American Joumnal of Pathology, Vol. 141, No. 2, August 1992 Copyright X American Association of Pathologists
Instillation of Chemotactic Factor to Silica-injected Lungs Lowers Interstitial Particle Content and Reduces Pulmonary Fibrosis 1. Y. R. Adamson, H. Prieditis, and D. H. Bowden From the Department of Pathology, University of Manitoba, Winnipeg, Canada
Silica-induced pulmonary fibrosis usually follows exposure to increased levels of this particulate and its retention in interstitial macrophages of the lung It is suggested that accelerated clearance ofparticles from the pulmonary interstitium may ameliorate subsequentfibrosis. To test this bypothesis, one group of mice received 2-mg intratracheal (IT) silica, some particles were phagocytized and cleared during the subsequent inflammatory response, other particles were translocated across the epithelium to reach interstitial macrophages by 2 weeks. These mice later showed increased fibroblast growth, a doubling of lung collagen levels and large silicotic nodules by 16 weeks when much of the silica was stillpresent in the lung. A second group of mice received IT silica, then 2 and 3 weeks later received IT injections of N-formyl-L-methionyl-leucyl-phenylalanine (FMLP), a leukocyte chemoattractant. Subsequently, a significant increase in inflammatory cells was seen and silica was observed mostly in phagocytes within the alveolar spaces. Few interstitial particles were found at 4 weeks; and extensive fibrosis did not develop by 16 weeks; only a few small nodules were seen and little silica was present in the lung, The results indicate that clearance of interstitial particles by a controlled inflammatory response is possible, and that removal of silica from the interstitium decreases the fibrotic response. (Am JPathol 1992, 141:319-326)
Exposure of the lung to an increased dose of silica particles is associated with the development of pulmonary fibrosis.13 Activation of macrophages is believed to be a key event in this process and various growth factors for fibroblasts have been identified in secretions of isolated pulmonary macrophages.3 Most of these studies have used alveolar macrophages (AM) lavaged from fibrotic
lungs, whereas in vivo, the fibrotic process occurs within the interstitium that also contains a population of macrophages. Previous studies from our laboratory indicate that penetration of particles into lung tissue and their retention by interstitial macrophages results in direct stimulation of the adjacent fibroblast population.78 At increased dose levels, particles such as silica cross the epithelium to reach the interstitium where clearance appears to be slow. Most particles are phagocytized by macrophages, some of which may migrate to the alveoli or to lymph nodes; other particles remain in the interstitium and may be trapped by the fibrotic response as granulomas form. We hypothesize that clearance of interstitial particles at an early stage would reduce macrophage activation within the interstitium and so reduce any subsequent pulmonary fibrosis. In this study, mice were injected intratracheally first with silica, and later with 2 doses of a leukocyte chemoattractant in an attempt to increase the phagocytic cell traffic across the pulmonary interstitium and clear any translocated silica. The inflammatory response, cell kinetics, hydroxyproline production, and retained silica were compared in the various treatment groups over the subsequent 16 weeks.
Materials and Methods A group of 50 Swiss-Webster mice (25-g males) received an intratracheal (IT) injection of 2-mg silica (crystalline quartz, diameter 0.3 ± 0.1 ,um, from Dowson and Dobson, South Africa) in 0.1 ml sterile water while under mild nembutal anesthesia.2 After 2 weeks and 3 weeks, these animals were given IT injections of 100 ,ug of N-formyl-Lmethionyl-leucyl-phenylalanine (FMLP) in 0.1 ml albumin.9 Other groups of mice received silica only, FMLP only, or had no treatment. The mice in groups of four were killed at day 0 then at weeks 1, 2, 3, 4, 6, 8, 12, and 16 from the time of the silica injection. Each mouse received Accepted for publication January 31, 1992. Address reprint requests to Dr. I. Adamson, Department of Pathology, University of Manitoba, 236-770 Bannatyne Avenue, Winnipeg, MB, Canada, R3E OW3.
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2 ,uCi/g tritiated thymidine by intraperitoneal injection 1 hour before death. The animals were killed by barbiturate injection, tracheotomies were performed, and the tracheas were cannulated. The lungs were washed four times in situ with 1.0 ml of saline. The lavage fluid was pooled for each animal, and the total number of cells was counted on a hemocytometer. The cell suspension was centrifuged, and a cytospin preparation was made and stained by the Giemsa method; differential counts of polymorphonuclear leukocytes (PMN) and AM were made on 500 cells per slide. The total number of cells of each type was calculated for each time studied, and the mean ± SE of the total cells per lung in the four mice per group was calculated. The supernatant of the lavage fluid from each mouse was used to determine total protein content and the level of glucosaminidase.10 After lavage, the bronchus leading to the right lung was clamped; this lung was removed, weighed, and frozen for biochemical analysis. The left lung was inflated with 0.5 ml of 2% buffered glutaraldehyde and removed; most of the tissue was processed for embedding in glycol methacrylate. Sections (0.75 ,um thick) from three random blocks per animal were prepared for autoradiography with the use of Kodak NTB2 emulsion. We determined the percentages of 3H-thymidine-labeled cells at each timepoint by counting 3000 lung cells per animal. The means and standard error were calculated for each group. The plastic sections were thin enough to allow identification of pulmonary cell types, and differential counts of labeled cells were performed on 300 labeled cells per animal. The product of the differential percentage and the total labeling percentage gave the labeling index for each cell type. The index for epithelial, interstitial, and endothelial cells was calculated at each time studied. A small sample of tissue was also processed for electron microscopy. The right lung of each mouse was homogenized in water and biochemical assays were performed on duplicate samples. Determinations of DNA and total protein were performed by conventional methods.11 12 As an index of collagen content, hydroxyproline (Hyp) levels were determined after hydrolysis with hydrochloric acid.13 To obtain an estimate of silica retention in the lungs, a residue after tissue digestion was prepared. At 16 weeks, six extra mice from each group were killed, and the lungs removed immediately.7 These were chopped up and incubated in 40% potassium hydroxide (KOH) overnight in a 800C waterbath. When the tissue was completely digested, the solution was centrifuged at 1500 rpm for 15 minutes, and a residue was obtained. This was washed twice in distilled water, resuspended in water, and transferred to a weighed tube. The residue was dried to con-
stant weight. Mean + standard error was calculated for each group of six and the significance of any difference from control mice was determined using the Student's t-test.
