Resistance to Acute Silicosis in Senescent Rats: Role of Alveolar

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Chem. Res. Toxicol. 2003, 16, 1520-1527

Resistance to Acute Silicosis in Senescent Rats: Role of Alveolar Macrophages Emanuela Corsini,*,† Alessandra Giani,† Laura Lucchi,† Sergio Peano,‡ Barbara Viviani,† Corrado L. Galli,† and Marina Marinovich† Laboratory of Toxicology, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy, and RBM, Colleretto Giacosa (TO), Italy Received July 2, 2003

We have previously demonstrated in alveolar macrophages that aging is associated with a decline in lipopolysaccharide-induced tumor necrosis factor-R production. The purpose of the present study was to investigate the immunotoxicological consequences of this defective activation in an experimental model of acute silicosis. Young (3 months old) and old (>18 months old) rats were intratracheally instilled with silica or saline as control. In young animals, as expected, silica induced a significant increase in bronchoalveolar lavage fluid of tumor necrosis factor-R, lactate dehydrogenase, and cell numbers, which correlated with increased collagen deposition and silicotic nodule formations. On the contrary, in old rats, no changes in bronchoalveolar lavage fluid or lung parameters were observed, indicating that senescent rats are resistant to the acute effects of silica. These in vivo results were confirmed in vitro, where silica-induced tumor necrosis factor-R release was drastically reduced in alveolar macrophages obtained from old animals. This could be explained with a defective protein kinase C βII translocation in aged macrophages, due to decreased expression of its anchoring protein RACK1. Furthermore, a decrease in FAS-L expression and silica-induced apoptosis in old macrophages was observed, supporting the idea that age-associated alterations in signal transduction pathways contribute to decreased sensitivity to silica-induced acute lung fibrosis in old animals.

Introduction At present, in the immunotoxicological evaluation, extremes of ages, meaning very young and old individuals, are considered as more sensitive subpopulations. Aging is associated with a progressive decline in both immune and pulmonary functions, which contribute to increased impact and severity of infections in aged individuals (1, 2). Many factors contribute to immunosenescence, including stem cell defects, thymus involution, aging of resting immune cells, replicative senescence of clonally expanding cells, defects in antigen-presenting cells, and dysfunction in several signal transduction pathways (3, 4). Age-related alterations in the functional capacity of macrophages is likely to contribute significantly to the increased risk of illness in the elderly (5). The aging process depresses chemotaxis and phagocytosis, induces a deficient respiratory burst, and is associated with a decline in the ability of these cells to present antigens and to produce cytokines (6-9). We have previously demonstrated that aging is associated with a progressive decline in the ability of rat alveolar macrophages to produce TNF-R1 in response to lipopolysaccharide (10). A significant decrease in accessory cell function and in release of TNF-R from human alveolar macrophages has also been reported (11). We demonstrated that the decline in alveolar macrophage * To whom correspondence should be addressed. Tel: +39 02 50318368. Fax: +30 02 50318284. E-mail: [email protected]. † University of Milan. ‡ RBM. 1 Abbreviations: PKC, protein kinase C; RACK-1, receptor for activated C-kinase-1; TNF-R, tumor necrosis factor-R.

functions reflects an impaired PKC signal transduction pathway and correlates with a defective PKC anchoring system (10). The activation of PKC results in redistribution (translocation) of the enzyme from the cytosol to membrane compartments (12). A family of proteins that interact with PKC has been described (13). These receptors for activated C kinase (RACKs) are 30-36 kDa proteins located in various subcellular compartments. RACK-1, a 36 kDa protein cloned from rat brain, is the best-characterized member of the RACK family. It has been shown that RACK-1 preferentially interacts with PKC β as compared to other PKC isoforms. We have shown that a deficit in RACK-1, even when there were no differences in the expression of lipopolysaccharide receptor CD14 or total PKC, contributes to the functional impairment in aged alveolar macrophages (10). Silicosis is an interstitial fibrosis resulting from workplace exposure to silica (32). Both acute and chronic exposure to silica particles leads to the formation in the lung parenchima of silicotic nodules, which are composed of inflammatory cells, mainly macrophages, and fibroblast within a whorl of collagen fibrils (14). Also, a single intratracheal instillation of silica induced by 7 days loosely organized granulomas and collagen deposition in the interstitium (15). In vitro and in vivo animal studies, as well as human investigations, strongly support the role of alveolar macrophage products in the development and progression of silicosis (16). Among the products released by macrophages in response to silica, a critical role of TNF-R in the pathogenesis of silicosis has been demonstrated (17, 18). In an experimental model of silicosis, the authors demonstrated that silica-induced collagen

