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Clusterin Decreases Oxidative Stress in Lung Fibroblasts. Exposed to Cigarette Smoke. Stefano Carnevali*, Fabrizio Luppi*, Domenico D'Arca, Andrea Caporali ...
Clusterin Decreases Oxidative Stress in Lung Fibroblasts Exposed to Cigarette Smoke Stefano Carnevali*, Fabrizio Luppi*, Domenico D’Arca, Andrea Caporali, Maria Paola Ruggieri, Maria Vittoria Vettori, Andrea Caglieri, Serenella Astancolle, Francesca Panico, Pierpaola Davalli, Antonio Mutti, Leonardo M. Fabbri, and Arnaldo Corti Section of Respiratory Diseases, Department of Oncology, Haematology, and Pulmonology, and Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena; Department of Experimental Medicine; ISPESL Research Center; Laboratory of Industrial Toxicology; and Department of Clinical Medicine, Nephrology of Health Sciences, University of Parma, Italy

(Received in original form December 1, 2005; accepted in final form May 18, 2006 )

flammatory response through the synthesis and release of several molecules, water-soluble components of cigarette smoke pass through the basement membrane and directly interact with fibroblasts (7, 9–11), the main cell type in the lung interstitium that is involved in tissue repair and remodeling (12). In vitro, cigarette smoke and its aqueous extracts affect various lung fibroblast functions, including inhibition of their recruitment and proliferation (13) and induction of apoptosis (14). Clusterin (also called apolipoprotein J and SGP-2, among many other names) is a heterodimeric glycoprotein, encoded by a single-copy gene, that contains 449 amino acids and generates a precursor form with a predicted molecular mass of 60 kD. Mature clusterin is glycosylated and secreted as a protein of 76–80 kD (15, 16). The protein is cleaved into one ␣ chain and one ␤ chain of about 40 kD each and held together by a unique 5-disulfide-bond motif (17). The wide distribution and sequence conservation of clusterin suggest that this protein performs functions of fundamental importance both inside and outside cells. Clusterin has been implicated in a variety of physiologic and pathologic processes, including cancer (18–20) and experimental models of pathologic stress (21). Clusterin-deficient mice exhibit enhanced inflammatory severity and sequelae in an autoimmune myocarditis model, suggesting that clusterin can have an antiinflammatory role under some conditions (22). Several studies suggest that clusterin may also confer protection against various cytotoxic agents, particularly on induction of oxidative stress, in several cell lines, including renal epithelial cells and human epidermoid carcinoma cells (23, 24). Also, in epithelial cells exposed to H2O2, clusterin protected cells from exogenous oxidant injury (23). This raises the possibility that clusterin may be encoded by a gene that exerts a protective function on surviving cells. Furthermore, clusterin appears to display a chaperone-like activity, similar to that of small heat shock proteins, which is important for cytoprotection in various diseases (25, 26). Although clusterin is involved in a variety of diseases, its role in cigarette smoke–related lung diseases has not been yet examined. The aim of the present study was to evaluate the effect of cigarette smoke on clusterin expression and the protective effect of this molecule on cigarette smoke–induced oxidative stress in lung fibroblasts. Some of the results of these studies have been previously reported in the form of abstracts (27, 28).

Supported by Associazione Ricerca Cura Asma, Consorzio Ferrara Ricerche, PRIN 2003 (2003062087_005).

METHODS

Rationale: Cigarette smoke causes injury to lung fibroblasts, partly by means of oxidative stress, and oxidative stress can lead to various lung diseases, such as chronic obstructive pulmonary disease. Clusterin is a widely distributed protein with many functions, including cellular protection in response to oxidative stress. Objectives: To determine whether clusterin is involved in the defense of the lung against cigarette smoke, we investigated the effects of cigarette smoke extract on clusterin expression and its protective effect, if any, against oxidative stress. Methods: Fibroblasts were coincubated with conditioned medium and cigarette smoke extract, and bronchial biopsy specimens obtained from nonsmokers, smokers, and ex-smokers were analyzed by immunohistochemistry. Measurements and Main Results: At concentrations of 2.5 and 5.0%, cigarette smoke extract induced oxidative stress. It also markedly increased the expression of two clusterin isoforms (60 and 76–80 kD) and the 76–80-kD isoform was secreted in the incubation medium. Coincubation of fibroblasts with conditioned medium significantly decreased the cellular oxidation caused by the cigarette smoke extract. Immunohistochemical analysis of clusterin on bronchial biopsy specimens obtained from smokers and ex-smokers showed localization of clusterin mainly in the submucosa. Conclusions: We conclude that clusterin may have a protective effect against cigarette smoke–induced oxidative stress in lung fibroblasts. Keywords: chronic bronchitis; chronic obstructive pulmonary disease; emphysema; inflammation; lung injury