Results Bronchoalveolar Lavage (BAL) The number of AM recovered by lavage increased sharply in silica-injected mice (Figure 1). Although the value decreased after 1 week, it remained above normal through most of the experimental period. The injection of FMLP alone, which is known to induce a rapid inflammatory response,9 produced a small increase in AM recovered at 3 weeks. When FMLP was injected to mice previously injected with silica, there was a significant increase in AM recovered by BAL from 3 to 12 weeks (Figure 1). A similar pattern was seen when the PMN response was quantitated. FMLP alone induced a brief efflux of PMN while silica alone induced an increased and prolonged PMN response (Figure 2). However, when FMLP was injected IT to silica-bearing mice as their PMN response was subsiding, an additional and significant increase in lavaged PMN was found during a subsequent 8-week period. Silica particles were identified in both AM and PMN in cytospin preparations. As a marker of lung injury, particularly a permeability change at the air-blood barrier, protein levels were measured in BAL fluid. Normal values were low at around 50 ,ug/ml, and the FMLP injection caused a brief doubling (Figure 3). Silica instillation induced an increased protein level that decreased slowly but never reached the control value. The injection of FMLP to silica-bearing mice caused a subsequent significant increase in BAL protein for a 6-week period. Levels of glucosaminidase followed Injection
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Figure 1. Numbers (mean ± SE) of alveolar macrophages (AM) recovered by lavage at intervals after silica instillation. FMLP injection times are shown by arrows. Crosses, FMLP alone; closed circles, silica only; open circles, silica followed by FMLP. *Silica + FMLP > silica only, P < 0.01. (Valuesfor nontreated controls were identical to time zero values).
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a similar pattern with silica alone inducing a brief increased level of enzyme activity that fell after 1 week, but never returned to control values (Figure 4). This proteolytic enzyme is associated with degranulation of PMN and is known to be elevated during silica-induced lung injury.2 However the injection of FMLP to silica-bearing mice caused only a temporary increase in the enzyme level over that seen in the silica-only group.
Lung Morphology
Figure 4. Glucosaminidase levels in bronchoalveolar lavagefluid in each group. Symbols as in Figure 1.
weeks large fibrotic granulomas were found (Figure 7), resembling those we have described previously for silica-induced fibrosis in mice.2'7 When animals that received silica were injected with FMLP after 2 weeks, a secondary inflammatory response was seen and at 4 weeks many alveoli were filled with inflammatory cells containing particles (Figure 8). The alveolar walls appeared thin and less cellular than after silica alone, and by electron microscopy, most of the silica present in the lung was in phagosomes of free AM and PMN (Figure 9); few particles were seen in the interstitium. In later weeks, as the AM and PMN became
Mice that received FMLP showed only a mild inflammatory exudate at 1 and 2 weeks after injection. The lung structure appeared normal at all other times. Animals that received silica only showed a large number of AM and PMN in the alveoli at 1 week. Silica particles were seen in these cells as well as free in the alveolar spaces. By 2 weeks, particles were seen in AM and in macrophages within the pulmonary interstitium, which was more cellular than normal (Figure 5). At 4 weeks, the interstitial tissue contained mixed macrophages and fibroblasts particularly in association with silica particles (Figure 6). Subsequently the fibrotic response progressed so that at 16 Injection
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Figure 3. Protein content of bronchoalveolar lavagefluid in each group. Symbols as in Figure 1.
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seen in some macrophages (arrows) in the more cellular than normal, X900.
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Figure 6. Electron micrograph oJ lung 4 weeks ajter silica alone. Interstitial macrophages contain silica particles (arrows) are adjacent to cells resembling fibroblasts. A = alveolus, x 4,800
Figure 7 Section of lung 16 weeks after silica alone Large interstitial granulomas are formed and contain macrophages with silica among the fibrobas and collagen, X 450.
fewer, less silica was seen. The alveolar walls were mostly thin and resembled normal lung; only an occasional small granuloma was found in the silica-FMLP group at 16 weeks (Figure 10).
after silica alone. Interstitial labeling in particular returned to a low level and few labeled fibroblasts were seen in the
8-16 week period.
