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Downloaded from ajplung.physiology.org on March 18, 2008 J. Wu, W. Liu, K. Koenig, S. Idell and V. C. Broaddus Am J Physiol Lung Cell Mol Physiol 279:916-923, 2000. You might find this additional information useful... This article cites 35 articles, 16 of which you can access free at: http://ajplung.physiology.Org/cgi/content/full/279/5/L916#BIBL This article has been cited by 1 other HighWire hosted article: SV40-Dependent AKT Activity Drives Mesothelial Cell Transformation after Asbestos Exposure P. Cacciotti, D. Barbone, C. Porta, D. A. Altomare, J. R. Testa, L. Mutti and G. Gaudino Cancer Res., June 15, 2005; 65 (12): 5256-5262. [Abstract] [Full Text] [PDF] Medline items on this article's topics can be found at http://highwire.stanford.edu/lists/artbytopic.dtl on the following topics: Physiology .. Phagocytosis Medicine .. Environmental Issues Earth Sciences .. Asbestos Earth Sciences .. Crocidolite Earth Sciences .. Chrysotile Physiology .. Apoptosis Updated information and services including high-resolution figures, can be found at: http://ajplung.physiology.org/cgi/content/full/279/5ZL916 Additional material and information about AJP - Lung Cellular and Molecular Physiology can be found at: http://www.the-aps.org/publications/ajplung This information is current as of March 18, 2008 . AJP - Lung Cellular and Molecular Physiology publishes original research covering the broad scope of molecular, cellular, and integrative aspects of normal and abnormal function of cells and components of the respiratory system. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2005 by the American Physiological Society. ISSN: 1040-0605, ESSN: 1522-1504. Visit our website at http://www.the-aps.org/. Am J Physiol Lung Cell Mol Physiol 279: L916-L923, 2000. Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis and toxicity for mesothelial cells Downloaded from ajplung.physiology.org on March 18, 2008 J. WU,1 W. LIU,1 K. KOENIG,2 S. IDELL,2 AND V. C. BROADDUS1 1Lung Biology Center, San Francisco General Hospital, University of California, San Francisco, California 94143-0854; and 2The University of Texas Health Center at Tyler, Tyler, Texas 75708 Received 23 February 2000; accepted in final form 23 May 2000 Wu, J., W. Liu, K. Koenig, S. Idell, and V. C. Broaddus. Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis and toxicity for mesothelial cells. Am J Physiol Lung Cell Mol Physiol 279: L916-L923, 2000.--Biological mod ification of asbestos fibers can alter their interaction with target cells. We have shown that vitronectin (VN), a major adhesive protein in serum, adsorbs to crocidolite asbestos and increases fiber phagocytosis by mesothelial cells via integrins. Because chrysotile asbestos differs significantly from crocidolite in charge and shape, we asked whether VN would also adsorb to chrysotile asbestos and increase its toxicity for mesothelial cells. We found that VN, either from purified solutions or from serum, adsorbed to chrysotile but at a lower amount per surface area than to crocidolite. Nev ertheless, VN coating increased the phagocytosis of chrysotile as well as of crocidolite asbestos. VN coating of both chrysotile and crocidolite, but not of glass beads, increased intracellular oxidation and apoptosis of mesothelial cells. The additional apoptosis could be blocked by integrin-ligand blockade with arginine-glycine-aspartic acid peptides, con firming a role for integrins in the fiber-induced toxicity. We conclude that VN increases the phagocytosis of chrysotile as well as of crocidolite asbestos and that phagocytosis is im portant in fiber-induced toxicity for mesothelial cells. crocidolite; apoptosis; integrin; arginine-glycine-aspartic acid peptides; dichlorofluorescein assay asbestos fibers are considered to be a complete carcin ogen for the formation of the mesothelium-derived tu mor mesothelioma. The interaction of asbestos with mesothelial cells that leads to cancer is still unknown; however, it appears that fiber phagocytosis by the target cell may be an important step. Phagocytosis brings the long thin asbestos fiber in close contact with the nucleus, probably enhancing the toxic effect of reactive oxygen species (ROS) on the DNA and allow ing the fiber to damage chromosomes during mitosis. In an animal study (34), the