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Effect of Coating with Lung Lining Fluid on the Ability of Fibres to Produce a Respiratory Burst in Rat Alveolar Macrophages
Toxicology in Vitro 12 (1998) 15±24
Eect of Coating with Lung Lining Fluid on the Ability of Fibres to Produce a Respiratory Burst in Rat Alveolar Macrophages D. M. BROWN*, N. K. ROBERTS and K. DONALDSON Department of Biological Sciences, Napier University, Edinburgh EH10 5DT, UK (Accepted 10 June 1997) AbstractÐThe aim of the study was to develop a simple short-term in vitro assay which would allow us to predict the pathogenicity of ®bres based on data already available from in vivo studies. Fibres were used naked (uncoated) or coated with rat IgG, or rat or sheep surfactant. The ®bres were used to stimulate superoxide anion release by rat alveolar macrophages. Binding of ®bres to rat alveolar macrophages was assessed by optical microscopy. Fibres used in the naked state produced little or no stimulation of superoxide anion from rat alveolar macrophages. When ®bres were coated with rat IgG there was a signi®cant increase in superoxide release for all ®bre types with the exception of RCF4 and Code 100/475. When ®bres were coated with rat or sheep surfactant, there was suppression of the respiratory burst for all ®bre types. The observed suppression was not due to a scavenging eect by the surfactant itself, because xanthine/xanthine oxidase generated superoxide was unaected by surfactant. The suppressive eect was shown to act directly on the macrophages. Comparing naked and coated ®bres for their ability to bind to macrophages, it was shown that in general more coated ®bres were bound and that increased binding was associated with suppressed superoxide release for both types of surfactantcoated ®bres. It was concluded that the nature of the ®bre coating is the main factor in¯uencing the interaction between ®bres and macrophages. The type of binding through dierent receptors may either stimulate or switch o the respiratory burst. The assay used here does not, however, allow any predictions to be made regarding the pathogenicity of ®bres. # 1998 Elsevier Science Ltd Abbreviations: BAL = bronchoalveolar lavage; MMVF = man-made vitreous ®bres; PBS = phosphate buered saline; PMA = phorbol myristate acetate; RCF = refractive ceramic ®bres; SOD = superoxide dismutase. Keywords: ®bres; superoxide anion; surfactant.
INTRODUCTION
It is widely recognized that the mechanisms of ®bre-induced lung ®brosis and cancer depend on several factors such as dimension (Donaldson et al., 1989 and 1993b), chemical nature (Hart et al., 1994; Hesterberg and Barrett, 1984), solubility (Morgan and Holmes, 1986) and durability (Donaldson et al., 1993a). Interaction between alveolar macrophage surface receptors and foreign particles including ®bres leads to phagocytosis and possibly a secretory response which is enhanced in the presence of an opsonin such as IgG (Donaldson et al., 1992; Hill et al., 1996; Nyberg and Klockars, 1990a; Perkins et al., 1991; Scheule and Holian, 1989). Phagocytosis is a stimulus for superoxide anion release, and it has been shown that when long ®bre *Author for correspondence.
crocidolite asbestos is phagocytozed, superoxide is released to the outside of the cell (Goodlick and Kane, 1986; Hill et al., 1996). In other studies (Roney and Holian, 1989), guinea pig macrophages did not produce a respiratory burst when treated with amosite asbestos, although chrysotile was stimulatory. The release of reactive oxygen species by macrophages may be the initiating factor in the pathogenesis of lung disease after deposition of particles (Kamp et al., 1992; Vallyathan et al., 1992). Superoxide anion release from stimulated cells through protein kinase C activation or by crosslinking of Fc receptors by IgG-coated ®bres has previously been demonstrated (Aida and Onoue, 1984; Scheule and Holian, 1989). As with other particles, ®bres that deposit on the lung surface become coated with lung lining material, a consequence of which is likely to be
0887-2333/98 $19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(97)00093-3
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modi®cation of the ®bre surface reactivity, and hence a change in the oxidant generating ability and possibly phagocytosis of the ®bres. In this study, we have examined the ability of a range of ®bres of diering pathogenicity, including asbestos (long ®bre amosite), refractory ceramic ®bres (RCFs) and man-made vitreous ®bres (MMVFs) to produce a respiratory burst in rat alveolar macrophages. The ®bres used were allocated to categories of pathogenic or non-pathogenic, depending on whether they caused tumours and ®brosis in recent inhalation studies (Davis et al., 1996; Glass et al., 1995; Hesterberg et al., 1993). Pathogenic ®bres were Long Fibre Amosite, Silicon carbide and RCF1; non-pathogenic ®bres were RCF4, Code 100/475 and MMVF10. The purpose of the present study was (1) to compare ®bres as to their ability to produce an oxidative burst in rat alveolar macrophages; (2) to determine whether this could be modulated by coating the ®bres with lung lining material; and (3) to determine whether any of the eects were related to the pathogenicity of the ®bres.
MATERIALS AND METHODS
Fibres The size characteristics of the ®bres used in this study are shown in Table 1. All assays were carried out at equal ®bre number of 3 106 ®bres/ml. Preparation of surface-active material Sheep surfactant. Lungs from freshly killed sheep were obtained and the left and right lobes separated at the bifurcation. Each lung was lavaged with 500 ml sterile saline. The lung was gently massaged and the lavageate collected into a sterile container. Approximately 700 ml of solution was collected after this procedure. The lavageate was transferred to 100-ml centrifuge tubes and spun at 1500 rpm for 10 min to remove cells and debris. The supernatant was transferred to clean 100-ml centrifuge tubes and spun at 23,000 rpm for 45 min. At the end of the centrifugation period, the supernatant from each tube was discarded and the remaining cell pellet resuspended in 10 ml sterile saline. This surfactant-enriched fraction which was termed 1 concentrated, was aliquotted into 5-ml volumes and stored at ÿ208C until required, in accordance with the method of Baughman et al. (1987). Table 1. Characterization of ®bres used in this study Cumulative % Fibre LFA SiC RCF1 RCF4 MMVF10 Code 100/475
>10 mm
>20 mm
64.75 60.86 77.36 59.35 85.24 50.00
35.25 27.64 45.27 17.96 67.17 19.32
Rat surfactant. Rat lungs were lavaged according to the procedure described in Bronchoalveolar lavage. The lavageate was pooled into a sterile 500-ml bottle. The procedure used for preparation of the sheep surfactant was then followed. Bronchoalveolar lavage Female Wistar rats, approximately 4 months old, were used throughout. Rats were killed with a single ip injection of pentobarbital, the lungs cannulated and removed and lavaged with 4 8 ml volumes of sterile saline. The lavageate was pooled into a single tube. Tubes were spun at 1200 rpm for 5 min at 48C, the supernatant collected for surfactant preparation, and the cell pellet resuspended in 1 ml phosphate buered saline (PBS) (Life Technologies, Paisley, UK). Cells were counted and resuspended in PBS at a concentration of 5 106 cells/ml for use in the superoxide assay. Cells were kept on ice throughout this procedure. BAL cells from normal rats are greater than 97% macrophages, the remainder being lymphocytes, with no neutrophils. Fibre coating The ®bres used in these experiments were a silicon carbide ®bre (Advanced Composite Materials Corporation) and long ®bre amosite asbestos (Davis et al., 1996). Refractive ceramic ®bres (RCF) from the TIMA repository and man-made vitreous ®bres (MMVF) and Code 100/475 glass ®bres, originally made by Johns Manville, were included. Fibres were suspended at a concentration of 2 mg/ ml in 1 concentrated rat or sheep surfactant or rat IgG (Sigma, Poole, Dorset, UK) at a concentration of 250 mg/ml in PBS. A separate group of ®bres designated `naked' ®bres, were suspended in PBS. The suspensions were sonicated brie¯y to disperse the ®bres and incubated in a rotary shaker for 1 hr at 378C. After the incubation period, the ®bres were washed twice with sterile PBS and resuspended in fresh PBS to give a ®nal ®bre number of 3 107 ®bres/ml. Fibre numbers were based on phase contrast optical microscopy counts relating ®bre number to a given mass of dust. Coated ®bres were used in the superoxide anion assay and in the macrophage binding assay. Coated and uncoated ®bres remained as single ®bres with no signs of clumping. Superoxide anion assay This assay was based on the reduction of cytochrome C (Johnston et al., 1978). The reaction mixture consisted of 50 mg cytochrome C (Sigma) 100 mg dextrose (BDH, Glasgow, UK) and 50 ml PBS. 900 mg cytochrome C reaction mixture was pipetted into triplicate groups of tubes and 100 ml of the appropriate ®bre suspension at a concentration of 3 107 ®bres/ml naked or coated, added. This gave a ®nal concentration of 3 106 ®bres/ml. 50 ml rat alveolar macrophages (5 106/ml) were added to each tube, mixed and incubated at 378C
Fibres and the macrophage respiratory burst
for 1 hr. Control tubes consisted of a group containing no ®bres and a series containing 0.1 mg/ml phorbol myristate acetate (PMA; Sigma) to test for macrophage triggerability. A superoxide dismutase (SOD) control (Sigma) was also included consisting of cytochrome C mixture containing 0.1 mg/ml PMA and 150 units SOD/ml. After incubation, samples were read at 550 nm and 468 nm to measure reduced and oxidized forms of cytochrome C. The optical density at 468 nm was subtracted from that obtained at 550 nm and multiplied by a factor of 47.6 to express results as nmol O2ÿ/ 0.25 105 cells/hr. Finally, the percentage of the SOD-inhibitable O2ÿ production was calculated. Results were ®nally expressed as nmol O2ÿ/million cells/hr. Trypan blue studies reveal that there was no cytotoxicity caused by any of the ®bre treatments.
