Br. J. exp. Path. (1983) 64, 66 FAILURE OF MACROPHAGE ACTIVATION TO INDUCE PULMONARY FIBROSIS IN ASBESTOS-EXPOSED GUINEA-PIGS R. J. EMERSON* AND P. J. COLE Fromt the Host Defence Unit, Department of ]ledicine, Cardiothoracic Institute, Brompton Hospital, Fulham Road, London, SW3 6HP Received for publication August 26, 1982 Summary.-Inhalation of asbestos by specific-pathogen-free (SPF) guinea-pigs the macrophages of which were systemically activated with Freund's complete adjuvant and/or M. tuberculosis (Strain H37Ra) induced a diffuse pulmonary mononuclearcell infiltration of the interstitium and air space. The severity of the reactions to the various insults was dependent on the treatment given. Although there was no histological evidence of increased collagen, there was a diffuse increase in reticulin in animals which were exposed to asbestos and the macrophages of which were activated. However, even in the case where severe pulmonary inflammatory changes occurred complete resolution of the response took place within 1 year. Therefore in this model activation of macrophages does not have a detrimental effect after inhalation of asbestos. THE RESPONSE OF THE LUNG to fibrogenic minerals may be altered considerably by prior colonization of the respiratory tract with various specific pathogens. For example, chronic inflammation and pulmonary fibrosis is a common lesion in conventionally reared animals exposed to asbestos for short periods of time (Holt, Mills and Young, 1966). However, inhalation of asbestos by specific-pathogen-free (SPF) animals for as long as 4 h initiates a chronic inflammatory response, but fails to induce fibrotic lesions (Botham and Holt, 1972). SPF animals are taken by caesarean section from germ-free foster mothers and barrier-maintained to prevent colonization of the respiratory tract by pathogenic organisms. Conventionally reared animals are carriers of pathogens which are believed to predispose the animal to respiratory infections which possibly alter the response of the lung to asbestos. Alveolar macrophages of SPF animals exhibit a reduced state of activation in comparison to conventionally maintained animals (Sorokin, 1977). Consequently, one might ask whether or not activated macrophages play a role in the pathogenesis of the fibrosis associated with asbestos inhalation. The present experiments were designed to study the histopathological features of the pulmonary response to asbestos in guinea-pigs in which alveolar macrophages were activated. This was accomplished by exposing these animals to an asbestos aerosol that was sufficient to cause an inflammatory reaction but not fibrosis (Holt et al., 1966). Although a characteristic pattern of parenchymal inflammation was found, the systemic activation of alveolar macrophages failed to promote the development of pulmonary fibrosis. MATERIALS AND METHODS Experimental groups and model design. Female, 3-35 g (SPF) Dunkin-Hartley guinea-pigs (Redfern Animal Breeders Ltd, Tunbridge Wells, England) were divided into 8 * Present Address: Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 545. (82) 656-221. Correspondence and reprint requests to Dr R. J. Emerson.
Group N AF M FM AM AF AFM MACROPHAGES IN ASBESTOS-INDUCED LUNG INJURY Asbestos* Day 14 14 TABLE I.-Treatment regime Freund's complete adjuvantt (FCA) Day M. tuberculosist Day * Aerosol exposure for 16 h. t FCA (Difco Laboratories, Detroit, Mich.). -1 ml injected in right and left hind footpad. t Strain H37Ra (Trudeau Mycobacterial Culture Collection, Trudeau Institute, Saranac Lake, New York); -1 ml of a suspension of 1 x 17 viable organisms per ml s.c. injected in right and left inguinal region. 21 21 21 67 groups of 3 animals each. The treatment regime is summarized in Table I. After treatment to initiate activation of macrophages, 3 members of each group were killed 2 weeks after treatment and at 2-week intervals for 3 months and then every 3 months for 9 more months. Asbestos inhalation. UICC Rhodesian chrysotile A asbestos was kindly supplied by Dr V. Timbrell of the M.R.C. Pneumoconiosis Unit, Llandough Hospital, Penarth, South Glamorgan. The exposure apparatus (Holt et al., 1966) and facilities for asbestos fibre inhalation were kindly provided by Professor P. F. Holt, University of Reading. Animals were exposed to asbestos (5, fibres/cc air) by placing 2 groups of 9 animals each in the inhalation chamber for a period of 8 h on 2 consecutive days, with the dust generator operating. During this time the dust concentration within the chamber was continually monitored by a recording tyndallometer. This instrument was calibrated by quantitative dust sampling, as described by Harness (1973). The animals remained in the inhalation chamber overnight and their own movements generated a further dust cloud during this period. However, the fibre concentration in the chamber was not determined during this time. Histology. After exsanguination the thoracic cavity was opened and the trachea exposed and cannulated with a stainless steel cannula attached to a 2ml syringe containing 5% cacodylate-buffered glutaraldehyde. The lungs were inflated slowly with approximately 1-12 ml of fixative, the trachea ligated and the thoracic contents removed en bloc and placed in fixative for 3 or more days. After fixation the lung was first dissected into its respective lobes, right and left upper (apical), right and left lower (diaphragmatic), and the small right and left middle (accessory). The individual lobes then were prepared for light and transmission electron microscopy using standard methods. A grading system based on visual comparative standards was used to provide an - assessment of the degree of inflammation from (no inflammation) to grade 4+ (severe inflammation). Macrophage activation.