Lung Chemistry Autoradiography Injection of FMLP resulted in only a brief increase in DNA synthesis in the lung and this was confined to the interstitial cell population (Figure 1 1). Silica instillation resulted in approximately a six-fold increase in thymidine-labeled cells by 1 week, then this level declined to 16 weeks but was always greater than controls. From differential counts of labeled cells, the major contributing factor was increased proliferation in the interstitial compartment. Labeled cells in the first 4 weeks morphologically resembled interstitial macrophages (Figure 12); labeled AM were rarely seen. After 4 weeks, labeled interstitial cells were mainly identified as fibroblasts. Animals that received both silica and FMLP showed a further increase in labeled lung cells to 6 weeks as compared with the silica only group (Figure 11). The increase in labeled cells was confined to the interstitial cell population and mostly resembled macrophages similar to these shown in Figure 12. After 6 weeks, cell labeling decreased considerably to a level less than that seen
Fibrosis was quantitated by measuring total Hyp per right lobe. In control (nontreated) mice, this value increased slowly with age, and these levels were not different in animals that received FMLP only (Figure 13). After silica only, tissue Hyp was significantly higher than normal beginning at 4 weeks, and fibrosis increased progressively over the 16-week period. In mice that received FMLP after silica, the tissue Hyp rose slightly by 4 weeks but then remained significantly lower than the increased level found in animals that received silica alone. Retention of silica in the lung at the end of the 1 6-week period was estimated by quantitating the tissue residue after KOH digestion. A light-brown residue weighing around 0.2 mg was obtained from control and FMLPtreated mice (Figure 14). This was taken as the nonparticulate, baseline organic residue. In mice that received 2 mg of silica, the residue appeared gray, particulate, and weighed about 0.7 mg. However, in the group that received FMLP after silica treatment, most of the silica had been cleared from the lung and the weight of a pale
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Figure 8 Section oflung 4 weeks after silica with FMLP injections at weeks 2 and 3. The alveoli are filled with large AM and small PMN (arrows), the interstitium appears near normal, x800.
Figure 9. Electron micrograph of lung shown in Figure 8. Dense silica particles are present in both AM and in PMN (arrows), the alveolar walls are thin and contain no particles, x4,800.
residue was significantly lower than that of the silica only group.
monary response to asbestos,814 and to other particles particularly in an overload situation in which more particles are translocated to the interstitium.15,16 In this study, the pulmonary reaction to silica alone followed the pattern described previously. There was a rapid response of PMN and AM with accompanying increases in protein leakage to alveoli and in the BAL content of lysosomal enzymes such as glucosaminidase. Some silica particles crossed the Type 1 epithelium and were phagocytized by interstitial macrophages. In the later stages, fibroblast proliferation and collagen deposition occurred at sites of silica retention producing large interstitial nodules. An increased level of silica was trapped in these areas and was quantitated in the lung residue at 16 weeks. The relationship between the retention of interstitial silica and the generation of localized regions of fibrosis led to the hypothesis that early clearance of silica from the interstitium might reduce macrophage activation at that site thereby reducing the subsequent fibrotic reaction. Interstitial macrophages may be cleared from the lung by migration to lymphatic channels or to the alveoli. Particle clearance to the lymphatics has been quantitated as a small percentage of the total load,16 but clearance from interstitium to alveoli is more difficult to measure. Interstitial macrophages have been shown to migrate to the alveoli in normal adult lung,17 developing lung,18 and in
Discussion Small quantities of particles deposited in the lung are mostly phagocytized and cleared by alveolar macrophages. As the number of particles is increased, an inflammatory response occurs and the efflux of PMN with additional macrophages enhances clearance. In the case of silica, particle interaction with AM is known to produce a variety of fibroblast growth factors. This has been shown in vitro and in vivo using AM lavaged from fibrotic lungs.36 Although the resultant pulmonary fibrosis is interstitial, most studies have concentrated on factors produced by the readily obtainable AM,5'6 which is believed to mirror the function of the interstitial macrophage. However, in a series of in vivo experiments, we demonstrated that pulmonary fibrosis is increased when the translocation of silica from the alveoli to the interstitium is enhanced.7 This suggests that direct macrophagefibroblast interaction within the pulmonary interstitium may be more effective in transferring a fibroblast growth factor than a process involving secretion by AM into the alveolar space. The role of the interstitial macrophage in promoting fibrosis has also been emphasized in the pul-
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Figure 10. Section of lung 16 weeks after silica with FMLP injections at weeks 2 and 3. A small granuloma is seen but most of the lung tissue appears normal, x 450.
Figure 12. Autoradiograph of lung section 2 weeks after silica only. Labeled interstitial cells (arrows) at this time resemble macrophages, x800.
particle-laden lungs.2'7 In an earlier experiment, we instilled chemotactic factor to mouse lung and demonstrated an early, large PMN efflux to the alveoli, followed by division and migration of interstitial mononuclear cells to the air sacs.9 The inflammatory response had subsided by 1 week with no injury to the lung9 and no subsequent fibrosis. We have used this same agent (FMLP) to induce an acute inflammatory response in a particleladen lung in an attempt to clear interstitial particles. This could occur by direct migration of interstitial macrophages containing silica, or through phagocytosis and clearance of free interstitial silica by PMN and monocytes
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12 8 16 4 6 WEEKS AFTER SILICA Figure 11. Percentage of thymidine-labeled lung cells (upper panel) and radiographic index (RI) of interstitial cells (lower panel) over the 16-week period. Solid circles, silica alone; crosses, FMLP alone; open circles, silica plus FMLP injected at times shown. (Nontreated controls equal to time zero values). *Silica + FMLP > silica only, P < 0.01. aSilica and FMLP < silica only, P < 0.01.