long thin shape of asbestos was found to be critical to its carcinogenicity, suggest ing that mechanical effects within cells might be of crucial importance. In in vitro studies (18, 19, 35, 36) of asbestos, phagocytosis of fibers has been correlated with toxic effects in certain cell types. Determining the role of phagocytosis in asbestos-induced injury to me- sothelial cells may hold clues to the important mecha nisms of asbestos toxicity. Biological modification of asbestos fibers may in crease fiber phagocytosis. In a previous study, Boylan et al. (3) exposed crocidolite asbestos fibers to serum, pleural liquid, bronchoalveolar fluid, or purified vitro nectin (VN) and showed that the fibers became coated with VN and were more readily phagocytosed by mesothelial cells. This phagocytosis was mediated via integrins capable of recognizing VN, the major adhe sive protein in serum. Liu et al. (26) have recently shown that phagocytosis of crocidolite asbestos is im portant for mesothelial cell toxicity. Therefore, biolog ical modification via VN adsorption may increase phagocytosis and possibly enhance cytotoxicity of the fibers in vivo. Chrysotile shares many toxic effects with crocidolite, although the fibers have striking differences, including that of surface charge. Chrysotile is positively charged in physiological solutions, whereas crocidolite is nega tively charged (25). The positive charge on chrysotile is known to account for one major difference with crocidolite; the hemolysis induced by chrysotile is a result of the interaction of the positive charge with sialic acid moieties on the surface of the erythrocyte (6). The differences in charge between the two fibers may also account for differences in protein adsorption and thus in the biological modification of each fiber in the body (11). VN, which contains a heparin-binding region, appears to bind preferentially to negatively charged materials (1) and might not be expected to adsorb as readily to chrysotile as to crocidolite. Although chrysotile fibers have been shown to be taken up by mesothe lial and other cells as early as 15 min after exposure (22), the mechanism of uptake of this fiber has not been explored. The differences in surface charge between crocidolite and chrysotile may herald different mecha nisms of entry of these fibers into cells. Therefore, we asked whether chrysotile would ad sorb VN and, if so, whether that adsorption would alter phagocytosis of chrysotile by mesothelial cells. To mea sure phagocytosis of chrysotile, a fiber too thin and variably shaped to be recognized by our other assays, Address for reprint requests and other correspondence: V. C. Broaddus, Lung Biology Center, Box 0854, Univ. of California, San Francisco, CA 94143-0854 (E-mail: sfcourt@itsa.ucsf.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. L916 1040-0605/00 $5.00 Copyright 2000 the American Physiological Society http://www.ajplung.org VITRONECTIN ADSORPTION TO CHRYSOTILE ASBESTOS L917 Downloaded from ajplung.physiology.org on March 18, 2008 we developed a novel assay using radiolabeled VN and albumin. Finally, we asked whether adsorption of VN onto chrysotile would also alter its toxicity for mesothelial cells. METHODS Reagents and proteins. National Institute of Environmen tal Health and Safety (NIEHS) asbestos fibers were used for all experiments (7). Union Internationale Contre le Cancer (UICC) asbestos fibers were obtained from Dr. Marie-Claude Jaurand (Institut National de la Sante et de la Recherche Medicale, Paris, France) (37) and used together with the NIEHS asbestos fibers in experiments of protein adsorption and phagocytosis. Asbestos fibers were stored in 1-ml ali quots of 100 ^g in PBS at --20C and were used within 1 mo of preparation. NIEHS crocidolite had a mean length of 10 ^m and a mean width of 0.3 ^m; NIEHS chrysotile had a mean length of 7 ^m and a mean width of 0.2 ^m (7). UICC crocidolite had a mean length of 2.1 3.6 ^m and a mean diameter of 0.2 0.1 ^m, and UICC chrysotile had a mean length of 1.7 2.2 ^m and a mean diameter of 0.05 0.04 ^m (41). In some experiments, NIEHS chrysotile fibers were rigorously sonicated to shorten mean fiber length (100 W for 10 min; Branson Ultrasonics, Danbury, CT) as confirmed by examination with dark-field microscopy. Glass beads (mean diameter 1.6 0.3 ^m; Duke