Cigarette smoke, the major risk factor for various lung diseases, such as chronic obstructive pulmonary disease (1–3), contains more than 4,500 chemical compounds, including high concentrations of oxygen free radicals and nitric oxide (NO) in the gas phase, and organic compounds such as semiquinone radicals in the tar phase, which result in the generation of reactive oxygen (ROS) and nitrogen species (4, 5), that induce lung damage by a variety of mechanisms. For example, generation of free radicals and ROS (6, 7) is increased in smokers (8). External stimuli first target epithelium, which acts as a mechanical barrier and contributes to the development of the in-

* These authors contributed equally to this work Correspondence and requests for reprints should be addressed to Leonardo M. Fabbri, M.D., Department of Oncology, Haematology, and Pulmonology, University of Modena & Reggio Emilia, Via del Pozzo 71, 41100 Modena, Italy. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 174. pp 393–399, 2006 Originally Published in Press as DOI: 10.1164/rccm.200512-1835OC on May 18, 2006 Internet address: www.atsjournals.org

A more detailed description of the methods is available in the online supplement.

Cell Culture and Treatments Human fetal lung fibroblasts (HFL-1; lung, diploid, human) were obtained from the American Type Culture Collection (Rockville, MD). HFL-1 fibroblasts were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and penicillin/streptomycin, and used for experiments from passage 15 to 20. Cigarette smoke extract

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(CSE) was prepared by a modification of the method of Carp and Janoff (29) as previously described. Subconfluent cells were exposed to either 2.5 or 5.0% CSE.

Western Blot Analysis Total cells were homogenized in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.25% Na-deoxycholate, 1% NP-40, 50 ␮g/ml DNase, 50 ␮g/ml DNase, and protease inhibitor cocktail. Protein was quantified with Bradford reagent (Sigma Chemicals, Milan, Italy). Clusterin-immunoreactive bands were detected with chemiluminescence blotting substrate (Roche Diagnostic, Milan, Italy) using anti-human clusterin monoclonal antibody (Upstate Biotechnology, Lake Placid, NY) followed by horseradish peroxidase–conjugated anti-mouse secondary antibody (Sigma Chemicals, Milan, Italy). ␤-Actin (Upstate Biotechnology) was used as a loading control.

Conditioned Medium HFL-1 fibroblasts were incubated with 5.0% CSE and conditioned medium harvested after 72 h. The supernatant was concentrated, and the resulting suspension was stored after the presence of clusterin was determined by Western blot analysis.

Statistical Analysis Statistical analysis was performed using analysis of variance and Student’s t test. A p value of less than 0.05 was considered significant. All values are reported as the mean ⫾ SD of independent experiments. Results obtained by flow cytometry were evaluated using KolmogorovSmirnov statistical analysis for comparisons between frequency distributions of fluorescence intensity of cells (35, 36). Statistical differences between distributions of events were accepted with D/s(n) ⬎ 8.50, where D/s(n) is an index of similarity between two curves.

cDNA Synthesis and Real Time–Polymerase Chain Reaction cDNA preparations were amplified by polymerase chain reaction (PCR). The real-time PCR data were normalized by the ⌬Ct method (31) using hydroxymethylbilane synthase (HMBS) as housekeeping gene (32).

