular Ca²⁺ Elevation Induced by Silica. The intracellular Ca²⁺ ([Ca²⁺]_i) level was assayed by measuring the fluorescence change of fura-2 due to the complex formation with Ca²⁺. Elevation of [Ca²⁺]_i is an early event in the response of phagocytic cells to stimulating agents, including chemoattractants (Luscinskas et al., 1990). When the alveolar macrophages loaded with fura-2 were treated with 100 µg/ml silica, the [Ca²⁺]_i was slowly increased in agreement with previous reports (Gleva et al., 1990; Chen et al., 1991). The present study observed the effect of ambroxol on the change in [Ca²⁺]_i in activated macrophages. As shown in Fig. 7, ambroxol (10 and 100 µM) attenuated the elevation of [Ca²⁺]_i in silica-activated macrophages.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effect of ambroxol on silica-induced increase in [Ca2+]i. Intracellular calcium level was measured as a fluorescence change of fura-2. Alveolar macrophages (8 × 106 cells/ml) were stimulated by addition of 100 µg/ml silica in the presence of ambroxol (10 or 100 µM). The traces are representative of five replicates in two separate experiments.

Effects of Ambroxol and Various Inhibitors on Cell Viability in Macrophages. The cytotoxicity of ambroxol, silica, and inhibitors on alveolar macrophages was measured using an MTT assay. As shown in Fig. 8, when macrophages were incubated with 100 µM genistein, 5 mM EGTA, and 100 µM trifluoperazine for 6 h, cell viability significantly decreased. In contrast, ambroxol, staurosporine, combined addition of ambroxol and protein kinase inhibitors, and inhibitors of nitric-oxide synthase did not induce a cell death. The cytotoxic effect of silica on macrophages was examined with cell viability and caspase-3 activity. When macrophages were treated with silica for 6 h, cell viability significantly decreased in a concentration-dependent manner, and 100 µg of silica induced 41% cell death (Fig. 9). Ambroxol decreased the cytotoxic effect of silica, and at 100 µM, 25% inhibition was observed. The apoptosis of alveolar macrophages induced by fibrogenic particulates is suggested as an initiating event in the onset of lung fibrosis (Iyer and Holian, 1997). The present study assayed the silica-induced apoptotic cell death by measuring the caspase-3 activity that is known to be involved in the apoptotic process (Stennicke and Salvesen, 1999). The incubation of alveolar macrophages with silica for 6 h showed an increase in caspase-3 activity in a concentration-dependent manner (Fig. 10A). Control macrophages showed an activity of 1.84 ± 0.34 U/2 × 10⁶ cells (n = 6) for 6 h of incubation, and in the presence of 100 µg of silica, the caspase-3 activity increased to 5.81 ± 0.69 U/2 × 10⁶ cells. The depressant effect of ambroxol on the silica-induced cytotoxicity in macrophages was observed with the caspase activity. Figure 10B shows that ambroxol decreased the silica-induced increase in caspase-3 activity, and at 100 µM, the increased activity was decreased by 53%.

View larger version (70K): [in this window] [in a new window]   Fig. 8.   Effect of ambroxol and inhibitors on cell viability. Alveolar macrophages (3 × 105 cells/200 µl) were incubated with 100 µM ambroxol (AMB), 100 nM staurosporine (STA), 10 µM genistein (GEN), 5 mM EGTA, 100 µM trifluoperazine (TFP), 500 µM NMMA, and L-NIL for 6 h. Data represent mean ± S.E.M. of six replicate values in two separate experiments. Statistically different from intact macrophages (percentage of control, P < 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Effect of ambroxol on silica-induced cell death. Alveolar macrophages (3 × 105 cells/200 µl) were incubated either with variable concentrations of silica (A) or with 100 µg/ml silica in the presence of variable concentrations of ambroxol (B) for 6 h. Data represent mean ± S.E.M. of six replicate values in two separate experiments. Statistically different from intact macrophages or silica alone (P < 0.05).