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each group. group were
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while crossing the interstitium. In either case, the interstitial burden of particles would be reduced. Instilling FMLP to the lung was carried out 2 and 3 weeks after silica, when particles had been phagocytized by AM or translocated to the interstitium, and the inflammatory response was subsiding. The chemotactic agent promoted a supplementary inflammatory reaction with increased PMN, AM, and associated enzymes in BAL fluid. Thymidine uptake by interstitial mononuclear cells was also increased over the level found after silica alone. It appears that the response to FMLP induced both division and migration of interstitial phagocytes to the alveoli. The lungs of these mice showed an increased number of free alveolar cells containing silica, along with reduced cellularity of the pulmonary interstitium. From the morphologic evaluation, most particles seen in the silica plus FMLP group were found in the AM and PMN in the alveolar spaces up to 8 weeks, though increased migration of macrophages to regional lymph nodes could also have occurred. Although quantitative data on particle distribution in airspaces, lung tissue, and lymphatics were not obtained serially, enhanced clearance of interstitial silica was confirmed at the end of the experiment when the weight of lung residue in the silica-FMLP group was substantially lower than that measured from the silica-only animals. In addition, mice that received FMLP after silica showed lower fibroblast labeling and much lower lung Hyp content. Fibrosis was limited to a few small foci, whereas after silica alone, large confluent silicotic nodules and high Hyp levels were found. These combined results confirm the importance of particle translocation in stimulating the fibrotic process and suggest that reducing the interstitial particle burden also reduces macrophage activation at that site. This may then lower the production of cytokines within the interstitium, resulting in a diminished fibrotic reaction. There is little information available on particle retention or clearance from an abnormal lung. There is evidence that fewer inhaled particles are retained in regions of the
lung where fibrosis has already developed, likely due to altered ventilation and reduced elastic recoil of those portions of the lung.19 In a study of particle clearance during acute pulmonary inflammation induced by parainfluenza virus, clearance was inhibited and more particles reached the interstitium.20 In this case however, the virus caused alveolar and bronchiolar epithelial necrosis, which removed the barrier to transport of particles directly to the interstitium; inhibited macrophage function by the virus may also have affected particle clearance.20 In the present study, any minor alveolar epithelial injury induced by silica would be repaired well within the 2-week period before injecting FMLP.2 The administration of a chemotactic agent to the lung with an interstitial particulate burden appears to be an effective way to cleanse the interstitium and may have clinical application. No long-term sequelae to the injection of FMLP were found in this study, and in a recent article, repeated instillation of the PMN chemoattractant C5a did not induce injury or lead to pulmonary fibrosis.21 There is some clinical evidence that an inflammatory response may induce particle clearance from human lung. In a classic article on studies of human anthracotic lungs at autopsy, Klotz noted that, in cases of industrially exposed workers who died of acute pneumonia, particles were present in interstitial regions but much less pigment was seen in areas of the lung showing an inflammatory reaction.22 Particles were seen in the exudate and in cells in lymphatic channels, leading him to conclude that "tsome of the pigment in the lung tissue became dislodged during the active migration of cells."22 We have used an experimental system to support this hypothesis and have shown that reducing the interstitial silica content is possible and is associated with amelioration of any subsequent fibrotic reaction.
References 1. Davis GS: Pathogenesis of silicosis: Current concepts and
hypotheses. Lung 1986,164:139-154 2. Adamson IYR, Bowden DH: Role of polymorphonuclear leukocytes in silica-induced pulmonary fibrosis. Am J Pathol 1984,117:37-43 3. Goldstein RH, Fine A: Fibrotic reactions in the lung: The activation of the lung fibroblast. Exp Lung Res 1986, 11: 245-261 4. Dubois CM, Bissonnette E, Rola-Pleszczynski M: Asbestos fibers and silica particles stimulate rat alveolar macrophages to release tumor necrosis factor. Am Rev Resp Dis 1989,139:1257-1264 5. Lugano EM, Dauber JH, Elias JA, Bosley RI, Jimenez SA, Daniele RP: The regulation of lung fibroblast proliferation by alveolar macrophages in experimental silicosis. Am Rev Resp Dis 1984,129:767-771
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6. Piguet PF, Collart MA, Grau GE, Sappino AP, Vassalli P: Requirement of tumor necrosis factor for development of silica-induced pulmonary fibrosis. Nature 1990, 344:245247 7. Adamson IYR, Letourneau HL, Bowden DH: Enhanced macrophage-fibroblast interactions in the pulmonary interstitium increases fibrosis after silica injection to monocytedepleted mice. Am J Pathol 1989,134:411-418 8. Adamson IYR, Letourneau HL, Bowden DH: Comparison of alveolar and interstitial macrophages in fibroblast stimulation after silica and long or short asbestos. Lab Invest 1991, 64:339-344 9. Adamson IYR, Bowden DH: Chemotactic and mitogenic components of the alveolar macrophage response to particles and neutrophil chemoattractant. Am J Pathol 1982, 109:71-7 10. Bossman HB, Lockwood T, Morgan HR: Surface biochemical changes accompanying primary infection with Rous sarcoma virus: II. Proteolytic and glycosidase activity and sublethal autolysis. Exp Cell Res 1974, 83:25-30 11. Burton K: A study of the conditions and mechanisms of the diphenylamine reaction to the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956, 62:315-320 12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951, 193:265-269 13. Woessner JF: Determination of hydroxyproline in connective tissue. Methodology of Connective Tissue Research. Edited by DA Hall. Oxford, Joynson and Bruwers, 1976, pp 227233
14. Adamson IYR, Bowden DH: Pulmonary reaction to long and short asbestos fibers is independent of fibroblast growth factor production by alveolar macrophages. Am J Pathol 1990, 137:523-529 15. Oberdorster G, Ferin J, Gelein R, Soderholm S, Cox C, Baggs R, Finkelstein J: Inhaled ultrafine particles: evidence of their increased pulmonary toxicity. Am Rev Resp Dis 1991, 143:A700 16. Ferin J, Feldstein ML: Pulmonary clearance and hilar lymph node content in rats after particle exposure. Environ Res 1978,16:342-348 17. Bowden DH, Adamson IYR: Role of monocytes and interstitial cells in the generation of alveolar macrophages: Kinetic studies of normal mice. Lab Invest 1980, 42:511-517 18. Sorokin SP, Hoyt RF, Jr.: Pure population of non-monocyte derived macrophages arising in organ cultures of embryonic rat lungs. Anat Rec 1987, 217:35-52 19. Sweeney TD, Brown JD, Tryka AF, Godleski JJ: Retention of inhaled particles in hamsters with pulmonary fibrosis. Am Rev Resp Dis 1983,128:138-143 20. Slauson DO, Lay JC, Castleman WL, Neilsen NR: Acute inflammatory lung injury retards pulmonary particle clearance. Inflammation 1989, 13:185-199 21. Harris JA, Hyde DM, Wang Q, Stovall MY, Giri SN: Repeated episodes of C5a-induced neutrophil influx do not result in pulmonary fibrosis. Inflammation 1991, 15:233-250 22. Klotz 0: Pulmonary anthracosis: A community disease. Am J Publ Health 1914, 4:887-916