Immunocytochemistry Cells were fixed with methanol-acetone solution. The cells were challenged with blocking solution and subjected to immunocytochemical analysis using mouse anti-human clusterin monoclonal antibody. Digital images were obtained with a digital camera (AxioCam; Carl Zeiss, Mu¨nchen-Hallbermoos, Germany) directly through the microscope.

Cytofluorometric Analysis Cellular oxidative stress was measured fluorometrically by monitoring the oxidation of intracellular dichlorodihydrofluorescein diacetate (Molecular Probes, Eugene, OR). Lung fibroblasts were exposed to fresh medium (control) or to 5.0% CSE for 24 h, and fluorescence intensity was measured by a flow cytometric assay as previously described (14). An isotype-matched control antibody (normal mouse IgG; Upstate Biotechnology) was used in control experiments. Cell cycle analysis was evaluated after staining the samples with propidium iodide (Sigma).

Lipid Peroxidation Cellular lipid peroxidation products were identified by measuring thiobarbituric acid reactive substances (TBARS) according to the method described by Jentzsch and colleagues (33), adapted for cells. TBARS concentration was measured using a Cary Eclipse fluorescence spectrophotometer (Varian, Inc., Cary, NC; excitation 515, emission 545). The same isotype-matched antibody used for cytofluorometric analysis was used for control experiments. For glutathione depletion, DL-buthionine sulphoximine (BSO; Sigma) was used.

Immunohistochemical Analysis Bronchial biopsy samples were obtained by fiberoptic bronchoscopy from six nonsmokers, three current smokers, and eight ex-smokers, and prepared for light microscopy as previously described (34).

RESULTS Effect of Cigarette Smoke on Clusterin Expression in HFL-1 Cell Lysates

At concentrations of 2.5 and 5.0%, CSE increased two clusterin bands of about 60 and 76–80 kD (because of the reducing experimental condition, the 76–80-kD form is seen as a 40-kD band) on Western blots after 24, 48, or 72 h of incubation with HFL-1 cell lysates (Figure 1A). The 60-kD form represents the unglycosylated, uncleaved precursor of the 76–80-kD isoform (32). Both concentrations of CSE increased the concentration of the 60kD clusterin isoform at all time intervals, but the strongest increase was with 5.0% CSE after 48 h. The secretion isoform of clusterin (76–80-kD band) was also increased after exposure to both concentrations of CSE at all time intervals studied, but the increase induced by 2.5% CSE was greater than that induced by 5.0% CSE. In both untreated cells and cells exposed to 2.5% CSE, the rate of expression of 76–80-kD clusterin increased with time. The amount of ␤-actin detected under the same conditions did not show any significant changes (Figure 1). We also used Western blot analysis to measure the concentration of clusterin in the cell culture–conditioned medium at the three incubation times. At 24 h, clusterin was undetectable in the medium, either in the presence or absence of CSE (Figure 1B). At 48 h, the 76–80-kD isoform was still undetectable in the supernatant of untreated fibroblasts, but it appeared in the medium of CSE-treated cells, and its intensity increased with the concentration of CSE. At 72 h, a small amount of clusterin was secreted into the medium of untreated cells, and a large amount of clusterin was secreted by cells exposed to 2.5 or 5.0% CSE (Figure 1B). The 60-kD isoform was never found in the medium (data not shown). We also performed fluorescence-activated cell sorting (FACS) analysis to determine the effects of CSE on cell cycle

Figure 1. A Western blot showing clusterin expression in cell lysates (A ) and conditioned medium (B ) of lung fibroblasts exposed to 2.5 or 5.0% cigarette smoke exposure for 24, 48, or 72 h. Representative results from three independent experiments are shown. ␤-Actin (42 kD) was used as a loading control. * Because of the reducing experimental condition, the 76–80-kD form is seen as a 40-kD band.