View larger version (14K): [in this window] [in a new window]   Fig. 10.   Effect of ambroxol on silica-induced increase in caspase-3 activity. Alveolar macrophages (2 × 106 cells/ml) were incubated either with variable concentrations of silica (A) or with 100 µg/ml silica in the presence of variable concentrations of ambroxol (B) for 6 h. Caspase-3 activity was measured using the assay kit. Data represent mean ± S.E.M. of six to eight replicate values in two separate experiments. Statistically different from intact macrophages or silica alone (P < 0.05).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Free radicals have been suggested to participate in the pulmonary inflammation and fibrosis associated with silicosis. Alveolar macrophages exposed to fractured silica discharged the superoxide anion and hydrogen peroxide, which was enhanced with increasing concentrations of silica. Protein kinases and calcium are known to be involved in the expression of functional responses in alveolar macrophages exposed to silica (Rojanasakul et al., 1993; Holian et al., 1994; Lim et al., 1997). In this respect, we examined the effects of protein kinase inhibitors and calcium chelator on the macrophage responses stimulated by silica. The inhibitory effects of staurosporine, genistein, EGTA, and trifluoperazine suggest the regulatory role of protein kinases and calcium in the silica-stimulated production of reactive oxygen species.

Nitric oxide, particularly peroxynitrite, has been shown to be involved in the silica-induced lung injury (Shi et al., 1998). The inhibitory effects of NMMA and L-NIL confirmed the nitric oxide production by nitric-oxide synthase, including inducible form, in the silica-activated macrophages. The effect of kinase inhibitors indicates the regulatory action of protein kinases on the nitric oxide production in macrophages activated by silica. Our report shows that N,N-dimethylsphingosine, an enhancer for epidermal growth factor receptor kinase, stimulates superoxide production by alveolar macrophages but attenuates nitric oxide production (Lee et al., 1999). This report suggests that the activation process involved in superoxide production may be different from that in nitric oxide production. The present finding also indicates that the nitric oxide production in macrophages appears to be differently affected by silica compared with the production of reactive oxygen species.

Ambroxol, a bromhexine metabolite, is demonstrated to exert a protective effect on oxidative tissue damage (Nowak et al., 1994; Cho et al., 1999) and attenuate the stimulated respiratory burst and lysosomal enzyme release in phagocytic cells activated by various stimulating agents (Lee et al., 1999; Park et al., 1999). However, the effect of ambroxol on the stimulated responses in alveolar macrophages exposed to silica has not been elucidated. Ambroxol attenuated the production of reactive oxygen and nitrogen species in alveolar macrophages treated or nontreated with silica. Ambroxol is reported to scavenge the superoxide anion released by activated phagocytic cells (Gillissen et al., 1997a). However, the reported scavenging effect of ambroxol on superoxide anion is weak and statistically insignificant (Gillissen et al., 1997b). In addition, the decomposing effect of ambroxol on hydrogen peroxide has not been investigated (Gillissen et al., 1997b; Cho et al., 1999). These findings suggest that the compound decreases the production of reactive oxygen and nitrogen species through the depressant action on macrophage responses rather than the scavenging action on reactive species. Ambroxol decreased the production of free radical production in silica-treated macrophages but did not enhance the inhibitory effect of protein kinase inhibitors. This finding suggests the involvement of protein kinase inhibition in the effect of ambroxol. The suggestion could also be supported by the finding that ambroxol inhibits the free radical production induced by phorbol-12-myristate-13-acetate, an activator of PKC, and by N,N-dimethylsphingosine, a PTK activator (Lee et al., 1999).

Phagocytic cells activated by stimulating agents effectively discharge granule enzymes that participate in tissue destruction. Alveolar macrophages treated with silica released acid phosphatase and lysozyme. Protein kinases and calcium are involved in the granule enzyme release by macrophages exposed to LPS and fMLP (Waga et al., 1993; Lee et al., 1999). The activation processes also were found in the degranulation in silica-activated macrophages. Compared with free radical production, the silica-induced release of granule enzymes was less decreased by protein kinase inhibitors, and the inhibitory effect of EGTA and trifluoperazine was not observed in nonstimulated macrophages. Therefore, the granule enzyme release from macrophages activated by silica may be differently regulated by the activation process compared with the respiratory burst, which also is observed in the fMLP-stimulated phagocytic cells (Kim et al., 2001). Ambroxol significantly attenuated the silica-induced degranulation in macrophages. The result further indicates that ambroxol shows a depressant effect on the silica-activated macrophage responses. From this effect, ambroxol may reduce a role of granule enzymes in tissue destruction.