containing 0.2% bovine serum albumin. 300 ml cell suspension and 33 ml of the appropriate ®bre suspension, naked or coated, at 3 107 ®bres/ml were dispensed into each well of eight-well chamber slides. The slides were incubated for 1 hr at 378C, after which the slides were washed in saline and stained with Diquick. The number of ®bres associated with each macrophage was determined microscopically at a magni®cation of 100. 100 cells/ treatment were counted. Fibres attached to plastic were not counted. Statistical analysis Data from the superoxide experiments were analysed using analysis of variance with Tukey multiple comparison test. Binding experiment data were examined using Student's t-test. RESULTS
Eect of surfactant on O2ÿ scavenging To test the ability of surfactant to scavenge O2ÿ, an O2ÿ generating system was used. Stock solutions of acetaldehyde, 40 mM in water; xanthine oxidase 20 mg/ml in water; cytochrome C, 50 mg in 5 ml PBS; and dextrose, 100 mg in 5 ml PBS were prepared. Each tube contained a mixture of the following:- acetaldehyde, 133 ml; xanthine oxidase, 133 ml; cytochrome C, 100 ml; dextrose, 100 ml; surfactant, 100 ml; PBS, 433 ml. Surfactant was added at various dilutions ranging from 1 concentrated to 1/8 concentrated. An SOD control consisting of 150 units/ml was included. Tubes were mixed and incubated at 378C for 30 min. Superoxide anion concentration was measured according to the method under Superoxide anion assay. Eect of surfactant on macrophage superoxide production The cytochrome C reaction mixture was prepared as previously described (Superoxide anion assay). 100 ml surfactant were added to 900 ml reaction mixture in triplicate groups. The surfactant was used at concentrations ranging from 1 to 1/8 dilution as described for the O2ÿ scavenging experiments. In three experiments, rat surfactant was used at 2.5 and 5 concentrated. 50 ml macrophage cell suspension (5 106 cells/ml) were added to each tube and mixed. A separate series of tubes consisted of cytochrome C reaction mixture and surfactant alone in addition to the control set with PMA and SOD as previously described (Superoxide anion assay). All tubes were incubated for 1 hr at 378C and read as before. The ®nal optical density obtained for cytochrome C mixture plus surfactant alone was subtracted from the readings obtained with the same mixture with macrophages. Binding of coated ®bres to macrophages Fibres were coated according to the method described under Fibre coating. Rat alveolar macrophages were obtained by BAL and resuspended at a concentration of 1 106 cells/ml in F-10 medium
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General All of the graphs are laid out with the three pathogenic ®bres long ®bre amosite, silicon carbide and RCF1 grouped on the left and the three nonpathogenic ®bres RCF4, MMVF10 and Code 100/ 475 on the right. This would make any eects speci®c for either category of ®bres evident. Superoxide production Adherence of rat alveolar macrophages to the plastic caused a stimulation in the respiratory burst of 38.93 nmol O2ÿ on average. Unless otherwise indicated all data are expressed as change relative to this background. IgG coating. Figure 1 compares the superoxide production by rat alveolar macrophages when treated with naked and IgG coated ®bres. By analysis of variance, there was no signi®cant dierence between control and naked ®bre treatment (F = 2.58; P > 0.05), probably because naked RCF4, MMVF10 and Code100/475 ®bres caused a mean reduction in superoxide production, with MMVF10 producing the largest decrease. By contrast, when the ®bres were coated with rat IgG, all ®bre types with the exception of RCF4 and Code100/475 caused more O2ÿ to be released compared with naked ®bres. By analysis of variance, there was a signi®cant eect of coating (F = 8.26; P < 0.05) compared with uncoated ®bres. The greatest increase between naked and coated ®bres was obtained with LFA (P < 0.05) and RCF1 and MMVF10 (P < 0.001). Ranking coated ®bres in order of increasing ability to stimulate O2ÿ production gave the following pro®le: RCF4 < Code100/475 < SiC < LFA < MMVF10 < RCF1. Sheep surfactant coating. In contrast to the enhanced production of O2ÿ by rat alveolar macrophages with IgG-coated ®bres, Fig. 2 shows that coating the ®bres with sheep surfactant caused a suppression of the respiratory burst. There was a signi®cant reduction in O2ÿ production when naked
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Fig. 1. The eect of rat IgG coating on the ability of ®bres to stimulate superoxide anion release by rat alveolar macrophages. The data represent the mean and SEM of triplicate groups in three separate experiments (**P < 0.01; ***P < 0.001).
®bres were compared for the ®bres RCF1 (P < 0.001), RCF4 and MMVF10 (P < 0.01) coated with sheep surfactant. In keeping with the data in Fig. 1, there was no signi®cant dierence when naked ®bres were compared with the medium control. Ranking coated ®bres in increasing order of suppressive activity gave the following pro®le: MMVF10 < RCF1 < RCF4 < Code100/475 < Si-
C < LFA. The RCF1 and RCF4 coated ®bres produced the same degree of response. Rat surfactant coating. Figure 3 shows the eect of coating ®bres with rat surfactant. As with ®bres coated with sheep surfactant, there was a signi®cant decrease in O2ÿ production when naked and coated ®bres were compared. The only ®bre type that showed a signi®cant dierence between naked and
Fig. 2. The eect of sheep surfactant coating on the ability of ®bres to stimulate superoxide anion release by rat alveolar macrophages. The data represent the mean and SEM of triplicate groups in three separate experiments (**P < 0.01; ***P < 0.001).
Fibres and the macrophage respiratory burst
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Fig. 3. The eect of rat surfactant coating on the ability of ®bres to stimulate superoxide anion release by rat alveolar macrophages. The data represent the mean and SEM of triplicate groups in three separate experiments (*P < 0.05).
coated ®bre was RCF1 (P < 0.05). Ranking coated ®bres in order of increasing ability to cause suppression of superoxide production gave the following pro®le: MMVF10 < RCF4 < RCF1 < LFA < Code 100/475 < SiC. RCF1 and RCF4 ®bres coated with rat surfactant produced a similar response to that seen with sheep surfactant.