-transmission electron microscopy was used to assess activation of alveolar macrophages in whole lung tissue. For the purpose of this study, activated macrophages were considered to be large cells which exhibited extensive pseudopod formation and increased numbers of cytoplasmic organelles (Carr and Daems, 198). RESULTS Numerous mononuclear cells were present in the alveolar spaces of all treated groups. Based on morphological criteria (Adams, 1975) lymphocytes, monocytes, immature and mature macrophages were the predominant cell types (Fig. 1). The majority of the macrophages exhibited numerous long pseudopods and an increase in cytoplasmic organelles, suggesting an activated state. It was difficult to find AM in the lungs of untreated controls (Group N); when present the cells appeared mature and lacked the morphological features of activation described above. It was not possible to distinguish one experimental group from another by light microscopic examination of the lungs. All treated groups differed from the untreated controls during the evolution of the inflammatory process. Regardless of the treatment regime, the histological changes differed only quantitatively throughout the course of the experiment (Table II). Although the lesions produced by combined treatment with asbestos, FCA and
68 R. J. EMERSON AND P. J. COLE FIG. 1.-Typical aggregate of alveolar macrophages seen in lungs of treated groups. Note numerous pseudopods, representative of activated cells, and the immature cell (arrow) among the group of mature activated macrophages. x 39. FIG. 4.-Near obliteration of lung architecture by massive increases in reticulin fibres which have become thickened and branched. Note argyrophilic material in remaining alveolar spaces. Gordon and Sweet's silver impregnation. x 16.
MTACROPHAGES IN ASBESTOS-INDUCED LUNG INJURY TABLE II.-Summary of histopathology after treatment* Group 2 wks 4 wks 6 wks 8 wks 1 wks 12 wks 26 wks 38 wks A + + ++ ++ ++ ++ + + F + ++ +++ ++ + + AF + + + + ++ + +++ +++ + + + + M + ++ +++ ++ +++ ++ +++ - AM + + + + + + + + + + ++ + + FM + + + + + + + + + + + + + + + AFM + + + ++++ + + + +++ + + + +++ + + + + * Severity of lesions graded from - (no inflammation) to + + + + (severe inflammation). 69 52 wks H37Ra (Group AFM) were similar to the lesions seen in other treated groups, they were more extensive. Not only did they appear earlier but they were slower to resolve. In general, combined treatment with any 2 agents (Groups FM, AM, AF) also accentuated the inflammatory response when compared to animals given only a single treatment (Groups M, F and A). It was not uncommon to find normal lung morphology in areas adjacent to the lesions. The peak of the inflammatory reaction for any given group was followed by complete resolution. Asbestos bodies were demonstrated after 4 weeks, and persisted for the entire year of the study but mycobacteria, amyloid deposits, and increases in collagen were not observed. Lesions in the treated animals had a sequential pattern of development. Initially they appeared as diffuse focal areas of interstitial and/or intraalveolar inflammmatory reactions consisting of mononuclear cells (Fig. 2). One stage merged with the next in which small sheets of loosely packed mononuclear cells began to obliterate alveolar lumens (Fig. 3). Thickened reticulin fibres appeared in the interstitial spaces and finer newly formed fibres developed around the mononuclear cells within alveoli (Fig. 4). Eventually, extensive areas of mononuclear cells arranged in large sheets and packed closely together caused widespread obliteration of the alveolar architecture (Fig. 5). This sequential pattern of parenchymal inflammation was seen throughout the 26-week observation period. By contrast, animals killed at 9 and 12 months after anv treatment demonstrated a resolution of the inflammatory response. Lesions gradually became smaller, reticulin was resorbed and the sheets of mononuclear cells reverted to the less differentiated interstitial reactions seen during the early stages of the inflammatory response. Eventually the lung tissue became normal in appearance. DISCUSSION Alveolar macrophages have been implicated as important participants in asbestosis since they secrete factors that are partly responsible for chronic inflammation and fibrosis (Allison, 1977; Davis and Allison, 1976). It has been suggested that accumulation of these cells in the lung is an initial step to fibrosis after asbestos inhalation. Although increased numbers of macrophages appear to be a prelude to fibrosis their presence alone may be insufficient as a further stimulus for fibrogenesis when interacting with asbestos fibres. The fibrosis seen may reflect a functional failure or alteration of the cell from the norm which may or may not be caused by asbestos. To elucidate further the role of alveolar macrophages in asbestosis the work presented here has focused on the histopathological lesions developing in guinea-pigs in which alveolar macrophages were activated before and after asbestos exposure. Although asbestos persisted in the lungs throughout the study, 4 factors may be responsible for its not eliciting a continuing inflammatory response leading to established fibrosis. First, the dose may not have been sufficient for induction of a rapid and selective release of lysosomal enzymes from the macrophages (Davies et
7 R. J. EMERSON AND P. J. COLE FIG. 2.-(a) Early lesion showing a diffuse interstitial mononuclear-cell infiltration. H. & E. x 123. (b) High-power view of Fig. 2a. x 312.