The present findings suggest the involvement of protein kinases and calcium in the processes of free radical formation and granule enzyme release in silica-activated macrophages. Therefore, we examined the change in protein kinase activities in silica-activated macrophages and the effect of ambroxol on the enzyme activities. When macrophages were treated with silica, PKC showed the peak of enzyme activity at 5 min of exposure time, whereas PTK activity was continuously increased up to 1 h of incubation. This finding indicates that macrophages exposed to silica appear to exhibit the different increase in the enzyme activities. The inhibitory effect of ambroxol on the protein kinases may contribute to the depressant effect of ambroxol on the silica-activated macrophage responses.

A change in [Ca²⁺]_i is thought to play an important role in the activation processes of macrophages and neutrophils (Luscinskas et al., 1990; Waga et al., 1993; Lee et al., 1999). Phagocytic cells exposed to chemoattractants show a rapid increase in [Ca²⁺]_i. Unlike the phagocytic cells treated with LPS or chemoattractant, the [Ca²⁺]_i in alveolar macrophages exposed to silica was slowly increased as demonstrated in previous reports (Gleva et al., 1990; Chen et al., 1991). Silica induces the elevation of [Ca²⁺]_i through the stimulation of both calcium influx and intracellular calcium release. The calcium transport in phagocytic cells is regulated by protein kinases, which affects the functional responses (Waga et al., 1993; Lee et al., 1999). Ambroxol is found to depress the elevation of [Ca²⁺]_i in both phases in macrophages stimulated by LPS (Lee et al., 1999). The depressant effect of ambroxol on the elevation of [Ca²⁺]_i also was observed in silica-activated macrophages. The present finding shows that the inhibitory effect of ambroxol on calcium transport may contribute to depression of the respiratory burst and degranulation in macrophages activated by silica.

We examined the cytotoxic effect of ambroxol and other compounds by using an MTT assay. The findings show that the cytotoxic effect as well as the inhibition of regulatory action of calcium may accomplish the depressant effects of EGTA and trifluoperazine on the stimulated macrophage responses. Decrease in the cytotoxic effect of genistein in the presence of ambroxol suggests the cytoprotective effect of ambroxol. Free radicals generated from silica have been shown to induce damage of cell components and cause cell injury. The disruption of intracellular calcium homeostasis may be involved in cell death, resulted from increase in [Ca²⁺]_i and opening of the membrane permeability pore in mitochondria (Gleva et al., 1990; Nicotera et al., 1990). Silica is suggested to cause cell injury in alveolar macrophages through the elevation of [Ca²⁺]_i (Rojanasakul et al., 1993). The cytotoxic effect of silica was observed in alveolar macrophages as decrease in the MTT reduction, which was decreased by addition of ambroxol. In addition to this finding, the depressant action on calcium transport may also indicate the cytoprotective effect of ambroxol on macrophages against the toxic action of silica.

Fibrogenic particulates, such as asbestos and silica, induce apoptosis in alveolar macrophages (Hamilton et al., 1996). The inhibition of protein tyrosine phosphatases is found to induce apoptosis in immunocompetent cells through the increase in tyrosine phosphorylation (Holsapple et al., 1996). This finding suggests that the toxicant-induced cell death may be modulated by PTK. Staurosporine induces caspase-3-like activity and apoptosis in J774 macrophages (Coxon et al., 1998). Genistein showed a cytotoxic effect on alveolar macrophages. Therefore, it is difficult to assay the role of protein kinases in the silica-induced apoptosis by using the protein kinase inhibitors. In the present study, the silica-induced apoptosis in macrophages was assayed by measuring caspase-3 activity. Silica at the concentration of more than 100 µg induced a significant increase in caspase-3 activity in macrophages that was significantly decreased by addition of ambroxol. The above-mentioned suggestion indicates that the decreasing effect of ambroxol on silica-induced cell death, including apoptosis, may be accomplished by the inhibitory action on PTK. Ambroxol appears to exhibit a protective effect on alveolar macrophages and pulmonary tissues against the toxic action of silica.

In conclusion, ambroxol showed a depressant effect on the silica-stimulated responses and cell death in rat alveolar macrophages, which may be accomplished by the inhibition of the activation processes, protein kinases and calcium transport. The inhibitory effect of ambroxol on the silica-induced cell death may provide the protective effect on pulmonary tissues against the toxic action of silica.