10.1021/tx034139+ CCC: $25.00 © 2003 American Chemical Society Published on Web 11/14/2003

Lack of Silicosis in Old Animals

deposition was almost completely prevented by antiTNF-R antibody, but it was significantly increased by continuos infusion of recombinant TNF-R, indicating the pivotal role of this cytokine in silicosis (17). Furthermore, polymorphisms in the promoter region of TNF, in particular at position -238, quantitatively affecting mRNA synthesis, have been associated with severe silicosis in humans (19, 20). There are indications that PKC is activated in particletreated alveolar macrophages (21, 22) and that PKC inhibitors can modulate silica-induced reactive oxygen species generation and TNF-R production, thus contributing to the regulation of key cellular events important in the pathogenesis of silicosis (23, 24). The purpose of the present study was to investigate the immunotoxicological consequences of the defective activation of alveolar macrophages associated with aging in an experimental model of acute silicosis. Silica was chosen as an experimental model due to demonstration of the pivotal role of alveolar macrophage products in the development and progression of silicosis. In this study, we compared, both in vitro and in vivo, the response to silica in young adult and old rats. We demonstrated that old animals are resistant to silica-induced lung toxicity. This reflects a defective PKC translocation in aged macrophages together with a decrease in FAS-L expression and silica-induced apoptosis, indicating that ageassociated alterations in signal transduction pathways contribute to decreased sensitivity to silica-induced lung fibrosis in old animals.

Experimental Procedures Caution: Silica is hazardous and must be handled carefully. Animals. The experiments were performed with male Sprague-Dawley rats of different ages (Charles River, Calco, Italy). Young adult rats were 3 months old, while old rats were at least 18 months old. All animal care procedures were in accordance with the local and Italian Animal Care Committee (Authorization n. 16/94-A). Before use, rats were quarantinated for 2 weeks and were acclimatized to a 12 h light-dark cycle. Food and water were ad libitum. Chemicals. Microcrystalline silica was obtained from Sigma (catalog no. S-5631; St. Louis, MO). The mean particle size range was 0.5-10 µm, with approximately 80% between 1 and 5 µm. Prior to use, silica was baked at 180 °C for 16 h to inactivate any possible endotoxin contamination. The silica suspension was sonicated for 60 s prior to exposure experiments. Recombinant murine TNF-R (specific activity, 4 × 107 U/mg) was obtained from R&D System (Minneapolis, MN). Antibodies against RACK-1, PKC βII, FasL, and β-actin were obtained from Transduction Laboratories (Affinity, Nottingham, U.K.) and Santa Cruz Biotechnology (Santa Cruz, CA). Salts were purchased from Sigma. All reagents were purchased at the highest purity available. Cells. Alveolar macrophages were collected by lavaging the lungs as described previously (25). The cell recovery was similar between young and old rats, and >98% of cells were macrophages as determined by the Giemsa staining. Once washed and resuspended to 106 viable alveolar macrophages/mL for functional assays, cells obtained from at least two animals were pooled and allowed to adhere to plastic plates in serum-free RPMI 1640 (Sigma) containing 2 mM l-glutamine, 0.1 mg/mL streptomycin, 100 IU/mL penicillin, and 50 ng/mL gentamicin (medium). After adherence for 1 h at 37 °C in 5% CO2, the plates were washed once with warm medium to remove nonadherent cells. Cells were then exposed to medium with 10% FCS (Sigma) and incubated with or without silica at time and concentration indicated in the figure legends. The experiment using the