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TABLE 1. CELL CYCLE Control

24 h 48 h 72 h

G0/G1

S

72.9 86.3 86.6

3.3 2.3 1.7

CSE 2.5%

CSE 5%

G2/M

G0/G1

S

G2/M

G0/G1

S

G2/M

5.7 4.9 5.1

73.7 73.5 85.9

4.2 1.9 1.6

10.5 7.2 8.4

65.4 70.9 80.9

3.1 2.7 2.0

11.3 12.6 13.0

Definition of abbreviation: CSE ⫽ cigarette smoke exposure. HFL-1 cells were incubated with control medium and CSE 2.5 and 5.0% for 24, 48, and 72 h. The values represent the percentage of the total cell population in each phase of the cell cycle. All assays were performed in triplicate, and all experiments were repeated at least three times.

parameters. As shown in Table 1, in untreated cells the percentage in G0/G1 phases increased with the time of incubation. Interestingly, the incubation of the cells with 5.0% CSE caused a twofold increase in the percentage of cells in the G2/M phases, and this effect was associated with a decrease of cells in the G0/G1 phases. We then measured the concentration of clusterin mRNA under the same conditions by real-time PCR. In contrast to the results at the protein level (Figure 1), incubation of untreated fibroblasts for up to 72 h did not significantly affect the levels of clusterin mRNA (Figure 2). Similarly, CSE did not affect clusterin mRNA levels after 24 or 48 h of incubation, but the mRNA level increased at both CSE concentrations after 72 h (Figure 2). We also studied the localization of clusterin in HFL-1 cells incubated for 24 h with 2.5 or 5.0% CSE (Figure 3). Consistent with the data obtained by Western blot analysis, the concentration of clusterin was very low in untreated cells, whereas it markedly increased in the cytoplasm of the fibroblasts after CSE stimulation, particularly after exposure to 5.0% CSE. To quantify the differences shown in Figure 3A, we performed a computer analysis of fluorescent signals in an equivalent region of interest, and the results were expressed as fluorescence per

Figure 2. Accumulation of clusterin mRNA analyzed by real-time polymerase chain reaction (PCR) after exposure of cells to either 2.5 or 5.0% cigarette smoke exposure (CSE) for 24 h (black circles), 48 h (black triangles), or 72 h (black squares). Real-time PCR data were normalized by mRNA level to hydroxymethylbilane synthase. * Significant increase in clusterin mRNA levels (p ⬍ 0.05).

1,000 pixels. As shown in Figure 3B, CSE caused a significant increase in fluorescence at concentrations of both 2.5% (p ⬍ 0.05) and 5.0% (p ⬍ 0.001). Clusterin-induced Decrease in Oxidative Stress Caused by CSE

On the basis of the results described above and previous data showing that CSE induces oxidative stress in human lung fibroblasts (14), we then investigated the protective effect of clusterin on human lung fibroblasts. We incubated HFL-1 cells for 24 h with 5.0% CSE and assessed levels of oxidative stress using flow cytometry (FACS) analysis of dichlorodihydrofluorescein diacetate–labeled cells. Figure 4A shows that exposure of fibroblasts to 5.0% CSE resulted in a significant increase in cellular oxidative stress (D/s(n) ⫽ 57.90; Kolmogorov-Smirnov statistical

Figure 3. (A ) Fluorescence microscopic imaging of clusterin localization in the cytoplasm of lung fibroblasts exposed to CSE (green fluorescence). Cell nuclei were visualized with 4⬘,6⬘-diamidino-2-phenylindole (blue). (B ) Fluorescence intensity (obtained by fluorescence microscopy) in HFL-1 cells exposed to 2.5 or 5.0% CSE. Analysis was performed with Adobe Photoshop (Adobe, San Jose, CA) for each cell and for each period of time over a similar region of interest and expressed as fluorescence per 1,000 pixels. * Significant increase in fluorescence after 24 h with 2.5% CSE (p ⬍ 0.05). ** Significant increase in fluorescence after 24 h with 5.0% CSE (p ⬍ 0.001).