Eect of rat surfactant on alveolar macrophage O2ÿ production Addition of rat surfactant to alveolar macrophages caused the respiratory burst to be suppressed in a dose-dependent manner. Figure 4 shows the eect on macrophage superoxide
Fig. 4. The eect of 5, 2.5 and 1 concentrated rat surfactant on superoxide anion release by rat alveolar macrophages. The data represent the mean and SEM of triplicate groups in three separate experiments.
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Table 2. The eect of various dilutions of rat and sheep surfactant on xanthine/xanthine oxidase-generated superoxide (control) Treatment Control Undiluted 1/2 1/4 1/8
Rat surfactant 25.00 26.29 27.88 27.78 26.97
(0.89) (1.17) (1.39) (1.29) (1.11)
Sheep surfactant 25.00 28.76 28.14 27.32 26.87
(0.89) (0.94) (1.01) (1.05) (1.18)
Data expressed as mean and SEM nmol O2ÿ.
production when rat surfactant was concentrated to 2.5 and ®ve times the original working concentration used to coat the ®bres. In both unstimulated and PMA-stimulated conditions, there was a signi®cant decrease in superoxide production compared with the controls when surfactant was concentrated. By analysis of variance, there was a signi®cant eect of surfactant concentration (F = 10.86; P < 0.01), and a signi®cant dierence between unstimulated and PMA-stimulated cells (F = 9.64; P < 0.05). The eect of surfactant on xanthine/xanthine oxidase-generated superoxide Table 2 shows the eect of rat and sheep surfactant on superoxide anion generated in a cell-free system. There was no scavenging eect of either surfactant type on superoxide.
ence between ®bre types (F = 22.43; P < 0.01) but no eect of coating (F = 8.73; P > 0.05). Ranking coated ®bres in order of increasing binding to rat alveolar macrophages produced the following: Code100/475 < LFA < MMVF10 < SiC < RCF1 < RCF4. Sheep surfactant-coated ®bres. For all ®bre types, with the exception of MMVF10, there was an increase in binding of coated ®bres to rat alveolar macrophages compared with the naked ®bres (Fig. 6). By analysis of variance, the eect of coating across all ®bre types was not statistically signi®cant (F = 1.13; P > 0.05). There was a signi®cant dierence between dierent ®bre types (F = 25.27; P < 0.01). Ranking coated ®bres in order of increasing binding to rat alveolar macrophages produced the following: Code100/475 < LFA < SiC < RCF4 < MMVF10 < RCF1. Rat surfactant-coated ®bres. Analysis of variance shows that for the six ®bre types, there was a signi®cant eect of rat surfactant coating in terms of increased binding to rat alveolar macrophages (F = 29.11; P < 0.05) (Fig. 7). There was, in addition, a signi®cant eect of ®bre type (F = 28.98; P < 0.01). Ranking coated ®bres in order of increasing binding to rat alveolar macrophages produced the following: Code100/475 < SiC< RCF1 < RCF4.
Binding of coated ®bres to rat alveolar macrophages The binding of ®bres coated with rat IgG, and sheep and rat surfactant to rat alveolar macrophages is shown in Figs 5±7. Rat IgG-coated ®bres. Coating ®bres with rat IgG produced increased binding to rat alveolar macrophages compared with the naked ®bres (Fig. 5). By analysis of variance there was a signi®cant dier-
DISCUSSION
Fibre pathogenicity has been examined in a number of dierent experimental systems and agreement has still to be reached regarding which methods provide the most accurate measure of ®bre-induced damage within the lung (Bignon et al., 1994). For
Fig. 5. Binding of rat IgG-coated ®bres to rat alveolar macrophages. Data is the mean and SEM of the number of ®bres associated with a single cell. 100 cells/experiment were counted and each experiment was performed three times.
Fibres and the macrophage respiratory burst
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Fig. 6. Binding of sheep surfactant-coated ®bres to rat alveolar macrophages. Data is the mean and SEM of the number of ®bres associated with a single cell. 100 cells/experiment were counted and each experiment was performed three times.
the most part, inhalation studies provide the most realistic model, although this is limited by high costs and time constraints and may not assist in the understanding of events at the single cell level because of intercellular interactions. Short-term in vitro studies based on an implied understanding of mechanisms have been a focus of ®bre testing in the hope that these tests may give a clear indication of the potential pathogenicity of dierent ®bres (McClellan et al., 1992).
Previous studies have con®rmed that dierent types of asbestos ®bres stimulate superoxide anion release from alveolar macrophages (Janssen et al., 1992). Hansen and Mossman (1987) compared ®bres and chemically similar non-®brous material in their ability to produce a respiratory burst and demonstrated that increased superoxide production appeared related to the geometry of the ®bres. With other known pathogenic dusts such as quartz, increased production of reactive oxygen metabolites
Fig. 7. Binding of rat surfactant-coated ®bres to rat alveolar macrophages. Data is the mean and SEM of the number of ®bres associated with a single cell. 100 cells/experiment were counted and each experiment was performed three times.
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has been shown compared with inert dusts such as TiO2 (Nyberg and Klockars, 1990b). As part of an Health and Safety Executive (HSE)-funded study into the ecacy of short-term tests we utilized a panel of ®bres, the pathogenicity of which is well known in long-term studies. We believe that this classi®cation is valid because the studies were carried out above exposure levels to long ®bres that would have shown pathogenicity if the ®bres had been pathogenic (Davis and Donaldson, 1993). For example, the studies on Code 100/475 (Davis et al., 1996). were carried out at a target concentration of 1000 ®bres/ml and the number of deposited long ®bres was similar for amosite asbestos and Code 100/475, but there were no tumours in the case of the latter. In the RCC (Research Consultancy Co., Geneva, Switzerland) studies, where MMVF10 and RCF4 were found to be non-pathogenic, the dosimetry again showed similar numbers of deposited long ®bres for all of the dierent ®bres tested, yet still some ®bres were tumorigenic (for example RCF1), and some were not (RCF4 and MMVF10) (Bunn et al., 1992; Glass et al., 1995; Hesterberg et al., 1993). These data strongly support a dierence in carcinogenic potency between the ®bres and not a dierence that can be explained as dierences in eective dose to the lung for the dierent ®bres. The endpoint of macrophage superoxide anion production was chosen because of evidence that oxidative stress occurs in vivo after asbestos exposure Janssen et al. (1992) and ®bres have been shown to stimulate macrophage respiratory burst (Hill et al., 1996; Perkins et al., 1991). We investigated the eect of coating with IgG, which is found normally in lung lining ¯uid and in increased levels during in¯ammation. Similarly, coating the ®bres with concentrated sheep or rat surfactant was carried out to detect any modifying eects the surfactant may have on the surface of the ®bre and its ability to stimulate superoxide anion release. In general, uncoated ®bres were inactive in causing O2ÿ release in rat alveolar macrophages as Hill et al. (1996) and others have previously reported, although silicon carbide showed minimal activity in this respect. Lung lining ¯uid is a complex mixture, and contains components other than surfactant and IgG that could contribute to modi®cation of the ®bre surface. Coating the ®bres with rat IgG produced a modest increase in O2ÿ for long-®bre amosite, as previously seen (Hill et al., 1996) while RCF1 and MMVF10 showed considerable increase in superoxide production after IgG opsonization. Code100/ 475 and RCF4 were unaected by IgG, in terms of ability to elicit a superoxide response. Silicon carbide ®bres, both naked and coated, stimulated almost identical amounts of superoxide, and MMVF10 caused inhibition in the naked state and stimulation in the coated state. These results suggest that there may be diering anity for rat IgG