Chem. Res. Toxicol., Vol. 16, No. 12, 2003 1521 antisense oligonucleotide against RACK-1 was performed as previously described (10). After oligonucleotide treatment, cells were treated for 24 h with silica 140 µg/mL and TNF-R content determined in the conditioned media. PKC-βII Translocation Assay. Alveolar macrophages (5 × 106) were incubated in a water bath at 37 °C for 30 min in medium with 10% FCS in 15 mL polypropylene tubes to acclimate them. Then, silica was added. After different times, cells were recovered by centrifugation for 5 min at 800g at 4 °C. Western blot analysis of PKC-βII immunoreactivity in cytosolic and membrane fractions was performed as previously described (10). The images were acquired with a Nikon CCD video camera module (Nikon, Melville, NY). The optical density of the bands was calculated, and the peak area of a given band was analyzed by means of the Image 1.62 program for digital image processing (Wayne Rasband, Research Service Branch, National Institute of Mental Health, NIH, Bethesda, MA). Western Blot Analysis. For RACK-1, FasL, and β-actin, 5 × 106 alveolar macrophages obtained from rats of different ages were lysed in 100 µL of homogenization buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA (pH 7.5), 0.5% Triton X-100, 50 µM PMSF, 2 µg/mL aprotinin, 1 µg/mL pepstatin, and 1 µg/mL leupeptin) and denatured in 100 µL of Laemmli sample buffer (26) for 5 min at 100 °C. The protein content of the cell lysate was measured using a commercial kit (Bio-Rad). Ten micrograms of protein was electrophoresed into a 12% SDS-polyacrylamide gel under reducing conditions and then transferred to nitrocellulose membrane. The different proteins were visualized with RACK-1 antiserum diluted at 1:2500, FasL antiserum diluted at 1:250, and β-actin at 1:5000 as primary antibodies and developed using enhanced chemoluminescence (ECL, Amersham, Buckinghamshire, U.K.). Assay for TNF-r. TNF-R content in culture supernatants was assayed by determining the cytotoxicity of TNF-R against sensitive L929 cells, as previously described (27). Rabbit antimurine TNF (R&D System) polyclonal antibody was used to demonstrate that the cytolytic activity was due to the presence of TNF-R in the conditioned media (data not shown). The results are expressed in pg/mL or as % of relative control. TNF-R concentration was calculated against a standard curve with known amounts of recombinant murine TNF-R. Apoptosis. For determination of apoptosis, 1 × 106 macrophages were incubated with silica 140 µg/mL or with media alone for 24 h in polypropylene tubes. Apoptosis was detected by a flow cytometer with propidium iodide as the fluorescence indicator essentially as described by Nicoletti et al. (28). Briefly, after incubation, cells were centrifuged and resuspended in 1 mL of PBS. Of this suspension, 0.2 mL was incubated for 30 min with 0.5 mL of RNase (0.5 mg/mL) at room temperature, 0.5 mL of propidium iodide was then added (5 µg/mL, in PBS), and the fluorescence of individual nuclei was measured (in the FL2 channel set for log scale) using FACScan flow cytometry (Becton Dickinson, Italy). Animal Treatment. A silica particle suspension (100 mg/ mL) in saline or saline alone as control was injected intratracheally (0.3 mL) in rats (n ) 5) 3 and 18 months old. Despite the increase in body weight in old animals, mainly due to fat accumulation, the weight of the lung was very similar in young and old rats (3.8 ( 0.2 vs 4.3 ( 0.3 g lung weights in young vs old rats, respectively). Furthermore, the number of alveolar macrophages recovered by bronchoalveolar lavage was identical in young and old rats. For these reasons, the same amount of silica was instilled. Animals were killed 14 days after instillation and lavaged in situ using a closed chest technique after cannulation of the trachea with a 19-gauge needle. Lungs were lavaged with 5 mL of Ca2+- and Mg2+-free HBSS eight times. The first bronchoalveolar lavage fluid was kept separated, centrifuged, and used for biochemical analysis (lactate dehydrogenase and TNF-R). Elevation of the cytoplasmic enzyme lactate dehydrogenase is a useful indicator of cytotoxicity, and its presence in the bronchoalveolar lavage fluid is a marker of lung damage and correlates with pulmonary fibrosis (29). The