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analysis). To test the hypothesis that clusterin exerts a protective role by reducing the oxidative stress caused by cigarette smoke, as shown in other biological systems (23, 24), we incubated HFL-1 cells with 5.0% CSE for 24 h in normal medium or in clusterin-enriched medium obtained from HFL-1 fibroblasts previously incubated for 72 h with 5.0% CSE (see Methods). As indicated in Figure 4B, conditioned medium significantly decreased cellular oxidation caused by cigarette smoke (D/s(n) ⫽ 34.73; Kolmogorov-Smirnov statistical analysis). To confirm the involvement of clusterin in the observed decrease of oxidative stress with conditioned medium, we incubated the conditioned medium overnight with anti-clusterin monoclonal antibody before using it for incubation of fibroblasts. As shown in Figure 4C, clusterin antibody significantly increased the level of oxidative stress (D/s(n) ⫽ 29.12; Kolmogorov-Smirnov statistical analysis) relative to that in fibroblasts incubated in the conditioned medium without added antibody. In contrast, control isotype antibody was not able to block oxidative stress inhibition caused by conditioned medium (Figure 4D). Lipid Peroxidation in CSE-exposed Fibroblasts

Figure 4. Flow cytometric analysis of the effect of CSE on oxidation in lung fibroblasts in vitro. The x axis represents the intensity of intracellular dichlorodihydrofluorescein diacetate fluorescence. HFL-1 fibroblasts were exposed to 5.0% CSE (A ), 5.0% CSE plus conditioned medium (B ), or 5.0% CSE plus conditioned medium and anti-clusterin monoclonal antibody (Ab) (C ). As shown in D, isotype-matched control antibody was not able to block the antioxidant effect of conditioned medium.

To evaluate the effect of CSE on lipid peroxidation, we measured the concentration of TBARS in lysates of control HFL-1 cells or cells exposed to 5.0% CSE for 24 h. CSE caused a significant enhancement of TBARS production (Figure 5), from 120.6 ⫾ 12.4 (nmol/g protein) in control cells to 212.8 ⫾ 10.4 (nmol/g protein) in cells exposed to 5.0% CSE (p ⬍ 0.01). When cells were incubated with 5.0% CSE in conditioned medium (Figure 5), the level of TBARS was markedly reduced (160.1 ⫾ 16.7 nmol/g protein) relative to CSE-treated fibroblasts incubated in regular medium (p ⬍ 0.05). As in the experiment described above, when we treated the conditioned medium with an anti-clusterin monoclonal antibody for 24 h, the protective effect of conditioned medium was partially abolished (TBARS, 189.5 ⫾ 15.7 nmol/g protein; p ⬍ 0.05). On the contrary, when anti-clusterin monoclonal antibody was replaced by an isotype-matched control antibody, the protective effect of conditioned medium was not abolished (Figure 5). To investigate the effect of clusterin on a more defined induced oxidative stress, in other experiments lung fibroblasts were preincubated with BSO for 3 h at a concentration of 125 ␮M, and then cultured 24 h with or without 5% CSE. As shown in Figure 6, glutathione depletion caused a significant increase of lipid peroxidation in lung fibroblasts exposed to 5% CSE (266.2 ⫾ 21.6, p ⬍ 0.05). This effect was consistently reduced when conditioned medium was added (224.7 ⫾ 15.1, p ⬍ 0.05).

Figure 5. Effect of CSE on thiobarbituric acid–reactive substance (TBARS) levels in HFL-1 cells. Cells were incubated for 24 h with normal medium, 5.0% CSE, 5% CSE plus conditioned medium, 5.0% CSE plus conditioned medium and anti-clusterin monoclonal antibody, or 5.0% CSE plus conditioned medium and isotype-matched control antibody, The levels of TBARS in cells from each incubation were measured by fluorescence spectrophotometry. ** Significantly different from control cells (p ⬍ 0.01). * Significantly different from cells exposed to 5% CSE (p ⬍ 0.05). § Significantly different from cells exposed to 5% CSE plus conditioned medium (p ⬍ 0.05). The error bars indicate the SD. This is the mean of at least three different experiments. ns ⫽ not significant.

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Figure 6. Effect of CSE on TBARS levels and its modulation by DL-buthionine sulphoximine (BSO). Cells were incubated for 24 h with normal medium, normal medium after a 3-h preincubation with 125 ␮M BSO, 5% CSE, or 5.0% CSE plus anti-clusterin monoclonal antibody after a 3-h preincubation with 125 ␮M BSO. The levels of TBARS in cells from each incubation were measured by fluorescence spectrophotometry. ** Significantly different from control cells (p ⬍ 0.01). * Significantly different from cells exposed to 5% CSE (p ⬍ 0.05). § Significantly different from cells preincubated with BSO and then exposed to 5% CSE (p ⬍ 0.05). The error bars indicate the SD. This is the mean of at least three different experiments.