Instillation of Chemotactic Factor to Silica-injected Lungs Lowers Interstitial Particle Content and Reduces Pulmonary Fibrosis 1. Y. R. Adamson, H. Prieditis, and D. H. Bowden From the Department of Pathology, University of Manitoba, Winnipeg, Canada
Silica-induced pulmonary fibrosis usually follows exposure to increased levels of this particulate and its retention in interstitial macrophages of the lung It is suggested that accelerated clearance ofparticles from the pulmonary interstitium may ameliorate subsequentfibrosis. To test this bypothesis, one group of mice received 2-mg intratracheal (IT) silica, some particles were phagocytized and cleared during the subsequent inflammatory response, other particles were translocated across the epithelium to reach interstitial macrophages by 2 weeks. These mice later showed increased fibroblast growth, a doubling of lung collagen levels and large silicotic nodules by 16 weeks when much of the silica was stillpresent in the lung. A second group of mice received IT silica, then 2 and 3 weeks later received IT injections of N-formyl-L-methionyl-leucyl-phenylalanine (FMLP), a leukocyte chemoattractant. Subsequently, a significant increase in inflammatory cells was seen and silica was observed mostly in phagocytes within the alveolar spaces. Few interstitial particles were found at 4 weeks; and extensive fibrosis did not develop by 16 weeks; only a few small nodules were seen and little silica was present in the lung, The results indicate that clearance of interstitial particles by a controlled inflammatory response is possible, and that removal of silica from the interstitium decreases the fibrotic response. (Am JPathol 1992, 141:319-326)
Exposure of the lung to an increased dose of silica particles is associated with the development of pulmonary fibrosis.13 Activation of macrophages is believed to be a key event in this process and various growth factors for fibroblasts have been identified in secretions of isolated pulmonary macrophages.3 Most of these studies have used alveolar macrophages (AM) lavaged from fibrotic
lungs, whereas in vivo, the fibrotic process occurs within the interstitium that also contains a population of macrophages. Previous studies from our laboratory indicate that penetration of particles into lung tissue and their retention by interstitial macrophages results in direct stimulation of the adjacent fibroblast population.78 At increased dose levels, particles such as silica cross the epithelium to reach the interstitium where clearance appears to be slow. Most particles are phagocytized by macrophages, some of which may migrate to the alveoli or to lymph nodes; other particles remain in the interstitium and may be trapped by the fibrotic response as granulomas form. We hypothesize that clearance of interstitial particles at an early stage would reduce macrophage activation within the interstitium and so reduce any subsequent pulmonary fibrosis. In this study, mice were injected intratracheally first with silica, and later with 2 doses of a leukocyte chemoattractant in an attempt to increase the phagocytic cell traffic across the pulmonary interstitium and clear any translocated silica. The inflammatory response, cell kinetics, hydroxyproline production, and retained silica were compared in the various treatment groups over the subsequent 16 weeks.
Materials and Methods A group of 50 Swiss-Webster mice (25-g males) received an intratracheal (IT) injection of 2-mg silica (crystalline quartz, diameter 0.3 ± 0.1 ,um, from Dowson and Dobson, South Africa) in 0.1 ml sterile water while under mild nembutal anesthesia.2 After 2 weeks and 3 weeks, these animals were given IT injections of 100 ,ug of N-formyl-Lmethionyl-leucyl-phenylalanine (FMLP) in 0.1 ml albumin.9 Other groups of mice received silica only, FMLP only, or had no treatment. The mice in groups of four were killed at day 0 then at weeks 1, 2, 3, 4, 6, 8, 12, and 16 from the time of the silica injection. Each mouse received Accepted for publication January 31, 1992. Address reprint requests to Dr. I. Adamson, Department of Pathology, University of Manitoba, 236-770 Bannatyne Avenue, Winnipeg, MB, Canada, R3E OW3.
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2 ,uCi/g tritiated thymidine by intraperitoneal injection 1 hour before death. The animals were killed by barbiturate injection, tracheotomies were performed, and the tracheas were cannulated. The lungs were washed four times in situ with 1.0 ml of saline. The lavage fluid was pooled for each animal, and the total number of cells was counted on a hemocytometer. The cell suspension was centrifuged, and a cytospin preparation was made and stained by the Giemsa method; differential counts of polymorphonuclear leukocytes (PMN) and AM were made on 500 cells per slide. The total number of cells of each type was calculated for each time studied, and the mean ± SE of the total cells per lung in the four mice per group was calculated. The supernatant of the lavage fluid from each mouse was used to determine total protein content and the level of glucosaminidase.10 After lavage, the bronchus leading to the right lung was clamped; this lung was removed, weighed, and frozen for biochemical analysis. The left lung was inflated with 0.5 ml of 2% buffered glutaraldehyde and removed; most of the tissue was processed for embedding in glycol methacrylate. Sections (0.75 ,um thick) from three random blocks per animal were prepared for autoradiography with the use of Kodak NTB2 emulsion. We determined the percentages of 3H-thymidine-labeled cells at each timepoint by counting 3000 lung cells per animal. The means and standard error were calculated for each group. The plastic sections were thin enough to allow identification of pulmonary cell types, and differential counts of labeled cells were performed on 300 labeled cells per animal. The product of the differential percentage and the total labeling percentage gave the labeling index for each cell type. The index for epithelial, interstitial, and endothelial cells was calculated at each time studied. A small sample of tissue was also processed for electron microscopy. The right lung of each mouse was homogenized in water and biochemical assays were performed on duplicate samples. Determinations of DNA and total protein were performed by conventional methods.11 12 As an index of collagen content, hydroxyproline (Hyp) levels were determined after hydrolysis with hydrochloric acid.13 To obtain an estimate of silica retention in the lungs, a residue after tissue digestion was prepared. At 16 weeks, six extra mice from each group were killed, and the lungs removed immediately.7 These were chopped up and incubated in 40% potassium hydroxide (KOH) overnight in a 800C waterbath. When the tissue was completely digested, the solution was centrifuged at 1500 rpm for 15 minutes, and a residue was obtained. This was washed twice in distilled water, resuspended in water, and transferred to a weighed tube. The residue was dried to con-
stant weight. Mean + standard error was calculated for each group of six and the significance of any difference from control mice was determined using the Student's t-test.