depending on the ®bre type, as previously described by Hill et al. (1996). For other ®bres, increased superoxide release induced by IgG-coated ®bres may have implications in people who have existing in¯ammatory lung disease where IgG levels are increased. Raised levels of IgG in the lung may lead to increased opsonization of inhaled ®bres, rendering them more active and consequently more pathogenic. It is clear from this data, however, that the endpoint of superoxide release in response to either naked or IgG-opsonized ®bres has no value in predicting pathogenicity. The surfactant lining the alveolar spaces of the lung is a complex mixture of phospholipids and protein material and serves to reduce surface tension in the alveoli during breathing (Caminiti and Young, 1991). Surfactant also plays a major role in pulmonary homoeostasis, having the ability to suppress lymphocyte proliferation (Roth et al., 1993; Woerndle and Bartmann, 1994), and endotoxinstimulated alveolar macrophage in¯ammatory cytokine secretion (Thomassen et al., 1992 and 1994). Other studies have reported that surfactant or its components can enhance or inhibit macrophage oxidative responses (Hayakawa et al., 1989; van Iwaarden et al., 1990). Experiments in which ®bres were coated with sheep or rat surfactant produced interesting and surprising results. With all ®bre types, coating the ®bres caused the background respiratory burst to be suppressed compared with the naked ®bres. The largest inhibitory eect was seen in both types of surfactant for the ®bres RCF1, RCF4 and MMVF10. This initially suggested that, as in the case of the IgG-coated ®bres, there were diering ®bre surface chemistry eects which may cause differing anities for the surfactant. The role of the surfactant alone was examined in terms of ability to cause superoxide suppression when not ®bre associated. Results from these experiments showed that surfactant alone could cause suppression of the respiratory burst in both untriggered and PMA-triggered macrophages. This eect was surfactant concentration dependent. Surfactant has been shown to have inhibitory activity, as shown by down regulation of endotoxin-stimulated in¯ammatory cytokine mRNA levels and tumour necrosis factor production in alveolar macrophages (Antal et al., 1996). We have demonstrated that a fungalderived inhibitor of the oxidative burst also inhibits TNF mRNA and protein production and we have hypothesized that this results from inhibition of protein kinases (Nicholson et al., 1996). Surfactant may work by a similar mechanism. The similarity in the eects of surfactant in solution and surfactant-coated ®bres in causing inhibition of the respiratory burst, suggests that surfactant-coated ®bres may have their eect by bringing surfactant into close contact with the cell membrane. Our own unpublished data (1996) suggests that ®bres incubated in normal BAL ¯uid, a diluted
Fibres and the macrophage respiratory burst
form of lung lining ¯uid, show slight inhibition of the oxidative burst compared with uncoated ®bres, suggesting that the surface is preferentially coated with surfactant. However, a full study of the modifying eects of other components of lung lining ¯uid, and how they compete for the ®bre surface, is warranted. The data shows that the ability to switch o the respiratory burst by coating with surfactant is not predictive of pathogenicity or non-pathogenicity. An additional possible reason for suppression of the respiratory burst was a scavenging eect of the surfactant itself. In experiments where superoxide anion was generated using the xanthine/xanthine oxidase system, both rat and sheep surfactant caused no scavenging eect. In another study, the antioxidant activity of surfactant has been demonstrated, although this eect was seen in arti®cial surfactant (Ghio et al., 1994). Binding to the cell membrane is an important step in the release of superoxide anion by alveolar macrophages triggering the NADPH oxidase enzyme assembly that catalyses the reduction of molecular oxygen to O2ÿ (Chanock et al., 1994). Therefore, the ability of the macrophage to bind ®bres may be an important factor. Increased binding and hence increased NADPH oxidase assembly result in an enhanced respiratory burst. In view of the inhibitory eects of surfactant-coated ®bres, we attempted to relate superoxide release to the degree of binding of ®bres to alveolar macrophages. Our results demonstrated that, in all cases except MMVF10, coating with sheep surfactant caused an increase in binding of ®bres to alveolar macrophages. In general, binding was inversely related to the superoxide release; that is, when coated with surfactant, the ®bre types that bound in highest numbers caused greatest inhibition of superoxide anion release. This was not a very robust relationship but clearly showed two populations of sheep surfactant-coated ®bresÐthose that bound more and produced inhibition and those that bound less and produced less inhibition. For sheep and rat surfactant, the ®bres that bound more and caused greater inhibition were MMVF10, RCF1 and RCF4. Long ®bre amosite, Code100/475 and SiC were bound less and produced less superoxide inhibition. The data demonstrate that the nature of ®bre coating is central to the biological outcome of interaction with macrophages. IgG-coated ®bres bind to a `receptor' that could stimulate superoxide release or had no eect. In contrast surfactant-coated ®bres engaged a `receptor' that inhibited superoxide anion release. However, there were clear and constant dierences between ®bres in the extent of stimulation or inhibition. No combinations of activities were predictive of pathogenicity. The purpose of the present study was to develop short-term in vitro assays which would allow us to predict the pathogenicity of ®bres. The results do not preclude superoxide anion from macrophages
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as a contributory factor in a multi-factorial sequence of events that culminate in disease in vivo. The results do, however, indicate that in vitro release of superoxide anion by rat alveolar macrophages in response to ®bres with various coatings does not predict pathogenicity as indicated by longterm inhalation studies with the same ®bres. The study has, however, shown that the bioactivity of ®bres can be altered by coating with surfactant and IgG, and that this should be considered in shortterm tests for assessing the eects of ®bres and in mechanistic studies. AcknowledgementsÐThis study was funded by the Health and Safety Executive. We also gratefully acknowledge an equipment grant from the British Occupational Health Research Foundation. REFERENCES
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Glass L. R., Brown R. C. and Hoskins J. A. (1995) Health eects of refractory ceramic ®bres: scienti®c issues and policy considerations. Occupational and Environmental Medicine 52, 433±440. Goodlick L. A. and Kane A. B. (1986) Role of reactive oxygen metabolites in crocidolite asbestos toxicity to mouse macrophages. Cancer Research 46, 5558±5566. Hansen K. and Mossman B. T. (1987) Generation of superoxide (O2ÿ ) from alveolar macrophages exposed to asbestiform and non®brous particles. Cancer Research 47, 1681±1686. Hart G. A., Kathman L. M. and Hesterberg T. W. (1994) In vitro cytotoxicity of asbestos and man-made vitreous ®bres: roles of ®bre length, diameter and composition. Carcinogenesis 15, 971±977. Hayakawa H., Myrvik Q. N. and St. Clair R. W. (1989) Pulmonary surfactant inhibits priming of rabbit alveolar macrophage: evidence that surfactant suppresses the oxidative burst of alveolar macrophages in infant rabbits. American Review of Respiratory Disease 140, 1390±1397. Hesterberg T. W. and Barrett J. C. (1984) Dependence of asbestos and mineral dust-induced transformation of mammalian cells in culture on ®bre dimension. Cancer Research 44, 2170±2180. Hesterberg T. W., Miller W. C., McConnell E. E., Chevalier J., Hadley J. G., Bernstein D. M., Thevanaz P. and Anderson R. (1993) Chronic inhalation toxicity of size-separated glass ®bers in Fisher-344 rats. Fundamental and Applied Toxicology 20, 464±476. Hill I. M., Beswick P. H. and Donaldson K. (1996) Enhancement of the oxidative burst by immunoglobulin coating of respirable ®bres: ®bre-speci®c dierences between asbestos and man-made ®bres. Experimental Lung Research 22, 133±148. Janssen Y. M. W., Marsh J. P., Absher M. P., Hemenway D., Vacek P. M., Leslie K. O., Borm P. J. A. and Mossman B. T. (1992) Expression of antioxidant enzymes in rat lung after inhalation of asbestos or silica. Journal of Biological Chemistry 267, 10625±10630. Johnston R. B., Godzik C. A. and Cohn Z. A. (1978) Increased superoxide by immunologically activated and chemically elicited macrophages. Journal of Experimental Medicine 148, 115±127. Kamp D. W., Gracea P., Pryor W. A. and Weitzman S. A. (1992) The role of free radicals in asbestos-induced diseases. Free Radical Biological Medicine 12, 293±315. McClellan R. O., Miller F. J., Hesterberg T. W., Warheit D. B., Bunn W. B., Kane A. B., Lippmann M., Mast T. W., McConnell E. E. and Reinhardt C. F. (1992) Approaches to evaluating the toxicity and carcinogenicity of man-made ®bres. Summary of a workshop held November 11±13, 1991. Durham, North Carolina. Regulatory Toxicology and Pharmacology 16, 321±364. Morgan A. and Holmes A. (1986) Solubility of asbestos and man-made mineral ®bres in vitro and in vivo: its signi®cance in lung disease. Environmental Research 39, 475±484.