1522 Chem. Res. Toxicol., Vol. 16, No. 12, 2003 recovery of bronchoalveolar lavage fluid was similar in all groups of rats. The cells obtained from complete lung lavage, after centrifugation, were suspended in 5 mL of RPMI 1640 culture medium (Sigma), and the total cell number was determined electronically (Coulter Counter). Aliquots of the cell suspension were used to prepare slides for differential counts using a cytospine apparatus. The cytospins were stained with Giemsa stain. Lactate Dehydrogenase. Lactate dehydrogenase was determined in the bronchoalveolar lavage fluid using a commercially available kit (Sigma). Results are expressed in mU/ mL. Hydroxyproline Content of the Lung. Collagen deposition was estimated by determining the total hydroxyproline content of the lung. The right lung lobes were excised, homogenized, and hydrolyzed in 6 N HCl overnight at 110 °C. Hydroxyproline content was assessed colorimetrically at 560 nm with p(dimethylamino)benzaldehyde (30). Data are expressed as µg OH-proline/g tissue. Histopathology. Following bronchoalveolar lavage, the left lung lobe was excised and fixed in 10% neutral buffered formalin. Paraffin-embedded tissue sections were prepared at 5-6 µm thickness and were stained with hematoxylin and eosin. Histological examination was done at RBM. Statistical Analysis. Each experiment was performed at least twice. The data are expressed as the mean ( SD; representative results are shown. Statistical significance was determined by Student’s t-test of Dunnett’s multiple comparison test where appropriated, after ANOVA. The p values less than 0.05 were considered statistically significant.

Results Defective PKC Activation Underlies the Decreased Production of TNF-r Following Silica Stimulation in Macrophages Obtained from Old Animals. To test the involvement of RACK-1 in silicainduced TNF production, alveolar macrophages, obtained from young rats, were treated for 48 h with 5 µM antisense oligonucleotide to decrease RACK-1 translation and then stimulated with 140 µg/mL silica for 24 h. As shown in Figure 1, under conditions in which RACK-1 expression was significantly decreased (∼40%, Figure 1A), RACK-1 antisense oligonucleotide prevented silicainduced TNF-R production, while the control sense oligonucleotide was ineffective (Figure 1B), demonstrating that RACK-1 is essential for silica-induced TNF-R production. We then evaluated the translocation of PKC following silica treatment in alveolar macrophages obtained from young and old rats. Alveolar macrophages were obtained by bronchoalveolar lavage from male Sprague-Dawley rats of different ages (3 and >18 months). The cells recovered, >98% of which were macrophages, were similar in number and morphology in the two groups. As shown in Figure 2, aging was associated with a defect in PKC translocation to the membrane compartment following silica stimulation, while in alveolar macrophage obtained from young animals, silica induces a timerelated PKC translocation. Alveolar macrophages obtained from young and old rats were then treated with increasing concentration of silica (0-280 µg/mL) and TNF-R production was assessed 24 h later. As shown in Figure 3A, alveolar macrophages obtained from old rats produced significantly less TNF-R in response to silica at all concentrations tested. This was not due to a different kinetic, as shown in Figure 3B in time-course experiments; alveolar macrophages obtained from old rats treated with silica 140 µg/mL released