Results Bronchoalveolar Lavage (BAL) The number of AM recovered by lavage increased sharply in silica-injected mice (Figure 1). Although the value decreased after 1 week, it remained above normal through most of the experimental period. The injection of FMLP alone, which is known to induce a rapid inflammatory response,9 produced a small increase in AM recovered at 3 weeks. When FMLP was injected to mice previously injected with silica, there was a significant increase in AM recovered by BAL from 3 to 12 weeks (Figure 1). A similar pattern was seen when the PMN response was quantitated. FMLP alone induced a brief efflux of PMN while silica alone induced an increased and prolonged PMN response (Figure 2). However, when FMLP was injected IT to silica-bearing mice as their PMN response was subsiding, an additional and significant increase in lavaged PMN was found during a subsequent 8-week period. Silica particles were identified in both AM and PMN in cytospin preparations. As a marker of lung injury, particularly a permeability change at the air-blood barrier, protein levels were measured in BAL fluid. Normal values were low at around 50 ,ug/ml, and the FMLP injection caused a brief doubling (Figure 3). Silica instillation induced an increased protein level that decreased slowly but never reached the control value. The injection of FMLP to silica-bearing mice caused a subsequent significant increase in BAL protein for a 6-week period. Levels of glucosaminidase followed Injection
160,-120-
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40
0
2
4
6 8 WEEKS AFTER SILICA
12
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Figure 1. Numbers (mean ± SE) of alveolar macrophages (AM) recovered by lavage at intervals after silica instillation. FMLP injection times are shown by arrows. Crosses, FMLP alone; closed circles, silica only; open circles, silica followed by FMLP. *Silica + FMLP > silica only, P < 0.01. (Valuesfor nontreated controls were identical to time zero values).
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a similar pattern with silica alone inducing a brief increased level of enzyme activity that fell after 1 week, but never returned to control values (Figure 4). This proteolytic enzyme is associated with degranulation of PMN and is known to be elevated during silica-induced lung injury.2 However the injection of FMLP to silica-bearing mice caused only a temporary increase in the enzyme level over that seen in the silica-only group.
Lung Morphology
Figure 4. Glucosaminidase levels in bronchoalveolar lavagefluid in each group. Symbols as in Figure 1.
weeks large fibrotic granulomas were found (Figure 7), resembling those we have described previously for silica-induced fibrosis in mice.2'7 When animals that received silica were injected with FMLP after 2 weeks, a secondary inflammatory response was seen and at 4 weeks many alveoli were filled with inflammatory cells containing particles (Figure 8). The alveolar walls appeared thin and less cellular than after silica alone, and by electron microscopy, most of the silica present in the lung was in phagosomes of free AM and PMN (Figure 9); few particles were seen in the interstitium. In later weeks, as the AM and PMN became
Mice that received FMLP showed only a mild inflammatory exudate at 1 and 2 weeks after injection. The lung structure appeared normal at all other times. Animals that received silica only showed a large number of AM and PMN in the alveoli at 1 week. Silica particles were seen in these cells as well as free in the alveolar spaces. By 2 weeks, particles were seen in AM and in macrophages within the pulmonary interstitium, which was more cellular than normal (Figure 5). At 4 weeks, the interstitial tissue contained mixed macrophages and fibroblasts particularly in association with silica particles (Figure 6). Subsequently the fibrotic response progressed so that at 16 Injection
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Figure 3. Protein content of bronchoalveolar lavagefluid in each group. Symbols as in Figure 1.
Figure 5. Section of lung 2 weeks after silica alone; particles are interstitium, which is
seen in some macrophages (arrows) in the more cellular than normal, X900.
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Figure 6. Electron micrograph oJ lung 4 weeks ajter silica alone. Interstitial macrophages contain silica particles (arrows) are adjacent to cells resembling fibroblasts. A = alveolus, x 4,800
Figure 7 Section of lung 16 weeks after silica alone Large interstitial granulomas are formed and contain macrophages with silica among the fibrobas and collagen, X 450.
fewer, less silica was seen. The alveolar walls were mostly thin and resembled normal lung; only an occasional small granuloma was found in the silica-FMLP group at 16 weeks (Figure 10).
after silica alone. Interstitial labeling in particular returned to a low level and few labeled fibroblasts were seen in the
8-16 week period.