Nicholson W. J., Slight J. and Donaldson K. (1996) Inhibition of cytokine gene transcription by a diusable component from the spores of the fungus Aspergillus fumigatus; role of the transcription activating factors NF-(B and AP-1). American Journal of Respiratory Cell and Molecular Biology 15, 88±96. Nyberg P. and Klockars M. (1990a) Eect of immunoglobulins on mineral dust-induced production of reactive oxygen metabolites by human macrophages. In¯ammation 14, 671±679. Nyberg P. and Klockars M. (1990b) Measurement of reactive oxygen metabolites produced by human monocytederived macrophages exposed to mineral dusts. International Journal of Experimental Pathology 71, 537±544. Perkins R. C., Scheule R. K. and Holian A. (1991) In vitro bioactivity of asbestos for the human alveolar macrophage and its modi®cation by IgG. American Journal of Respiratory Cell and Molecular Biology 4, 532±537. Roney P. L. and Holian A. (1989) Possible mechanism of chrysotile asbestos-stimulated superoxide anion production in guinea pig alveolar macrophages. Toxicology and Applied Pharmacology 100, 132±144. Roth M. D., Pinto M., Golub S. H. and Shau H. (1993) Pulmonary surfactant inhibits interleukin-2-induced proliferation and the generation of lymphokine-activated killer cells. American Journal of Respiratory Cell and Molecular Biology 9, 652±658. Scheule R. K. and Holian A. (1989) IgG speci®cally enhances chrysotile asbestos-stimulated superoxide anion production by the alveolar macrophage. American Journal of Respiratory Cell and Molecular Biology 1, 313±318. Thomassen M. J., Antal J. M., Connors M. J., Meeker D. P. and Wiedemann H. P. (1994) Characterization of Exosurf (surfactant)-mediated suppression of stimulated human alveolar macrophages cytokine responses. American Journal of Respiratory Cell and Molecular Biology 10, 399±404. Thomassen M. J., Meeker D. P., Antal J. M., Connors M. J. and Wiedemann H. P. (1992) Synthetic surfactant (Exosurf) inhibits endotoxin-stimulated cytokine secretion by human alveolar macrophages. American Journal of Respiratory Cell and Molecular Biology 7, 257±260. Vallyathan V., Mega J. F., Xianglin S. and Dalal N. S. (1992) Enhanced generation of free radicals from phagocytes induced by mineral dusts. American Journal of Respiratory Cell and Molecular Biology 6, 404±413. van Iwaarden F., Welmers B., Verhoef J., Haagsman H. P. and Van Golde L. M. (1990) Pulmonary surfactant protein A enhances the host defence mechanism of rat alveolar macrophages. American Journal of Respiratory Cell and Molecular Biology 2, 91±98. Woerndle S. and Bartmann P. (1994) The eect of three surfactant preparations on in vitro lymphocyte functions. Journal of Perinatal Medicine 22, 119±128.
Eect of Coating with Lung Lining Fluid on the Ability of Fibres to Produce a Respiratory Burst in Rat Alveolar Macrophages D. M. BROWN*, N. K. ROBERTS and K. DONALDSON Department of Biological Sciences, Napier University, Edinburgh EH10 5DT, UK (Accepted 10 June 1997) AbstractÐThe aim of the study was to develop a simple short-term in vitro assay which would allow us to predict the pathogenicity of ®bres based on data already available from in vivo studies. Fibres were used naked (uncoated) or coated with rat IgG, or rat or sheep surfactant. The ®bres were used to stimulate superoxide anion release by rat alveolar macrophages. Binding of ®bres to rat alveolar macrophages was assessed by optical microscopy. Fibres used in the naked state produced little or no stimulation of superoxide anion from rat alveolar macrophages. When ®bres were coated with rat IgG there was a signi®cant increase in superoxide release for all ®bre types with the exception of RCF4 and Code 100/475. When ®bres were coated with rat or sheep surfactant, there was suppression of the respiratory burst for all ®bre types. The observed suppression was not due to a scavenging eect by the surfactant itself, because xanthine/xanthine oxidase generated superoxide was unaected by surfactant. The suppressive eect was shown to act directly on the macrophages. Comparing naked and coated ®bres for their ability to bind to macrophages, it was shown that in general more coated ®bres were bound and that increased binding was associated with suppressed superoxide release for both types of surfactantcoated ®bres. It was concluded that the nature of the ®bre coating is the main factor in¯uencing the interaction between ®bres and macrophages. The type of binding through dierent receptors may either stimulate or switch o the respiratory burst. The assay used here does not, however, allow any predictions to be made regarding the pathogenicity of ®bres. # 1998 Elsevier Science Ltd Abbreviations: BAL = bronchoalveolar lavage; MMVF = man-made vitreous ®bres; PBS = phosphate buered saline; PMA = phorbol myristate acetate; RCF = refractive ceramic ®bres; SOD = superoxide dismutase. Keywords: ®bres; superoxide anion; surfactant.
INTRODUCTION
It is widely recognized that the mechanisms of ®bre-induced lung ®brosis and cancer depend on several factors such as dimension (Donaldson et al., 1989 and 1993b), chemical nature (Hart et al., 1994; Hesterberg and Barrett, 1984), solubility (Morgan and Holmes, 1986) and durability (Donaldson et al., 1993a). Interaction between alveolar macrophage surface receptors and foreign particles including ®bres leads to phagocytosis and possibly a secretory response which is enhanced in the presence of an opsonin such as IgG (Donaldson et al., 1992; Hill et al., 1996; Nyberg and Klockars, 1990a; Perkins et al., 1991; Scheule and Holian, 1989). Phagocytosis is a stimulus for superoxide anion release, and it has been shown that when long ®bre *Author for correspondence.
crocidolite asbestos is phagocytozed, superoxide is released to the outside of the cell (Goodlick and Kane, 1986; Hill et al., 1996). In other studies (Roney and Holian, 1989), guinea pig macrophages did not produce a respiratory burst when treated with amosite asbestos, although chrysotile was stimulatory. The release of reactive oxygen species by macrophages may be the initiating factor in the pathogenesis of lung disease after deposition of particles (Kamp et al., 1992; Vallyathan et al., 1992). Superoxide anion release from stimulated cells through protein kinase C activation or by crosslinking of Fc receptors by IgG-coated ®bres has previously been demonstrated (Aida and Onoue, 1984; Scheule and Holian, 1989). As with other particles, ®bres that deposit on the lung surface become coated with lung lining material, a consequence of which is likely to be
0887-2333/98 $19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(97)00093-3
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modi®cation of the ®bre surface reactivity, and hence a change in the oxidant generating ability and possibly phagocytosis of the ®bres. In this study, we have examined the ability of a range of ®bres of diering pathogenicity, including asbestos (long ®bre amosite), refractory ceramic ®bres (RCFs) and man-made vitreous ®bres (MMVFs) to produce a respiratory burst in rat alveolar macrophages. The ®bres used were allocated to categories of pathogenic or non-pathogenic, depending on whether they caused tumours and ®brosis in recent inhalation studies (Davis et al., 1996; Glass et al., 1995; Hesterberg et al., 1993). Pathogenic ®bres were Long Fibre Amosite, Silicon carbide and RCF1; non-pathogenic ®bres were RCF4, Code 100/475 and MMVF10. The purpose of the present study was (1) to compare ®bres as to their ability to produce an oxidative burst in rat alveolar macrophages; (2) to determine whether this could be modulated by coating the ®bres with lung lining material; and (3) to determine whether any of the eects were related to the pathogenicity of the ®bres.