Scientific, Palo Alto, CA) were used as control particles. Purified proteins included VN, purified as described (3) with the technique of Yatohgo et al. (40), and BSA (fraction V; Sigma, St. Louis, MO) that was confirmed to be VN free by immunoblot (3). Human serum was freshly prepared. Radio labeled proteins included 125I-BSA (ICN Pharmaceuticals) and 125I-VN, iodinated by the IODO-GEN method (14). IODO-GEN (Pierce, Rockford, IL) was prepared so that 10 ^g of IODO-GEN were dried in a 10 X 75-mm glass tube. VN (50 ^g) was added to the tubes with 5 ^l of 20 mM HEPES buffer (pH 7.45, 150 mM NaCl and 0.5 mCi of Na125I) to a final total volume of 100 ^l. This was allowed to react on ice for 5 min. Then 25 ^l of 1% KI in water were added to the tube to stop the reaction, and 25 ^l of 1% ovalbumin in water were added. Free iodine was separated from the 125I-labeled VN by gel filtration in a 12-ml column of Sephadex G-25 equilibrated with HEPES-buffered saline containing 1% BSA. GRGDSP, the control GRGESP peptides, trypsin-EDTA, and mouse laminin were obtained from GIBCO BRL (Life Technologies, Gaithersburg, MD). Propidium iodide was ob tained from Sigma. SDS-PAGE and Western blot analysis of eluted proteins from serum-coated fibers. Asbestos fibers (750 ^g) were incu bated with undiluted human serum (100 ^l; ~6.0 mg of serum protein) for 1 h in clean Eppendorf tubes. After three washes with PBS, the asbestos fibers were sonicated (100 W for 8 s; Branson Ultrasonics) to disperse the fibers. After sonication, the fiber solutions were transferred to clean Eppendorf tubes and spun at 14,000 rpm for 10 min. Approxi mately 900 ^l of supernatant were discarded, 5X Laemmli sample buffer (20 ^l) and p-mercaptoethanol (2 ^l) were added, and samples were boiled at 100C for 10 min to elute the proteins bound to the fibers. Eluted protein (~120 ^l) was analyzed by SDS-PAGE and Western blot analysis. SDS-PAGE. Eluted proteins at equal volumes of eluate were analyzed by electrophoresis. Eluted proteins (~60 ^l) were loaded onto 10% SDS-polyacrylamide gels and run at 100 V in the stacking gel and 150-200 V in the separating gel for 3 h. Gels were stained with Coomassie blue. Western blotting. Proteins from the SDS-polyacrylamide gel were transferred onto Immobilon-P transfer membrane (Millipore, Bedford, MA). Powdered milk [5% in Tris-buffered saline (TBS)] was used as the blocking solution throughout. The membrane was blocked for 1 h and washed with TBS four times. The membrane was incubated with the primary antibody mouse anti-human VN (Chemicon International, Temecula, CA), at 1:500 in blocking solution for 1 h. After a wash with TBS, the membrane was treated with the second ary antibody (goat anti-mouse Ig conjugated to horseradish peroxidase; Amersham Life Science, Piscataway, NJ) at 1:2,000 for 1 h and washed again with TBS. Proteins were detected with chemiluminescence (Amersham). Coating offibers. Fibers were coated with purified, radio labeled BSA or VN at 10 ^g/ml, similar to the concentrations of VN in lung lining fluid (29). The incubation time of 1 h was sufficient for maximal adsorption (11), as we confirmed. The following method is a general description of fiber coating with purified proteins. Eppendorf tubes were coated with 1 ml of 1% BSA for 1 h to minimize adsorption of radiolabeled pro tein to the tubes. In the BSA-coated tubes, 100 ^g of asbestos fibers in 100 ^l of PBS were incubated with 1 ^g of radiola beled protein (VN or BSA) for 1 h on a vortex at room temperature (RT). The fibers were then washed three times by adding 900 ^l of PBS, centrifuging at 14,000 rpm for 10 min, aspirating 900 ^l, and then adding 900 ^l of fresh PBS. After removal of the supernatant from the third wash, the 125I-protein-asbestos solution was transferred into a new set of Eppendorf tubes to minimize the presence of unbound proteins. Fresh 1X PBS was added, bringing the final volume to 1 ml. All washes were saved for radioactivity determina tion to assess recovery of total radioactivity. Washes were spun at 14,000 rpm for 10 min and found to contain no asbestos fibers. The radioactivity of fibers plus wash was always at least 90% of the radioactivity of a comparable amount of radiolabeled protein spiked in a separate Eppendorf tube. The radioactivity of fibers and the wash was counted in a gamma counter to calculate