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Figure 1. RACK-1 is essential for silica-induced TNF-R production. Alveolar macrophages (106/mL) obtained from young rats (3 months old) were treated with RACK-1 antisense or sense oligonucleotide (5 µM) as the control for 48 h, and then, 140 µg/mL silica was added. Twenty-four hours later, TNF-R production was assessed. (A) RACK-1 immunoreactivity following antisense and sense oligonucleotides treatment. β-Actin immunoreactivity was used as the control of protein loading. (B) TNF-R release. Each value represents the mean ( SD of four samples. ANOVA followed by Dunnett’s t-test, with *p < 0.01 vs relative control; §p < 0.01 vs silica and silica sense. Antisense ) RACK-1 antisense oligonucleotide; sense ) RACK-1 sense oligonucleotide. Table 1. Aging Is Associated with a Progressive Decline in the Ability of Alveolar Macrophage to Produce TNF-r in Response to Silicaa age (months)

TNF-R (% of control)

RACK-1/β-actin

3 6 12

620 ( 77* 430 ( 135* 286 ( 54*,§

1.81 ( 0.24 1.62 ( 0.12 0.90 ( 0.30§

a Alveolar macrophages (106/mL) obtained from animals of different ages were treated for 24 h with 140 µg/mL silica. Immunoreactivity of RACK-1 and β-actin in cell homogenate was assessed by Western blot analysis. Each value represents the mean ( SD of 3-4 animals. ANOVA followed by Dunnett’s t-test, *p < 0.01 vs relative control; §p < 0.01 vs 3 month old animals treated with silica.

significantly less TNF-R as compared to alveolar macrophages obtained from young adult rats at all times tested. Furthermore, a progressive decline with aging in the ability of alveolar macrophages to respond to silica in terms of TNF-R release was observed, which was associated with a progressive reduction in RACK-1 expression. As shown in Table 1, in alveolar macrophages obtained from 3, 6, and 12 month old rats, a gradual decline was observed in silica-induced TNF-R release, which paralleled a decline in RACK-1 expression. By the age of 12 months, a 50% reduction in RACK-1 expression was associated with a 48% reduction in silica-induced TNF-R release. Lack of Fibrosis in Old Rats in an Acute Model of Silicosis. Young (3 months old) and old (18 months

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Figure 2. Silica fails to induce PKC translocation in alveolar macrophages obtained from old rats. Alveolar macrophages (106/mL) obtained from young (3 months old) and old (18 months old) rats were treated with 140 µg/mL silica. PKCβII translocation was assessed after 15 and 30 min. (A) Representative Western blot analysis of PKCβII immunoreactivity in the membrane compartments. β-Actin immunoreactivity was used as the control of protein loading. (B) Densitometric analysis. The data for both PKCβII in cytosol and membrane compartments were normalized for the relative β-actin immunoreactivity. Each data represents the mean ( SD of three independent experiments. ANOVA followed by Dunnett’s t-test, with *p < 0.01 vs time 0. Table 2. Bronchoalveolar Lavage Fluid Parameters Following Silica Instillation in Young and Old Ratsa

treatment

total cells × 106

TNF-R (pg/mL)

lactate dehydrogenase (mU/mL)

saline silica (30 mg)

young (3 months old) 8.8 ( 3.8 2.9 ( 1.1 61.3 ( 26.4* 11.2 ( 4.7*

29 ( 12 101 ( 51*

saline silica (30 mg)

old (18 months old) 12.2 ( 3.7 2.7 ( 1.1 8.0 ( 2.8§ 2.6 ( 0.5§

32 ( 12 38 ( 8§

a Animals were intratracheally instilled with 30 mg of silica or saline as the vehicle control. Animals were terminated 14 days following instillation and cellularity, and TNF-R and lactate dehydrogenase were measured in the bronchoalveloar lavage fluid as described in the Experimental Procedures. Each value represents the mean ( SD of five animals. ANOVA followed by Dunnett’s t-test, *p < 0.05 vs relative control; §p < 0.05 vs young treated with silica.