Lung Chemistry Autoradiography Injection of FMLP resulted in only a brief increase in DNA synthesis in the lung and this was confined to the interstitial cell population (Figure 1 1). Silica instillation resulted in approximately a six-fold increase in thymidine-labeled cells by 1 week, then this level declined to 16 weeks but was always greater than controls. From differential counts of labeled cells, the major contributing factor was increased proliferation in the interstitial compartment. Labeled cells in the first 4 weeks morphologically resembled interstitial macrophages (Figure 12); labeled AM were rarely seen. After 4 weeks, labeled interstitial cells were mainly identified as fibroblasts. Animals that received both silica and FMLP showed a further increase in labeled lung cells to 6 weeks as compared with the silica only group (Figure 11). The increase in labeled cells was confined to the interstitial cell population and mostly resembled macrophages similar to these shown in Figure 12. After 6 weeks, cell labeling decreased considerably to a level less than that seen
Fibrosis was quantitated by measuring total Hyp per right lobe. In control (nontreated) mice, this value increased slowly with age, and these levels were not different in animals that received FMLP only (Figure 13). After silica only, tissue Hyp was significantly higher than normal beginning at 4 weeks, and fibrosis increased progressively over the 16-week period. In mice that received FMLP after silica, the tissue Hyp rose slightly by 4 weeks but then remained significantly lower than the increased level found in animals that received silica alone. Retention of silica in the lung at the end of the 1 6-week period was estimated by quantitating the tissue residue after KOH digestion. A light-brown residue weighing around 0.2 mg was obtained from control and FMLPtreated mice (Figure 14). This was taken as the nonparticulate, baseline organic residue. In mice that received 2 mg of silica, the residue appeared gray, particulate, and weighed about 0.7 mg. However, in the group that received FMLP after silica treatment, most of the silica had been cleared from the lung and the weight of a pale
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Figure 8 Section oflung 4 weeks after silica with FMLP injections at weeks 2 and 3. The alveoli are filled with large AM and small PMN (arrows), the interstitium appears near normal, x800.
Figure 9. Electron micrograph of lung shown in Figure 8. Dense silica particles are present in both AM and in PMN (arrows), the alveolar walls are thin and contain no particles, x4,800.
residue was significantly lower than that of the silica only group.
monary response to asbestos,814 and to other particles particularly in an overload situation in which more particles are translocated to the interstitium.15,16 In this study, the pulmonary reaction to silica alone followed the pattern described previously. There was a rapid response of PMN and AM with accompanying increases in protein leakage to alveoli and in the BAL content of lysosomal enzymes such as glucosaminidase. Some silica particles crossed the Type 1 epithelium and were phagocytized by interstitial macrophages. In the later stages, fibroblast proliferation and collagen deposition occurred at sites of silica retention producing large interstitial nodules. An increased level of silica was trapped in these areas and was quantitated in the lung residue at 16 weeks. The relationship between the retention of interstitial silica and the generation of localized regions of fibrosis led to the hypothesis that early clearance of silica from the interstitium might reduce macrophage activation at that site thereby reducing the subsequent fibrotic reaction. Interstitial macrophages may be cleared from the lung by migration to lymphatic channels or to the alveoli. Particle clearance to the lymphatics has been quantitated as a small percentage of the total load,16 but clearance from interstitium to alveoli is more difficult to measure. Interstitial macrophages have been shown to migrate to the alveoli in normal adult lung,17 developing lung,18 and in
Discussion Small quantities of particles deposited in the lung are mostly phagocytized and cleared by alveolar macrophages. As the number of particles is increased, an inflammatory response occurs and the efflux of PMN with additional macrophages enhances clearance. In the case of silica, particle interaction with AM is known to produce a variety of fibroblast growth factors. This has been shown in vitro and in vivo using AM lavaged from fibrotic lungs.36 Although the resultant pulmonary fibrosis is interstitial, most studies have concentrated on factors produced by the readily obtainable AM,5'6 which is believed to mirror the function of the interstitial macrophage. However, in a series of in vivo experiments, we demonstrated that pulmonary fibrosis is increased when the translocation of silica from the alveoli to the interstitium is enhanced.7 This suggests that direct macrophagefibroblast interaction within the pulmonary interstitium may be more effective in transferring a fibroblast growth factor than a process involving secretion by AM into the alveolar space. The role of the interstitial macrophage in promoting fibrosis has also been emphasized in the pul-
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Figure 10. Section of lung 16 weeks after silica with FMLP injections at weeks 2 and 3. A small granuloma is seen but most of the lung tissue appears normal, x 450.
Figure 12. Autoradiograph of lung section 2 weeks after silica only. Labeled interstitial cells (arrows) at this time resemble macrophages, x800.
particle-laden lungs.2'7 In an earlier experiment, we instilled chemotactic factor to mouse lung and demonstrated an early, large PMN efflux to the alveoli, followed by division and migration of interstitial mononuclear cells to the air sacs.9 The inflammatory response had subsided by 1 week with no injury to the lung9 and no subsequent fibrosis. We have used this same agent (FMLP) to induce an acute inflammatory response in a particleladen lung in an attempt to clear interstitial particles. This could occur by direct migration of interstitial macrophages containing silica, or through phagocytosis and clearance of free interstitial silica by PMN and monocytes
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12 8 16 4 6 WEEKS AFTER SILICA Figure 11. Percentage of thymidine-labeled lung cells (upper panel) and radiographic index (RI) of interstitial cells (lower panel) over the 16-week period. Solid circles, silica alone; crosses, FMLP alone; open circles, silica plus FMLP injected at times shown. (Nontreated controls equal to time zero values). *Silica + FMLP > silica only, P < 0.01. aSilica and FMLP < silica only, P < 0.01.