the percentage of the total protein adsorbed by the fibers. Fibers were then used in the phagocytosis assays. Cells and culture. Rabbit mesothelial cells were harvested as described (3) and maintained in standard medium: RPMI 1640 medium and DMEM (1:1), 10% fetal bovine serum (GIBCO BRL), 2 nM L-glutamine (GIBCO BRL), 100 U/ml of penicillin, and 100 ^g/ml of streptomycin. Cells between passages 3 and 7 were used in all studies. The experimental medium was the same as that described above but was serum free to avoid additional protein adsorption. Fiber phagocytosis protocol. The cells were incubated with the radiolabeled VN-coated or BSA-coated fibers at two dif ferent temperatures for 4 h and then washed free of nonad herent fibers. At 4C, with phagocytosis blocked, the associ ation of fibers with the cell monolayer indicated adherence of fibers. At 37C, with normal phagocytosis, fiber association with the cell monolayer indicated both adherence and phago cytosis. Eight-well chamber tissue culture slides (Nunc Interna tional, Naperville, IL) were coated with mouse laminin (200 ^l, 10 ^g/ml) for 1 h at 37C, washed with PBS, and plated with 25,000 cells/well the night before the experiment, with a goal of 90% confluence. On the day of the experiment, cells were washed once with PBS and incubated with 250 ^l of serum-free medium. The slides were placed at 37C or 4C for 15 min and triturated, radiolabeled VN- or BSA-coated fibers equilibrated at either 37C or 4C were added at 7.5 ^g/cm2. Cells and fibers were incubated at either 37C or 4C for 4 h. The cells were gently L918 VITRONECTIN ADSORPTION TO CHRYSOTILE ASBESTOS Downloaded from ajplung.physiology.org on March 18, 2008 washed to remove nonadherent fibers. Cell counts showed that at 4C, no cells were lost with washing, whereas at 37C, ~10% of the cells were lost with washing. Fortunately, the cell loss was equal whether cells were exposed to VN-coated or BSA-coated fibers, allowing comparison between them at 37C. Adsorbed protein remained tightly adherent to the fibers during the time of the assay; when VN- or BSA-coated fibers were incubated without cells for 4 h at either 37C or 4C, <1% of adsorbed protein could be found in the superna tant. All wash fluids were saved for counting of nonadherent fibers. Adherent cells were then detached with trypsin (0.25% wt/vol) and EDTA (0.5 mM) and saved for counting of adherent plus internalized fibers. Radioactivity was detected with the Beckman gamma 5500 counting system (Beckman Instruments, Fullerton, CA). The percentage of fibers associated with cells was calcu lated as [radioactivity of adherent + internalized fibers (e.g., cells after washing) X 100]/total radioactivity (e.g., cells + wash). The recovery of radioactivity was determined by compar ing the total recovered experimental counts with the total counts of standard vials to which identical amounts of pro tein-coated fibers had been added. The total recovery was >90%. Dichlorofluorescein assay of intracellular oxidation. To de termine whether VN coating of chrysotile fibers increased intracellular oxidation, cells exposed to protein-coated fibers or glass beads were incubated with an oxidation-sensitive fluorescent probe, 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene, OR). Intra cellular deacylation of DCFH-DA results in the formation of the nonfluorescent 2',7'-dichlorofluorescein (DCFH); with ox idative stress, DCFH is oxidized to the fluorescent dichlorofluorescein (DCF) by a variety of ROS and reactive nitrogen species (10) and is sensitive to a general level of oxidative stress (2). DCFH-DA, divided into aliquots in DMSO from a stock solution of 5 M and stored in a dessicator in the dark at --20C, was diluted in PBS immediately before the experi ment. After their exposure to BSA- or VN-coated fibers or glass beads (5 ^g/cm2) for 4 h, the mesothelial cells were detached with trypsin-EDTA, which was then neutralized, combined with the floating cells, and incubated with DCFH-DA (5 ^M) for 1 h before and continuously during flow cytometric analysis. Propidium iodide (15 ^g/ml) was added before flow cytometric analysis to allow exclusion of cells that were permeable and thus would not retain the fluorescent probe. Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA), and acquisition and data analysis were performed with the use of CELLQuest soft ware (Becton Dickinson). At least 5,000 cells were analyzed for adequate statistical collection. Intracellular oxidative shift was measured as the percentage of cells with fluores cence greater than that of 95% of the control, unexposed cells. Finally, maximal cell fluorescence was measured after excess H2O2 was added to confirm equivalent loading of DCF. Max imal fluorescence exceeded 85% in all cases. Annexin V assay for apoptosis. Apoptosis was measured by the binding of green fluorescent protein (GFP)-annexin V to the phosphatidylserine residues on the outer leaflet of the apoptotic cellular membrane with a GFP-annexin V fusion protein constructed as described by Ernest et al. (13). After exposure to BSA- or VN-coated fibers or glass beads (5 ^g/ cm2) for 18 h, mesothelial cells were collected and centrifuged (1,000 rpm for 10 min). In some experiments, arginine-gly cine-aspartic acid (RGD)- or arginine-glycine-glutamic acid (RGE)-containing peptides (0.5 mg/ml) were added to the cells 1 h before the fibers. The cell pellet was resuspended in serum-free RPMI 1640 medium-DMEM buffer and stained with GFP-annexin V fusion protein (3 ^g/ml in HEPES buffer) for 10 min on ice. Propidium iodide (15 ^g/ml) was added just before analysis by flow cytometry. Early apoptotic cells, i.e., those with positive staining for annexin V but negative staining for propidium iodide, were measured. Cells were analyzed with the FACScan flow cytometer, with acqui sition and data analysis as described above. Five thousand events per sample were acquired to ensure adequate mean data. Statistics. Data were analyzed for significance with SuperANOVA (Abacus Concepts, Berkeley, CA) with ANOVA with Tukey's test. Data are means SD unless otherwise noted. A difference was regarded as significant if P < 0.05. RESULTS SDS-PAGE and Western blot analysis of eluted pro teins from fibers. Elution of serum proteins adsorbed to asbestos fibers showed that, in general, a wide range of serum proteins adsorbed to both fibers. Consistently, a larger amount of protein adsorbed to chrysotile fibers than to crocidolite fibers (n = 4 experiments; Fig. 1). The similarity in elution pattern for both crocidolite and chrysotile fibers suggested that the larger surface area of chrysotile was an important factor in its greater protein binding ability. Of the eluted proteins from both fibers, the most intense band(s) comigrated with purified VN and/or albumin (Fig. 1, lanes 2 and 3). By Western blotting of the eluted serum proteins, VN was confirmed to adsorb to both fibers from serum (Fig. 2). Adsorption of purified proteins to fibers. To quanti tate adsorption of proteins to fibers, radiolabeled puri fied VN or BSA (1 ^g) was incubated with fibers (100 ^g), and the percentage of adsorption was quantitated. Purified VN adsorbed to both fibers. For crocidolite, significantly more VN than BSA bound to the fibers (P < 0.001; Fig. 3). No preferential adsorption of VN compared with BSA was found for chrysotile. The sim- 220 kDa -- -- 97 kl)a -- 66 kDa -- 46 kDa -- 30 kDa -- 21 kDa -- s 1 2 3 45 Fig. 1. SDS-PAGE analysis of serum proteins eluted from crocidolite and chrysotile fibers from the National Institute of Environmental Health Sciences (NIEHS). Lanes 1-3, human serum (25 |xg), 5 |xg of purified vitronectin (VN), and 5 |xg of purified BSA, respectively. Lanes 4 and 5, proteins eluted from crocidolite and chrysotile fibers (750 |xg), respectively, incubated for 1 h with human serum. In general, darker bands can be observed in lane 5 compared with those in lane 4. Nos. at left, molecular mass. VITRONECTIN ADSORPTION TO CHRYSOTILE ASBESTOS L919 Table 1. Surface area and relative adsorption of vitronectin per surface area of crocidolite and chrysotile asbestos Fig. 2. Western blot of VN in serum proteins eluted from crocidolite and chrysotile fibers (NIEHS). Lanes 1 and 2, 5 and 1 p.g of purified VN, respectively. Lanes 3 and 4, VN eluted from crocidolite and chrysotile fibers (750 p.g), respectively, incubated with human se rum. The 75- and 65-kDa bands are the major bands of VN. Bands < 65 kDa may represent fragments; bands > 75 kDa likely represent multimers. Vitronectin Adsorbed, (jug protein/ 100 p,g fiber Surface Area, m2/g Specific Vitronectin Adsorption, (jug protein/m2 fiber Crocidolite (NIEHS) Crocidolite (UICC) Chrysotile (NIEHS) Chrysotile (UICC) 0.39 0.05 0.16 0.04 0.41 0.09 0.27 0.08 9.8 8.3 54.2 21.3 39851 19353 76 16* 12840* Values are means SD of 3 experiments. Vitronectin adsorption was determined by amount of radiolabeled vitronectin (1 p.g) ad sorbed by 100 p.g of fibers over 1 h. Surface area data are means of surface area determined by nitrogen adsorption for Union Interna tionale Contre le Cancer (UICC; 37) and National Institute of Envi ronmental Health Sciences (NIEHS; 7). * Significant difference be tween specific vitronectin adsorption by chrysotile and crocidolite (P < 0.05). ilar binding of VN and BSA to chrysotile suggested that the adsorption was nonspecific and that differ ences in total binding were because of the greater surface area of chrysotile. Indeed, when published val ues of the surface area of each fiber were used to calculate the specific adsorption, it was shown that chrysotile adsorbed less VN per surface area than crocidolite (Table 1). The differences in VN binding to the fibers led us to examine whether VN binding al tered fiber phagocytosis and toxicity for mesothelial cells. Fiber adherence and phagocytosis. When fibers were incubated with mesothelial cells for 4 h at 4C to inhibit phagocytosis, BSA- and VN-coated fibers had a similar adherence to the cells (Fig. 44.). At 37C, how- ever, VN-coated fibers displayed a significantly greater association with the cells than BSA-coated fibers for both chrysotile and crocidolite (Fig. 4B). Because fiber cell association is due to adherence plus phagocytosis, increases in fiber cell association at 37C that were not seen at 4C were interpreted to be a result of fiber phagocytosis. By this assay, then, VN was shown to enhance phagocytosis of both chrysotile and crocidolite Downloaded from ajplung.physiology.org on March 18, 2008 Crocidolite Chrysotile Fig. 3. Percent of adsorption of radiolabeled BSA and VN to crocidolite and chrysotile fibers. Fibers were incubated with purified, radio labeled BSA or VN (1 p.g) for 1 h and washed thoroughly. UICC, Union Internationale Contre le Cancer. Values are means SD; n = 3 experiments. VN adsorption was greater than that of BSA to crocidolite but not to chrysotile. *Significantly different from fibers with BSA adsorption, P < 0.01. Crocidolite Chrysotile Fig. 4. Effect of protein adsorption on the percentage of fibers asso ciated with rabbit pleural mesothelial cells. Radiolabeled BSA- or VN-coated fibers (7.5 p.g/cm2) were incubated with rabbit mesothelial cells for 4 h either at 4C to prevent phagocytosis (A) or at 37C to allow phagocytosis (B). After cells were washed, the radioactivity associated with the cells indicated fibers that were either adherent only (A) or adherent plus phagocytosed (B). Values are means SD; n = 10 experiments. * Significantly different from fibers with BSA adsorption, P < 0.01. L920 VITRONECTIN ADSORPTION TO CHRYSOTILE ASBESTOS fibers, as has been previously shown (3) only for crocidolite. Intracellular oxidation. Crocidolite and chrysotile fi bers (NIEHS) both induced an increase in intracellular oxidation as measured by a shift in fluorescence of the intracellular probe DCF (Fig. 5). Compared with BSA coating, VN coating of fibers led to greater intracellular oxidation for crocidolite but not for the standard chrysotile. VN coating of chrysotile did increase oxidation when the chrysotile was sonicated to reduce mean length, suggesting that the shape or length of the standard chrysotile fiber interfered with cell interac tions in this assay. Glass beads were also phagocytosed by cells (VN coated, 80 6% cells with >4 intracellular beads; BSA coated, 71 11% with >4 intracellular beads; P > 0.05). However, VN coating of glass beads did not affect intracellular oxidation, suggesting that the process of phagocytosis alone did not contribute to intracellular oxidation. Apoptosis. VN-coated fibers induced more apoptosis than BSA-coated fibers for both crocidolite and chrysotile (NIEHS) in mesothelial cells (Fig. 6). BSA-coated asbestos induced the same amount of apoptosis as uncoated asbestos (chrysotile: 20 7% BSA-coated, 18 8% uncoated, n = 4; crocidolite: 26 5% BSAcoated, 24 7% uncoated, n = 4 experiments). VN itself was shown to have no effect because VN-coated glass beads did not induce apoptosis. RGD peptides, but not control RGE