old) rats were intratracheally instilled with 30 mg of silica or saline as vehicle control. Thirty milligrams was chosen based on an average of the doses used in studies published in the literature, which varies from less than 1 mg up to 50 mg, as well as considering that commercially available microcristalline silica, not freshly grounded (likely to be less reactive), was used. Animals were terminated 14 days following instillation. This time point was chosen because it still allows the detection of biochemical markers of activation in the bronchoalveolar lavage fluid and the collagen deposition already started. Cell number, TNF-R, and lactate dehydrogenase were examined in bronchoalveolar lavage fluids. As shown in Table 2, a signifcant increase in cellularity, TNF-R release, and lactate dehydrogenase leakage was observed in young adult rats following silica exposure. As expected, the inflammatory cells recovered in bronchoalveolar lavage fluid of silica-exposed young adult rats consisted primarily of neutrophils (70%, data not shown). On the

contrary, no changes in all parameters considered were observed in old rats exposed to silica. Furthermore, silica exposure of young adult rats resulted in a statistically significant increase in lung hydroxyproline content (Figure 4B), indicative of collagen deposition. Once again, no change was observed in old rats exposed to silica. The histology of the lungs supported the biochemical changes. Microscopic changes relating to silica exposure were observed only in young adult rats, consisting mainly of inflammatory cell infiltration associated with silicotic nodules in the interstitium (Figure 4A). No changes in lung histology were observed in old rats following silica exposure, indicating that senescent rats are resistant to acute silicosis. Decreased in Vitro Responsiveness of Alveolar Macrophages Obtained from Old Rats to SilicaInduced Apoptosis. The absence of response to silica in old animals was striking. This prompted us to investigate if other mechanism(s) relevant in the induction of silicosis may contribute to the decreased responsiveness in old animals. A pivotal role for FasL in a mouse model of pulmonary silicosis has recently been shown as follows: Borges et al. demonstrated the lack of silicosis in FasL deficient mice and located its action upstream of TNF-R (31). This, together with evidences of a decreased Fas-mediated apoptosis in the immune system of elderly individual (32, 33), led us to investigate FasL in our experimental model. As shown in Figure 5A, in alveolar macrophages obtained from old rats, there is a statistically significant reduction in FasL immunoreactivity, which leads to a significant reduction in silica-induced apoptosis in the macrophages obtained from old rats (Figure 5B). At the age of 18 months, alveolar macrophages showed >30% reduction in FasL expression (0.73 ( 0.07 vs 0.48 ( 0.10 FAS-L/β-actin ratio in young and old rats, respectively), which was associated with a 45% reduction in silica-induced apoptosis.

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inflammation and tissue destruction, followed by tissue repair leading to fibrosis. TNF-R plays a central role in pulmonary inflammation and fibrosis induced by silica deposition (17, 34), and Borges et al. demonstrated the lack of silicosis in FasL deficient mice and located its action upstream of TNF-R (31). The authors demonstrated that Fas ligand deficient mice [gld] did not develop silicosis, had markedly reduced neutrophil extravasation into bronchoalveolar space, and did not show increased TNF-R production. Furthermore, the administration of a neutralizing anti-FasL antibody in wildtype mice blocked induction of silicosis, indicating that FasL has a central role in induction of silicosis. A scenario has been proposed as follows: oxidant injury provoked by silica induces FasL expression (35) in macrophages, which is followed by apoptosis and the release of chemotactic factors for neutrophils from the dying macrophages. FasL itself has also been shown to be chemotactic for neutrophils: forced expression of FasL can induce a dramatic inflammatory response by promoting the production of proinflammatory mediators by preapoptotic resident tissue macrophages (36). Neutrophils in turn would phagocytose dying cells and, together with macrophages, damage the lung parenchyma by releasing reactive oxygen species, proteolytic enzymes, and cytokines, initiating a vicious circle. Figure 3. Alveolar macrophages obtained from old rats produce less TNF-R in response to silica as compared to young rats. (A) Dose response. Alveolar macrophages (106/mL) obtained from young (3 months old) and old (18 months old) rats were treated with increasing concentrations of silica. TNF-R release was assessed 24 h later. (B) Time-course. Alveolar macrophages (106/mL) were treated with 140 µg/mL silica for different times (2, 4, 24, and 48 h). Results are expressed as percent of relative control. The release of TNF-R from control cells was 17 ( 6 and 17 ( 4 pg/mL in young vs old macrophages, respectively. Each value represents the mean ( SD of four samples. ANOVA followed by Dunnett’s t-test, with *p < 0.05 and **p < 0.01 vs relative control; §p < 0.01 vs young treated with silica.