MATERIALS AND METHODS
Fibres The size characteristics of the ®bres used in this study are shown in Table 1. All assays were carried out at equal ®bre number of 3 106 ®bres/ml. Preparation of surface-active material Sheep surfactant. Lungs from freshly killed sheep were obtained and the left and right lobes separated at the bifurcation. Each lung was lavaged with 500 ml sterile saline. The lung was gently massaged and the lavageate collected into a sterile container. Approximately 700 ml of solution was collected after this procedure. The lavageate was transferred to 100-ml centrifuge tubes and spun at 1500 rpm for 10 min to remove cells and debris. The supernatant was transferred to clean 100-ml centrifuge tubes and spun at 23,000 rpm for 45 min. At the end of the centrifugation period, the supernatant from each tube was discarded and the remaining cell pellet resuspended in 10 ml sterile saline. This surfactant-enriched fraction which was termed 1 concentrated, was aliquotted into 5-ml volumes and stored at ÿ208C until required, in accordance with the method of Baughman et al. (1987). Table 1. Characterization of ®bres used in this study Cumulative % Fibre LFA SiC RCF1 RCF4 MMVF10 Code 100/475
>10 mm
>20 mm
64.75 60.86 77.36 59.35 85.24 50.00
35.25 27.64 45.27 17.96 67.17 19.32
Rat surfactant. Rat lungs were lavaged according to the procedure described in Bronchoalveolar lavage. The lavageate was pooled into a sterile 500-ml bottle. The procedure used for preparation of the sheep surfactant was then followed. Bronchoalveolar lavage Female Wistar rats, approximately 4 months old, were used throughout. Rats were killed with a single ip injection of pentobarbital, the lungs cannulated and removed and lavaged with 4 8 ml volumes of sterile saline. The lavageate was pooled into a single tube. Tubes were spun at 1200 rpm for 5 min at 48C, the supernatant collected for surfactant preparation, and the cell pellet resuspended in 1 ml phosphate buered saline (PBS) (Life Technologies, Paisley, UK). Cells were counted and resuspended in PBS at a concentration of 5 106 cells/ml for use in the superoxide assay. Cells were kept on ice throughout this procedure. BAL cells from normal rats are greater than 97% macrophages, the remainder being lymphocytes, with no neutrophils. Fibre coating The ®bres used in these experiments were a silicon carbide ®bre (Advanced Composite Materials Corporation) and long ®bre amosite asbestos (Davis et al., 1996). Refractive ceramic ®bres (RCF) from the TIMA repository and man-made vitreous ®bres (MMVF) and Code 100/475 glass ®bres, originally made by Johns Manville, were included. Fibres were suspended at a concentration of 2 mg/ ml in 1 concentrated rat or sheep surfactant or rat IgG (Sigma, Poole, Dorset, UK) at a concentration of 250 mg/ml in PBS. A separate group of ®bres designated `naked' ®bres, were suspended in PBS. The suspensions were sonicated brie¯y to disperse the ®bres and incubated in a rotary shaker for 1 hr at 378C. After the incubation period, the ®bres were washed twice with sterile PBS and resuspended in fresh PBS to give a ®nal ®bre number of 3 107 ®bres/ml. Fibre numbers were based on phase contrast optical microscopy counts relating ®bre number to a given mass of dust. Coated ®bres were used in the superoxide anion assay and in the macrophage binding assay. Coated and uncoated ®bres remained as single ®bres with no signs of clumping. Superoxide anion assay This assay was based on the reduction of cytochrome C (Johnston et al., 1978). The reaction mixture consisted of 50 mg cytochrome C (Sigma) 100 mg dextrose (BDH, Glasgow, UK) and 50 ml PBS. 900 mg cytochrome C reaction mixture was pipetted into triplicate groups of tubes and 100 ml of the appropriate ®bre suspension at a concentration of 3 107 ®bres/ml naked or coated, added. This gave a ®nal concentration of 3 106 ®bres/ml. 50 ml rat alveolar macrophages (5 106/ml) were added to each tube, mixed and incubated at 378C
Fibres and the macrophage respiratory burst
for 1 hr. Control tubes consisted of a group containing no ®bres and a series containing 0.1 mg/ml phorbol myristate acetate (PMA; Sigma) to test for macrophage triggerability. A superoxide dismutase (SOD) control (Sigma) was also included consisting of cytochrome C mixture containing 0.1 mg/ml PMA and 150 units SOD/ml. After incubation, samples were read at 550 nm and 468 nm to measure reduced and oxidized forms of cytochrome C. The optical density at 468 nm was subtracted from that obtained at 550 nm and multiplied by a factor of 47.6 to express results as nmol O2ÿ/ 0.25 105 cells/hr. Finally, the percentage of the SOD-inhibitable O2ÿ production was calculated. Results were ®nally expressed as nmol O2ÿ/million cells/hr. Trypan blue studies reveal that there was no cytotoxicity caused by any of the ®bre treatments.
containing 0.2% bovine serum albumin. 300 ml cell suspension and 33 ml of the appropriate ®bre suspension, naked or coated, at 3 107 ®bres/ml were dispensed into each well of eight-well chamber slides. The slides were incubated for 1 hr at 378C, after which the slides were washed in saline and stained with Diquick. The number of ®bres associated with each macrophage was determined microscopically at a magni®cation of 100. 100 cells/ treatment were counted. Fibres attached to plastic were not counted. Statistical analysis Data from the superoxide experiments were analysed using analysis of variance with Tukey multiple comparison test. Binding experiment data were examined using Student's t-test. RESULTS
Eect of surfactant on O2ÿ scavenging To test the ability of surfactant to scavenge O2ÿ, an O2ÿ generating system was used. Stock solutions of acetaldehyde, 40 mM in water; xanthine oxidase 20 mg/ml in water; cytochrome C, 50 mg in 5 ml PBS; and dextrose, 100 mg in 5 ml PBS were prepared. Each tube contained a mixture of the following:- acetaldehyde, 133 ml; xanthine oxidase, 133 ml; cytochrome C, 100 ml; dextrose, 100 ml; surfactant, 100 ml; PBS, 433 ml. Surfactant was added at various dilutions ranging from 1 concentrated to 1/8 concentrated. An SOD control consisting of 150 units/ml was included. Tubes were mixed and incubated at 378C for 30 min. Superoxide anion concentration was measured according to the method under Superoxide anion assay. Eect of surfactant on macrophage superoxide production The cytochrome C reaction mixture was prepared as previously described (Superoxide anion assay). 100 ml surfactant were added to 900 ml reaction mixture in triplicate groups. The surfactant was used at concentrations ranging from 1 to 1/8 dilution as described for the O2ÿ scavenging experiments. In three experiments, rat surfactant was used at 2.5 and 5 concentrated. 50 ml macrophage cell suspension (5 106 cells/ml) were added to each tube and mixed. A separate series of tubes consisted of cytochrome C reaction mixture and surfactant alone in addition to the control set with PMA and SOD as previously described (Superoxide anion assay). All tubes were incubated for 1 hr at 378C and read as before. The ®nal optical density obtained for cytochrome C mixture plus surfactant alone was subtracted from the readings obtained with the same mixture with macrophages. Binding of coated ®bres to macrophages Fibres were coated according to the method described under Fibre coating. Rat alveolar macrophages were obtained by BAL and resuspended at a concentration of 1 106 cells/ml in F-10 medium
17
General All of the graphs are laid out with the three pathogenic ®bres long ®bre amosite, silicon carbide and RCF1 grouped on the left and the three nonpathogenic ®bres RCF4, MMVF10 and Code 100/ 475 on the right. This would make any eects speci®c for either category of ®bres evident. Superoxide production Adherence of rat alveolar macrophages to the plastic caused a stimulation in the respiratory burst of 38.93 nmol O2ÿ on average. Unless otherwise indicated all data are expressed as change relative to this background. IgG coating. Figure 1 compares the superoxide production by rat alveolar macrophages when treated with naked and IgG coated ®bres. By analysis of variance, there was no signi®cant dierence between control and naked ®bre treatment (F = 2.58; P > 0.05), probably because naked RCF4, MMVF10 and Code100/475 ®bres caused a mean reduction in superoxide production, with MMVF10 producing the largest decrease. By contrast, when the ®bres were coated with rat IgG, all ®bre types with the exception of RCF4 and Code100/475 caused more O2ÿ to be released compared with naked ®bres. By analysis of variance, there was a signi®cant eect of coating (F = 8.26; P < 0.05) compared with uncoated ®bres. The greatest increase between naked and coated ®bres was obtained with LFA (P < 0.05) and RCF1 and MMVF10 (P < 0.001). Ranking coated ®bres in order of increasing ability to stimulate O2ÿ production gave the following pro®le: RCF4 < Code100/475 < SiC < LFA < MMVF10 < RCF1. Sheep surfactant coating. In contrast to the enhanced production of O2ÿ by rat alveolar macrophages with IgG-coated ®bres, Fig. 2 shows that coating the ®bres with sheep surfactant caused a suppression of the respiratory burst. There was a signi®cant reduction in O2ÿ production when naked
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D. M. Brown et al.