peptides, reduced the apoptosis caused by VN-coated chrysotile fibers (Fig. 7). RGD Fig. 6. The effect of BSA- or VN-coated asbestos fibers on apoptosis of rabbit mesothelial cells. Mesothelial cells were exposed to BSA- or VN-coated fibers or glass beads (5 |xg/cm2) for 18 h, detached, com bined with floating cells, and stained with green fluorescent proteinannexin V (3 |xg/ml) and propidium iodide (15 |xg/ml). Values are means SD; n = 4 experiments. Apoptotic cells were early apoptotic, i.e., those staining positively for annexin V and negatively for pro pidium iodide. In all instances, cells incubated with VN-coated as bestos fibers exhibited more apoptosis than cells incubated with BSA-coated asbestos fibers. Apoptosis resulting from BSA-coated asbestos was no different from that resulting from uncoated asbestos (see results). * Significantly different from fibers with BSA adsorp tion, P < 0.01. peptides had no effect on BSA-coated chrysotile fibers, indicating that the effect of RGD was VN dependent. DISCUSSION In this study, we have shown that despite differences in the two fiber types, VN adsorbs to both chrysotile Downloaded from ajplung.physiology.org on March 18, 2008 Fig. 5. Intracellular oxidation in primary mesothelial cells induced by BSA- or VN-coated asbestos fibers. After a 4-h exposure to pro tein-coated fibers or glass beads (5 |xg/cm2), cells were collected and incubated with 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (5 |xM) for 1 h before and continuously during flow cytometric analysis. Intracellular oxidation was measured as the percentage of cells with a fluorescence > 95% of the control, unexposed cells in each experiment. DCF, dichlorofluorescein. Values are means SD; n = 3 experiments. Compared with that for BSA-coated fibers, greater intracellular oxidation was observed for cells exposed to VN-coated crocidolite (Croc) and chrysotile (Chrys) fibers sonicated to reduce mean length. A significant difference was not observed for standard chrysotile fibers. No increase in intracellular oxidation was detected by VN-coated glass beads despite an observed increase in phagocy tosis. * Significantly different from fibers with BSA adsorption, P < 0.05. BSAcoated Vitronectincoated Chrysotile (NIEHS) Fig. 7. The effect of arginine-glycine-aspartic acid (RGD)-containing peptides on the ability of BSA- and VN-coated chrysotile fibers to induce apoptosis in rabbit mesothelial cells. Cells were treated with RGD or control arginine-glycine-glutamic acid (RGE) peptides (0.5 mg/ml) 1 h before the addition of fibers. Values are means SD; n = 3 experiments. RGD but not RGE peptides decreased apoptosis for primary mesothelial cells treated with VN-coated chrysotile fibers. RGD peptide had no effect on apoptosis of cells incubated with BSA-coated chrysotile fibers. * Significantly different from no pep tide, P < 0.05. L922 VITRONECTIN ADSORPTION TO CHRYSOTILE ASBESTOS Downloaded from ajplung.physiology.org on March 18, 2008 biological effects because phagocytosis of glass beads (either BSA or VN coated) had no effect. The role of phagocytosis was particularly evident in the apoptosis studies in which VN coating led to more apoptosis by crocidolite and chrysotile, both sonicated and unsoni cated. The increased apoptosis could be blocked by RGD-containing peptides, known to block VN-dependent phagocytosis without altering fiber adherence (3). There were similar findings in the oxidation assay; in this assay, however, the VN coating increased intracel lular oxidation of crocidolite and the shorter sonicated chrysotile but not of the longer unsonicated chrysotile fibers. The difference in results for the longer chrysotile asbestos may arise because in the apoptosis stud ies, cells and fibers remained undisturbed during the entire assay, whereas in the oxidation study, cells were harvested before incubation with the fluoroprobe, a step that may have dislodged some of the longer fibers. Longer fibers may also take more time to phagocytose, allowing their toxicity to be demonstrated in the 18-h apoptosis assay but not in the 4-h oxidation assay. Although asbestos has been suspected of generating intracellular ROS, the evidence for intracellular oxida tion has been mostly indirect, such as the increase in cellular antioxidant enzymes, protection by antioxi dant enzymes, or