This together with decreased TNF-R release is likely to significantly contribute to the lack of responsiveness observed in the old animals following silica instillation. Neutralizing antibody against TNF-R failed to prevent silica-induced apoptosis in alveolar macrophages obtained from young animals (% of apoptotic cells 39.5 ( 5.3 vs 39.5 ( 3.0 in alveolar macrophages treated with silica 140 µg/mL in the absence or presence of anti TNF-R antibody 1:200 diluted, respectively), further supporting the disjunction of the two events (31).

Discussion The understanding of the molecular mechanisms underlying toxicity is crucial to define the influence of age on the toxic response and progression of the disease. At present, in immunotoxicology assessment, the extremes of age are considered as more sensitive subpopulations. Here, we demonstrated in an experimental model of acute silicosis that aged rats do not develop silicosis. A decrease in both silica-induced TNF-R production, FasL-mediated apoptosis, and subsequent neutrophil accumulation contributes to the lack of response in the aged animals. Both TNF-R and FasL have been reported to be crucial in the induction of silicosis (17, 31). Silicosis starts with

In macrophages obtained from old animals, we observed a decrease in both TNF-R production and silicainduced apoptosis, which significantly contributed to the lack of response observed following in vivo instillation. The decreased FasL expression in old macrophages helps to explain the lack of neutrophil inflammation in old animals instilled with silica. We demonstrated that the decrease in silica-induced TNF-R production, as previously shown for lipopolysaccharide (10), reflects impairment in PKC activation due to a decrease in its anchoring protein RACK-1. The decrease in FasL and FasL-mediated apoptosis does not seem, however, to be related to the decline in expression of the receptor for activated C kinase associated with aging. RACK-1 antisense oligonucleotide fails to modulate FasL expression in young alveolar macrophages (data not shown), while significantly reduces the ability of these cells to produce TNFR, suggesting that these two age-associated defects occur independently. In addition, neutralizing antibody against TNF-R failed to prevent silica-induced apoptosis in alveolar macrophages obtained from young animals, further supporting the disjunction of the two events. It is likely that other age-associated defects may also contribute to the lack of response to silica. Aging is indeed associated with profound alteration in the physiology of both the immune and the pulmonary system. It is possible to speculate a role of the antiinflammatory cytokine IL-10 or TGF-β, which has been demonstrated to be more frequently present in bronchoalveolar lavage from old normal individuals (37). In the context of silicosis, it has been shown that treatment with recombinant IL-10 reduces reactive oxygen species and nitric oxide in silica-exposed rats (38). Thus, IL-10 may eventually contribute limiting the extent and duration of inflammatory reaction in the old animals. Preliminary data, however, do not indeed indicate increased IL-10 levels in the bronchoalveolar lavage fluid obtained from old rats (data not shown).

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Figure 4. Old rats are resistant to silica-induced lung toxicity. Young (3 months old) and old (18 months old) rats were intratracheally instilled with 30 mg of silica or sterile saline as the control. Animals were terminated 14 days following instillation. (A) Histological section (H&E staining; magnification 40×) of the lungs from young and old rats instilled with saline or silica. (B) Lung hydroxyproline concentration determined colorimetrically after acid hydrolysis of the lungs. Results are expressed as µg OH-proline/g tissue. Each value represents the mean ( SD of five animals. ANOVA following Dunnett’s t-test, with *p < 0.01 vs relative saline control; §p < 0.01 vs young rats instilled with silica.