To evaluate the protective role, if any, of the cytoplasmic form of clusterin, in separate experiments we exposed HFL-1 cells to 5.0% CSE for 72 h (data not shown). At the end of the incubation, the medium was removed, and fresh 5.0% CSE was added for another 24 h, with or without clusterin-enriched conditioned medium. TBARS levels were then assessed in cells incubated with 5.0% CSE, showing a significant increase (235.6 ⫾ 14.8 nmol/g protein) relative to untreated cells (131.8 ⫾ 5.5 nmol/g protein; p ⬍ 0.01). However, when conditioned medium was added, a significant decrease in lipid peroxidation activity was observed (186.3 ⫾ 13.4 nmol/g protein; p ⬍ 0.05). These results demonstrated that extracellular but not intracellular clusterin was able to prevent the oxidative stress–mediated cell damage caused by cigarette smoke. Analysis of Clusterin Expression in Bronchial Biopsy Specimens

Finally, to test whether clusterin might be involved in the response to smoke in humans, we analyzed the expression of clusterin by immunohistochemistry in sections of bronchial biopsy specimens obtained by fiberoptic bronchoscopy from six nonsmokers, three current smokers, and eight ex-smokers. Patients’ characteristics are reported in Table 2. As shown in Figure 7, clusterin was mildly expressed in specimens obtained from nonsmokers (left panel). In contrast, clusterin immunostaining was markedly increased in biopsy specimens obtained from active smokers and appeared uniformly throughout the stromal compartment (center panel); it is of interest that, in ex-smokers, stromal clusterin was expressed at levels comparable to those in current smokers (right panel). Although the number of biopsies was small, the comparison of clusterin immunoreactivity showed a clear difference between the nonsmoker group and the other two groups.

DISCUSSION Our findings show that exposure of human lung fibroblasts to cigarette smoke results in a marked accumulation of clusterin,

TABLE 2. PATIENT CHARACTERISTICS Groups

Nonsmokers

Current Smokers

Ex-Smokers

Patients examined, n Age, yr Sex, M/F FEV1, % predicted

6 41.7 ⫾ 7.6 4/2 94.5 ⫾ 12.6

3 50.1 ⫾ 8.6 2/1 80.3 ⫾ 6.5

8 62.3 ⫾ 10.1 6/2 63.7 ⫾ 29.2

Definition of abbreviations: F ⫽ female; M ⫽ male. Values are expressed as mean ⫾ SD.

both in its precursor form (60 kD) and in its secreted form (76–80 kD); after accumulating within the cells, the 76–80-kD form is released into the incubation medium, where it appears to protect lung fibroblasts against cigarette smoke–induced oxidative stress. Previous studies have suggested that clusterin might be involved in the clearance of toxic compounds from the extracellular milieu through its ability to bind unfolded proteins, cell debris, or immune complexes, and that it may act as a secreted chaperone molecule functioning like heat shock proteins (25). Furthermore, overexpression of the secreted form of clusterin in three different cell lines inhibited cell death (37). There is a general consensus that secreted clusterin seems to protect cells against various kinds of insults, such as ionizing radiation (38) and oxidative stress (23, 24). Moreover, clusterin that is overexpressed in non–small cell lung cancer appears to confer protection against chemotherapeutic agents (39). We have shown that incubation of fibroblasts with CSE (2.5 and 5.0%) for 24, 48, or 72 h affected the levels of clusterin forms. However, at 48 and 72 h of incubation, it appeared that 5.0% CSE was less effective than the 2.5% extract in inducing the 76–80-kD secreted clusterin. Detection of the 76–80-kD protein in the incubation medium provided an explanation for this apparent discrepancy. At 24 h, despite an increased accumulation of the CSE-induced intracellular protein, none was found in the incubation medium; at 48 h, there was still no release of 76–80-kD clusterin from untreated cells, but during incubation with CSE, the protein was abundantly released into the medium— more so with 5.0% than with 2.5% extract. This accounted for the lower level of 76–80-kD clusterin found intracellularly with 5.0% CSE than with 2.5% CSE. At 72 h, a significant amount of protein was found in the medium of the fibroblasts at both CSE concentrations, and the clusterin that had accumulated in untreated cells started to be released. This release of clusterin from untreated cells with increasing incubation times was probably due to the cells’ approaching confluence. In fact, as we have previously shown to occur with human skin fibroblasts (40), the percentage of G0/G1 phases increases with the time of incubation. Furthermore, the incubation of the cells with CSE 5.0% caused a twofold increase in the percentage of cells in the G2/M phases. Thus, exposure to toxic components of cigarette smoke induces both the precursor and the mature forms of secreted clusterin. It is only after clusterin has accumulated to a certain level within the cells that it starts to be released into the extracellular space. The release process seems to be accelerated in the presence of CSE, as shown by the fact that, at 48 and 72 h of incubation, higher levels of 76–80-kD clusterin were found in the incubation medium (and lower levels within the fibroblasts) with 5.0% CSE than with 2.5% CSE.