detection of oxidized bases (8, 12, 15, 20). Attempts to measure ROS directly have failed to confirm increases due to asbestos (16, 24, 28), although asbestos-induced increases in reactive nitrogen species have been detected (9). Here, in using the DCF fluoroprobe, which can detect both reactive oxygen and ni trogen species (10), we have been able to confirm that both fibers can increase intracellular oxidation. The VN coating on both fibers increased asbestosinduced apoptosis of mesothelial cells. The increased apoptosis was likely a result of the increased intracel lular oxidation because Broaddus et al. (5) have previ ously shown that asbestos-induced apoptosis of mesothelial cells is mediated by ROS. Ultimately, we attribute the effect of VN on apoptosis to an increased phagocytosis of fibers because the cells were otherwise exposed to the same numbers of VN- and BSA-coated fibers during the assay. The fibers that settled on the cells at 5 ^g/cm2 would have been distributed as exter nal, both adherent and nonadherent, and as internal. BSA-coated fibers were phagocytosed at a basal rate similar to that of uncoated fibers. VN coating thus served to shift more of the asbestos fibers from the outside to the inside of the cell, where the fibers were able to induce greater damage. RGD peptides, by block ing the phagocytosis and not the adherence of fibers, therefore acted to block the shift of fibers from the outside to the inside of cells. Indeed, the effect of the RGD peptides was specific for VN-induced effects and did not alter BSA-coated fiber-induced apoptosis. Thus the role of integrin-dependent phagocytosis and its role in enhancing fiber-induced apoptosis are shown for both crocidolite and chrysotile. Protein adsorption to asbestos fibers has been ex plored in many previous studies (11, 21, 38), including some that have examined the biological consequences of serum and selective protein adsorption to asbestos on epithelial cells or macrophages (23, 31, 32). Our study differed from these in that it examined VN, a biologically important opsonin for phagocytosis, and examined the biological consequence of VN adsorption for two different fibers in mesothelial cells. The relative adsorption of serum proteins did not differ greatly between the two fibers (Fig. 1), unlike the observation of Desai and Richards (11), who attributed differences in serum protein binding to differences in fiber compo sition and charge. Instead, our findings were similar to those of Valerio et al. (38) in that protein adsorption was not heavily influenced by surface charge. Instead, other forces may play a more important role in protein adsorption, including hydrophobic interactions and protein charge density (38). The biological environment is complex and may have multiple effects on the fibers and the cells. Biological materials other than serum proteins, such as surfac tant proteins and lipids, antibodies, and DNA that may alter the fibers' interaction with cells, may adsorb to asbestos fibers. Protein adsorption, at least, is associ ated with partial denaturation of the protein by a conformational change that renders the adsorption nearly irreversible (33). Thus proteins can be expected to remain on the surface of fibers as they move and accumulate in the pleural space (27). In addition, the biological environment may alter the cellular response to asbestos. In the in vitro environment, increases in asbestos phagocytosis may lead to increased apoptosis, whereas in the in vivo environment with its growth factors, extracellular matrix, and cell-cell interactions, apoptosis may be inhibited. If so, in the biological setting, some of the damaged cells may avoid apoptosis and survive with their damaged DNA, thus increasing the likelihood of eventual malignant change (4). We speculate that the biological modification of asbestos that leads to increased phagocytosis could enhance carcinogenicity of asbestos in the in vivo setting where multiple factors tend to oppose apoptosis. In conclusion, although the crocidolite and chrysotile asbestos fibers are different in composition, charge, and shape, they each become coated with VN when exposed to purified protein or to serum. VN coating leads to increased phagocytosis of chrysotile as well as of crocidolite fibers. 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