Other factors, besides cytokines, may also influence the sensitivity to silicosis. The generation reactive oxygen species and nitric oxide in response to silica, as mediators of lung injury following silica exposure (18, 39), or the production and the level of surfactants in the lungs, as pulmonary phospholipidosis attenuates the acute damage associated with intratracheal instillation of silica (40, 41), may also contribute to the different responsiveness observed. One can speculate that a decrease in oxidative

stress in response to silica or an increase in surfactants in old rats may make old animals less sensitive to silicosis. This is supported by the observation that aging is associated in monocytes/macrophages with depressed chemotaxis, phagocytosis, and deficient respiratory burst (5). On the other hand, there are factors that could make old individuals more sensitive to toxicity, such as increased retention in the lung due to diminished muco-

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model of silicosis in old animals. Our findings may also give a biological explanation for previous epidemiological findings that the risk of contracting silicosis is higher at lower starting age (44). Silica was initially chosen as a reference compound, due to demonstration of the pivotal role of alveolar macrophage products in the development and progression of silicosis. However, we also have evidence that alveolar macrophages obtained from old rats are less responsive to other pulmonary toxicants, such as diesel-exhausted particles or carbon black particles. The influence of innate immunity on the sensitivity to a toxic insult is not limited to silicosis. There is also evidence that the impaired macrophage activation associated with aging contributes to attenuation of cadmiuminduced liver injury (45). Cadmium-mediated stimulation of Kupffer cell activity and elevation of hepatic cytokines were markedly reduced in old rats as compared with young adult rats, indicating that the attenuation of Cd-induced liver injury that occurs in old age is caused by an impairment in Kupffer cells activation. Overall, our data suggest that old individuals are not necessarily more sensitive to immunotoxic compounds, but the understanding of the mechanisms underlying toxicity is critical to define the impact of age on the toxic response and progression of the disease. Considering the demographic trend of the world population toward an increased incidence of aging related conditions, we believe that this paper further contributes to our understanding of immunosenescence and its consequences in term of age-dependent susceptibility and/or resistance to various environmental contaminants.

Figure 5. Decrease in FasL expression in alveolar macrophages obtained from old rats and in silica-induced apoptosis. (A) FasL and β-actin immunoreactivity in cell homogenate obtained from young (3 months old, n ) 5) and old (18 months old, n ) 4) rats and relative densitometric analysis. Each value represents the mean ( SD. Student’s t-test, with *p < 0.05 vs young animals. (B) Relative frequency of DNA fragmentation in alveolar macrophages induced by silica. Alveolar macrophages (106/mL) obtained from animals of different ages were treated for 24 h with 140 µg/mL of silica. Apoptosis was assessed by flow cytometer with propidium iodide as the fluorescence indicator. Histograms are from one representative experiment. The percentage of apoptotic cells is reported. Each value represents the mean ( SD of three samples. ANOVA followed by Dunnett’s t-test, *p < 0.01 vs relative control; §p < 0.01 vs 3 month old animals treated with silica.

ciliary clearance with advancing age (37), depressed glottic protective reflexes (42), and decreased activity of hydrogen peroxide scavenging enzymes (43). As the cells of the immune system are affected in many ways by aging, it is reasonable to propose that the influence of aging on the acute silicosis involves alteration in the inflammatory process. Our data indicate that critical mediators of silicosis derived from the macrophages are defective in the aged rats, making them resistant to silicosis. We cannot, however, exclude that older rats might develop evidence of silicosis at later time points, but we clearly demonstrated under the same experimental conditions the lack of response in an acute

Acknowledgment. This work was partially supported by FIRST funds from the University of Milan and by the Center for Risk Assessment of the University of Milan. We also thank Prof. H. van Loveren, Dr. A. J. Boere, and Dr. F. Cassee of the National Institute of Public Health and Environmental Health, Bilthoven, The Netherlands, for the training on intratracheal instillation.

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