Fig. 1. The eect of rat IgG coating on the ability of ®bres to stimulate superoxide anion release by rat alveolar macrophages. The data represent the mean and SEM of triplicate groups in three separate experiments (**P < 0.01; ***P < 0.001).
®bres were compared for the ®bres RCF1 (P < 0.001), RCF4 and MMVF10 (P < 0.01) coated with sheep surfactant. In keeping with the data in Fig. 1, there was no signi®cant dierence when naked ®bres were compared with the medium control. Ranking coated ®bres in increasing order of suppressive activity gave the following pro®le: MMVF10 < RCF1 < RCF4 < Code100/475 < Si-
C < LFA. The RCF1 and RCF4 coated ®bres produced the same degree of response. Rat surfactant coating. Figure 3 shows the eect of coating ®bres with rat surfactant. As with ®bres coated with sheep surfactant, there was a signi®cant decrease in O2ÿ production when naked and coated ®bres were compared. The only ®bre type that showed a signi®cant dierence between naked and
Fig. 2. The eect of sheep surfactant coating on the ability of ®bres to stimulate superoxide anion release by rat alveolar macrophages. The data represent the mean and SEM of triplicate groups in three separate experiments (**P < 0.01; ***P < 0.001).
Fibres and the macrophage respiratory burst
19
Fig. 3. The eect of rat surfactant coating on the ability of ®bres to stimulate superoxide anion release by rat alveolar macrophages. The data represent the mean and SEM of triplicate groups in three separate experiments (*P < 0.05).
coated ®bre was RCF1 (P < 0.05). Ranking coated ®bres in order of increasing ability to cause suppression of superoxide production gave the following pro®le: MMVF10 < RCF4 < RCF1 < LFA < Code 100/475 < SiC. RCF1 and RCF4 ®bres coated with rat surfactant produced a similar response to that seen with sheep surfactant.
Eect of rat surfactant on alveolar macrophage O2ÿ production Addition of rat surfactant to alveolar macrophages caused the respiratory burst to be suppressed in a dose-dependent manner. Figure 4 shows the eect on macrophage superoxide
Fig. 4. The eect of 5, 2.5 and 1 concentrated rat surfactant on superoxide anion release by rat alveolar macrophages. The data represent the mean and SEM of triplicate groups in three separate experiments.
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Table 2. The eect of various dilutions of rat and sheep surfactant on xanthine/xanthine oxidase-generated superoxide (control) Treatment Control Undiluted 1/2 1/4 1/8
Rat surfactant 25.00 26.29 27.88 27.78 26.97
(0.89) (1.17) (1.39) (1.29) (1.11)
Sheep surfactant 25.00 28.76 28.14 27.32 26.87
(0.89) (0.94) (1.01) (1.05) (1.18)
Data expressed as mean and SEM nmol O2ÿ.
production when rat surfactant was concentrated to 2.5 and ®ve times the original working concentration used to coat the ®bres. In both unstimulated and PMA-stimulated conditions, there was a signi®cant decrease in superoxide production compared with the controls when surfactant was concentrated. By analysis of variance, there was a signi®cant eect of surfactant concentration (F = 10.86; P < 0.01), and a signi®cant dierence between unstimulated and PMA-stimulated cells (F = 9.64; P < 0.05). The eect of surfactant on xanthine/xanthine oxidase-generated superoxide Table 2 shows the eect of rat and sheep surfactant on superoxide anion generated in a cell-free system. There was no scavenging eect of either surfactant type on superoxide.
ence between ®bre types (F = 22.43; P < 0.01) but no eect of coating (F = 8.73; P > 0.05). Ranking coated ®bres in order of increasing binding to rat alveolar macrophages produced the following: Code100/475 < LFA < MMVF10 < SiC < RCF1 < RCF4. Sheep surfactant-coated ®bres. For all ®bre types, with the exception of MMVF10, there was an increase in binding of coated ®bres to rat alveolar macrophages compared with the naked ®bres (Fig. 6). By analysis of variance, the eect of coating across all ®bre types was not statistically signi®cant (F = 1.13; P > 0.05). There was a signi®cant dierence between dierent ®bre types (F = 25.27; P < 0.01). Ranking coated ®bres in order of increasing binding to rat alveolar macrophages produced the following: Code100/475 < LFA < SiC < RCF4 < MMVF10 < RCF1. Rat surfactant-coated ®bres. Analysis of variance shows that for the six ®bre types, there was a signi®cant eect of rat surfactant coating in terms of increased binding to rat alveolar macrophages (F = 29.11; P < 0.05) (Fig. 7). There was, in addition, a signi®cant eect of ®bre type (F = 28.98; P < 0.01). Ranking coated ®bres in order of increasing binding to rat alveolar macrophages produced the following: Code100/475 < SiC< RCF1 < RCF4.
Binding of coated ®bres to rat alveolar macrophages The binding of ®bres coated with rat IgG, and sheep and rat surfactant to rat alveolar macrophages is shown in Figs 5±7. Rat IgG-coated ®bres. Coating ®bres with rat IgG produced increased binding to rat alveolar macrophages compared with the naked ®bres (Fig. 5). By analysis of variance there was a signi®cant dier-
DISCUSSION
Fibre pathogenicity has been examined in a number of dierent experimental systems and agreement has still to be reached regarding which methods provide the most accurate measure of ®bre-induced damage within the lung (Bignon et al., 1994). For
Fig. 5. Binding of rat IgG-coated ®bres to rat alveolar macrophages. Data is the mean and SEM of the number of ®bres associated with a single cell. 100 cells/experiment were counted and each experiment was performed three times.
Fibres and the macrophage respiratory burst
21
Fig. 6. Binding of sheep surfactant-coated ®bres to rat alveolar macrophages. Data is the mean and SEM of the number of ®bres associated with a single cell. 100 cells/experiment were counted and each experiment was performed three times.
the most part, inhalation studies provide the most realistic model, although this is limited by high costs and time constraints and may not assist in the understanding of events at the single cell level because of intercellular interactions. Short-term in vitro studies based on an implied understanding of mechanisms have been a focus of ®bre testing in the hope that these tests may give a clear indication of the potential pathogenicity of dierent ®bres (McClellan et al., 1992).
Previous studies have con®rmed that dierent types of asbestos ®bres stimulate superoxide anion release from alveolar macrophages (Janssen et al., 1992). Hansen and Mossman (1987) compared ®bres and chemically similar non-®brous material in their ability to produce a respiratory burst and demonstrated that increased superoxide production appeared related to the geometry of the ®bres. With other known pathogenic dusts such as quartz, increased production of reactive oxygen metabolites
Fig. 7. Binding of rat surfactant-coated ®bres to rat alveolar macrophages. Data is the mean and SEM of the number of ®bres associated with a single cell. 100 cells/experiment were counted and each experiment was performed three times.