MACROPHAGES IN ASBESTOS-INDUCED LUNG INJURY 71 ~~~r~ 4 FIG. 3.-(a) Typical small loose sheet of mononuclear cells partially obliterating alveoli. H. & E. x 123. (b) High-power view of Fig. 3a. x 312.
72 R. J. EMERSON AND P. J. COLE v r a...t -*; _- ss..;..- l FIG. 5.-(a) Large sheet of mononuclear cells causing obliteration of lung architecture with adjacent normal lung. H. & E. x 48. (b) High-powei view of Fig. 5a. x 78.
MACROPHAGES IN ASBESTOS-INDUCED LUNG INJURY 73 at., 1974). Lysosomal enzymes such as g- glucuronidase and /-galactosidase may be responsible for part of the tissue damage seen in chronic inflammation (Davies and Allison, 1976). Second, the formation of asbestos bodies (as seen by light microscopy) may be a protective mechanism of the lung against asbestos fibres (Selikoff and Lee, 1978), thus reducing the number of fibres able to interact with macrophages in a detrimental way. Third, asbestos fibres, particularly chrysotile, fragment and components are leached (Selikoff and Lee, 1978), again reducing the number of fibres with pathogenic potential. Fourth, interaction of asbestos fibres with activated macrophages may not be required for fibrogenesis. However, before this can be conclusively answered, asbestos dose-response studies with activated macrophages must be undertaken. Spector, Henson and Stevens (1968) have shown that persistence of an intracellular irritant is required for the maintenance of chronic inflammation. It is possible that, after inhalation, asbestos fibres which are not cleared from the lung eventually lose their capacity to maintain a chronic inflammatory response. Injury produced by any combination of treatment (i.e. AM, AF, FM, and AFM) resembled the histological changes seen in Groups A, F, and M, but lesions were more extensive, appeared earlier and were slower to resolve. Treatment with FCA is capable of eliciting an inflammatory response in the lung after injection in the hind footpad and, when given 21 days before a s.c. inoculation of live H37Ra, it potentiates the pulmonary inflammatory reaction to the s.c. inoculation of mycobacteria. The pulmonary reaction to these s.c. injected antigens resemble the delayed hypersensitivity reaction seen in the lung following inhalation of purified protein derivative (PPD) in guinea-pigs sensitized i.m. with FCA (Miyamoto, Kabe and Noda, 1971). This suggests that the reaction seem in the lung after treatment with these agents is immunologically mediated. In contrast, asbestos is an inorganic fibre that elicits an inflammatory reaction by physical interactions with macrophage cell membranes. Consequently, the lesions seen in the lung after combined treatment with asbestos, FCA and H37Ra are probably additive. However, once the intracellular irritants are eliminated or degraded there is no mechanism for a continued inflammatory response and resolution occurs. The results show that systemic macrophage activation does not enhance establishment of pulmonary fibrosis in guinea-pigs that have inhaled asbestos for a short time. Although the results indicate that macrophage activation may not be an important host factor in asbestos-induced fibrogenesis, one must consider that the amount of asbestos capable of interacting with activated cells may be too small to cause fibrosis, no matter how the cells were activated. Dose-response studies in a similar model would be needed to answer this question. We would like to thank Professor Bryan Corrin for his helpful discussion throughout this study and Drs John E. Craighead and Kenneth B. Adler for their helpful review of this manuscript. The work by Dr Robert J. Emerson was carried out in partial fulfilment of the requirements for a Ph.D. Degree of the University of London, and was supported by a grant from the Chest, Heart and Stroke Association. REFERENCES ADAMS, D.. (1975) The Structure of Mononuclear Phagocytes II. The Effects of Mycobacterium Tuberculosis. Am. J. Path., 8, 11. ALLISON, A. C. (1977) Mechanism of Macrophage Damage in Relation to the Pathogenesis of Some Lung Diseases. In Respiratory Defense Mechanisms, Eds J. D. Brain., D. F. Proctor and L. M. Reid. New York: Marcel Dekker. p. 175. BOTHAM, S. K. & Holt, P. F. (1972) The Effects of Inhaled Crocidolites from Transvaal and Southwest Cape Mines on the Lungs of Rats and Guinea Pigs. Br. J. exp. Path., 53, 612. CARR, I. & DAEMS, W. T. (Eds) (198) The Reticuloendothelial System: A Comprehensive Treatise. New York: Plenum Press. p. 57. DAVIES, P., ALLISON, A. C., ACKERMAN, J., BUTTER- FIELD, A. & WILLIAMS, S. (1974) Asbestos induced
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