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Figure 7. Clusterin expression in bronchial biopsy specimens obtained by fiberoptic bronchoscopy from a nonsmoker, a current smoker, and an exsmoker. Note the high levels of clusterin expression in specimens from the smoker and the ex-smoker.

Transcriptional activation does not seem to be involved in the induction of clusterin protein after 24 and 48 h of exposure to CSE. In fact, levels of clusterin mRNA, as assessed by realtime PCR, did not change significantly at the time intervals used, in the presence of either dose of extract. However, when incubation was extended to 72 h, clusterin mRNA was enhanced with both doses of CSE. The first response of clusterin expression to CSE exposure thus seems to occur at the post-transcriptional level and does not involve direct gene activation. Direct gene activation comes into play with prolonged exposure of the cells to the toxic mixture. Immunocytochemistry experiments provided visual confirmatory evidence of the expected cytoplasmic localization of the 76–80-kD clusterin and of the dose-dependent induction exerted by CSE. Computer analysis of fluorescent signals showed that the increase in fluorescence was significant, mainly at the higher CSE concentration (5.0%). We have previously reported that cigarette smoke induces oxidative stress in human lung fibroblasts (14). These data were confirmed by the results of the present study, in which we used both FACS and measurement of lipid peroxidation levels (TBARS assay) under the same conditions. Both measurements indicated that exposure of HFL-1 cells to 5.0% CSE for 24 h significantly enhanced oxidative stress activity. However, when cells were incubated in conditioned medium, obtained from HFL-1 fibroblasts exposed for 72 h to 5.0% CSE (see Methods), oxidative stress activity was significantly reduced. This effect was almost completely cancelled by treatment of the conditioned medium with anti-human clusterin monoclonal antibody before using it for fibroblast incubation. These in vitro results provide new insights into the pathogenesis of smoking-induced lung injury and, more specifically, into the fibroblast changes that may occur in smokers. Both these conditions are characterized by an abnormal inflammatory response of the lungs, mainly to cigarette smoke (2, 41). A large body of evidence shows increased oxidative stress in healthy smokers (8). Our in vitro results were supported by immunohistochemistry experiments performed on bronchial biopsy specimens showing that clusterin expression was very low in specimens obtained from nonsmoking subjects; in contrast, clusterin

was intensely and uniformly expressed in the submucosa of current smokers and ex-smokers. Thus, we may hypothesize an in vivo scenario in which, after accumulation within the fibroblasts in response to a toxic agent, such as cigarette smoke, clusterin secretion in submucosa has a cytoprotective effect against toxic compounds. In conclusion, our results demonstrate that lung fibroblasts respond to various doses of CSE by up-regulating clusterin expression. This finding suggests that clusterin may have a protective effect in the airways of smokers. Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgment : The authors thank Dr. Elisa Veratelli for her scientific secretarial support and Mary McKenney for editing the paper.

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