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D. M. Brown et al.
has been shown compared with inert dusts such as TiO2 (Nyberg and Klockars, 1990b). As part of an Health and Safety Executive (HSE)-funded study into the ecacy of short-term tests we utilized a panel of ®bres, the pathogenicity of which is well known in long-term studies. We believe that this classi®cation is valid because the studies were carried out above exposure levels to long ®bres that would have shown pathogenicity if the ®bres had been pathogenic (Davis and Donaldson, 1993). For example, the studies on Code 100/475 (Davis et al., 1996). were carried out at a target concentration of 1000 ®bres/ml and the number of deposited long ®bres was similar for amosite asbestos and Code 100/475, but there were no tumours in the case of the latter. In the RCC (Research Consultancy Co., Geneva, Switzerland) studies, where MMVF10 and RCF4 were found to be non-pathogenic, the dosimetry again showed similar numbers of deposited long ®bres for all of the dierent ®bres tested, yet still some ®bres were tumorigenic (for example RCF1), and some were not (RCF4 and MMVF10) (Bunn et al., 1992; Glass et al., 1995; Hesterberg et al., 1993). These data strongly support a dierence in carcinogenic potency between the ®bres and not a dierence that can be explained as dierences in eective dose to the lung for the dierent ®bres. The endpoint of macrophage superoxide anion production was chosen because of evidence that oxidative stress occurs in vivo after asbestos exposure Janssen et al. (1992) and ®bres have been shown to stimulate macrophage respiratory burst (Hill et al., 1996; Perkins et al., 1991). We investigated the eect of coating with IgG, which is found normally in lung lining ¯uid and in increased levels during in¯ammation. Similarly, coating the ®bres with concentrated sheep or rat surfactant was carried out to detect any modifying eects the surfactant may have on the surface of the ®bre and its ability to stimulate superoxide anion release. In general, uncoated ®bres were inactive in causing O2ÿ release in rat alveolar macrophages as Hill et al. (1996) and others have previously reported, although silicon carbide showed minimal activity in this respect. Lung lining ¯uid is a complex mixture, and contains components other than surfactant and IgG that could contribute to modi®cation of the ®bre surface. Coating the ®bres with rat IgG produced a modest increase in O2ÿ for long-®bre amosite, as previously seen (Hill et al., 1996) while RCF1 and MMVF10 showed considerable increase in superoxide production after IgG opsonization. Code100/ 475 and RCF4 were unaected by IgG, in terms of ability to elicit a superoxide response. Silicon carbide ®bres, both naked and coated, stimulated almost identical amounts of superoxide, and MMVF10 caused inhibition in the naked state and stimulation in the coated state. These results suggest that there may be diering anity for rat IgG
depending on the ®bre type, as previously described by Hill et al. (1996). For other ®bres, increased superoxide release induced by IgG-coated ®bres may have implications in people who have existing in¯ammatory lung disease where IgG levels are increased. Raised levels of IgG in the lung may lead to increased opsonization of inhaled ®bres, rendering them more active and consequently more pathogenic. It is clear from this data, however, that the endpoint of superoxide release in response to either naked or IgG-opsonized ®bres has no value in predicting pathogenicity. The surfactant lining the alveolar spaces of the lung is a complex mixture of phospholipids and protein material and serves to reduce surface tension in the alveoli during breathing (Caminiti and Young, 1991). Surfactant also plays a major role in pulmonary homoeostasis, having the ability to suppress lymphocyte proliferation (Roth et al., 1993; Woerndle and Bartmann, 1994), and endotoxinstimulated alveolar macrophage in¯ammatory cytokine secretion (Thomassen et al., 1992 and 1994). Other studies have reported that surfactant or its components can enhance or inhibit macrophage oxidative responses (Hayakawa et al., 1989; van Iwaarden et al., 1990). Experiments in which ®bres were coated with sheep or rat surfactant produced interesting and surprising results. With all ®bre types, coating the ®bres caused the background respiratory burst to be suppressed compared with the naked ®bres. The largest inhibitory eect was seen in both types of surfactant for the ®bres RCF1, RCF4 and MMVF10. This initially suggested that, as in the case of the IgG-coated ®bres, there were diering ®bre surface chemistry eects which may cause differing anities for the surfactant. The role of the surfactant alone was examined in terms of ability to cause superoxide suppression when not ®bre associated. Results from these experiments showed that surfactant alone could cause suppression of the respiratory burst in both untriggered and PMA-triggered macrophages. This eect was surfactant concentration dependent. Surfactant has been shown to have inhibitory activity, as shown by down regulation of endotoxin-stimulated in¯ammatory cytokine mRNA levels and tumour necrosis factor production in alveolar macrophages (Antal et al., 1996). We have demonstrated that a fungalderived inhibitor of the oxidative burst also inhibits TNF mRNA and protein production and we have hypothesized that this results from inhibition of protein kinases (Nicholson et al., 1996). Surfactant may work by a similar mechanism. The similarity in the eects of surfactant in solution and surfactant-coated ®bres in causing inhibition of the respiratory burst, suggests that surfactant-coated ®bres may have their eect by bringing surfactant into close contact with the cell membrane. Our own unpublished data (1996) suggests that ®bres incubated in normal BAL ¯uid, a diluted
Fibres and the macrophage respiratory burst
form of lung lining ¯uid, show slight inhibition of the oxidative burst compared with uncoated ®bres, suggesting that the surface is preferentially coated with surfactant. However, a full study of the modifying eects of other components of lung lining ¯uid, and how they compete for the ®bre surface, is warranted. The data shows that the ability to switch o the respiratory burst by coating with surfactant is not predictive of pathogenicity or non-pathogenicity. An additional possible reason for suppression of the respiratory burst was a scavenging eect of the surfactant itself. In experiments where superoxide anion was generated using the xanthine/xanthine oxidase system, both rat and sheep surfactant caused no scavenging eect. In another study, the antioxidant activity of surfactant has been demonstrated, although this eect was seen in arti®cial surfactant (Ghio et al., 1994). Binding to the cell membrane is an important step in the release of superoxide anion by alveolar macrophages triggering the NADPH oxidase enzyme assembly that catalyses the reduction of molecular oxygen to O2ÿ (Chanock et al., 1994). Therefore, the ability of the macrophage to bind ®bres may be an important factor. Increased binding and hence increased NADPH oxidase assembly result in an enhanced respiratory burst. In view of the inhibitory eects of surfactant-coated ®bres, we attempted to relate superoxide release to the degree of binding of ®bres to alveolar macrophages. Our results demonstrated that, in all cases except MMVF10, coating with sheep surfactant caused an increase in binding of ®bres to alveolar macrophages. In general, binding was inversely related to the superoxide release; that is, when coated with surfactant, the ®bre types that bound in highest numbers caused greatest inhibition of superoxide anion release. This was not a very robust relationship but clearly showed two populations of sheep surfactant-coated ®bresÐthose that bound more and produced inhibition and those that bound less and produced less inhibition. For sheep and rat surfactant, the ®bres that bound more and caused greater inhibition were MMVF10, RCF1 and RCF4. Long ®bre amosite, Code100/475 and SiC were bound less and produced less superoxide inhibition. The data demonstrate that the nature of ®bre coating is central to the biological outcome of interaction with macrophages. IgG-coated ®bres bind to a `receptor' that could stimulate superoxide release or had no eect. In contrast surfactant-coated ®bres engaged a `receptor' that inhibited superoxide anion release. However, there were clear and constant dierences between ®bres in the extent of stimulation or inhibition. No combinations of activities were predictive of pathogenicity. The purpose of the present study was to develop short-term in vitro assays which would allow us to predict the pathogenicity of ®bres. The results do not preclude superoxide anion from macrophages
23
as a contributory factor in a multi-factorial sequence of events that culminate in disease in vivo. The results do, however, indicate that in vitro release of superoxide anion by rat alveolar macrophages in response to ®bres with various coatings does not predict pathogenicity as indicated by longterm inhalation studies with the same ®bres. The study has, however, shown that the bioactivity of ®bres can be altered by coating with surfactant and IgG, and that this should be considered in shortterm tests for assessing the eects of ®bres and in mechanistic studies. AcknowledgementsÐThis study was funded by the Health and Safety Executive. We also gratefully acknowledge an equipment grant from the British Occupational Health Research Foundation. REFERENCES
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