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each group. group were
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0.8
*
0.6
LUNG RESIDUE
(mg)
0.4
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while crossing the interstitium. In either case, the interstitial burden of particles would be reduced. Instilling FMLP to the lung was carried out 2 and 3 weeks after silica, when particles had been phagocytized by AM or translocated to the interstitium, and the inflammatory response was subsiding. The chemotactic agent promoted a supplementary inflammatory reaction with increased PMN, AM, and associated enzymes in BAL fluid. Thymidine uptake by interstitial mononuclear cells was also increased over the level found after silica alone. It appears that the response to FMLP induced both division and migration of interstitial phagocytes to the alveoli. The lungs of these mice showed an increased number of free alveolar cells containing silica, along with reduced cellularity of the pulmonary interstitium. From the morphologic evaluation, most particles seen in the silica plus FMLP group were found in the AM and PMN in the alveolar spaces up to 8 weeks, though increased migration of macrophages to regional lymph nodes could also have occurred. Although quantitative data on particle distribution in airspaces, lung tissue, and lymphatics were not obtained serially, enhanced clearance of interstitial silica was confirmed at the end of the experiment when the weight of lung residue in the silica-FMLP group was substantially lower than that measured from the silica-only animals. In addition, mice that received FMLP after silica showed lower fibroblast labeling and much lower lung Hyp content. Fibrosis was limited to a few small foci, whereas after silica alone, large confluent silicotic nodules and high Hyp levels were found. These combined results confirm the importance of particle translocation in stimulating the fibrotic process and suggest that reducing the interstitial particle burden also reduces macrophage activation at that site. This may then lower the production of cytokines within the interstitium, resulting in a diminished fibrotic reaction. There is little information available on particle retention or clearance from an abnormal lung. There is evidence that fewer inhaled particles are retained in regions of the
lung where fibrosis has already developed, likely due to altered ventilation and reduced elastic recoil of those portions of the lung.19 In a study of particle clearance during acute pulmonary inflammation induced by parainfluenza virus, clearance was inhibited and more particles reached the interstitium.20 In this case however, the virus caused alveolar and bronchiolar epithelial necrosis, which removed the barrier to transport of particles directly to the interstitium; inhibited macrophage function by the virus may also have affected particle clearance.20 In the present study, any minor alveolar epithelial injury induced by silica would be repaired well within the 2-week period before injecting FMLP.2 The administration of a chemotactic agent to the lung with an interstitial particulate burden appears to be an effective way to cleanse the interstitium and may have clinical application. No long-term sequelae to the injection of FMLP were found in this study, and in a recent article, repeated instillation of the PMN chemoattractant C5a did not induce injury or lead to pulmonary fibrosis.21 There is some clinical evidence that an inflammatory response may induce particle clearance from human lung. In a classic article on studies of human anthracotic lungs at autopsy, Klotz noted that, in cases of industrially exposed workers who died of acute pneumonia, particles were present in interstitial regions but much less pigment was seen in areas of the lung showing an inflammatory reaction.22 Particles were seen in the exudate and in cells in lymphatic channels, leading him to conclude that "tsome of the pigment in the lung tissue became dislodged during the active migration of cells."22 We have used an experimental system to support this hypothesis and have shown that reducing the interstitial silica content is possible and is associated with amelioration of any subsequent fibrotic reaction.
References 1. Davis GS: Pathogenesis of silicosis: Current concepts and
hypotheses. Lung 1986,164:139-154 2. Adamson IYR, Bowden DH: Role of polymorphonuclear leukocytes in silica-induced pulmonary fibrosis. Am J Pathol 1984,117:37-43 3. Goldstein RH, Fine A: Fibrotic reactions in the lung: The activation of the lung fibroblast. Exp Lung Res 1986, 11: 245-261 4. Dubois CM, Bissonnette E, Rola-Pleszczynski M: Asbestos fibers and silica particles stimulate rat alveolar macrophages to release tumor necrosis factor. Am Rev Resp Dis 1989,139:1257-1264 5. Lugano EM, Dauber JH, Elias JA, Bosley RI, Jimenez SA, Daniele RP: The regulation of lung fibroblast proliferation by alveolar macrophages in experimental silicosis. Am Rev Resp Dis 1984,129:767-771
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6. Piguet PF, Collart MA, Grau GE, Sappino AP, Vassalli P: Requirement of tumor necrosis factor for development of silica-induced pulmonary fibrosis. Nature 1990, 344:245247 7. Adamson IYR, Letourneau HL, Bowden DH: Enhanced macrophage-fibroblast interactions in the pulmonary interstitium increases fibrosis after silica injection to monocytedepleted mice. Am J Pathol 1989,134:411-418 8. Adamson IYR, Letourneau HL, Bowden DH: Comparison of alveolar and interstitial macrophages in fibroblast stimulation after silica and long or short asbestos. Lab Invest 1991, 64:339-344 9. Adamson IYR, Bowden DH: Chemotactic and mitogenic components of the alveolar macrophage response to particles and neutrophil chemoattractant. Am J Pathol 1982, 109:71-7 10. Bossman HB, Lockwood T, Morgan HR: Surface biochemical changes accompanying primary infection with Rous sarcoma virus: II. Proteolytic and glycosidase activity and sublethal autolysis. Exp Cell Res 1974, 83:25-30 11. Burton K: A study of the conditions and mechanisms of the diphenylamine reaction to the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956, 62:315-320 12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951, 193:265-269 13. Woessner JF: Determination of hydroxyproline in connective tissue. Methodology of Connective Tissue Research. Edited by DA Hall. Oxford, Joynson and Bruwers, 1976, pp 227233
14. Adamson IYR, Bowden DH: Pulmonary reaction to long and short asbestos fibers is independent of fibroblast growth factor production by alveolar macrophages. Am J Pathol 1990, 137:523-529 15. Oberdorster G, Ferin J, Gelein R, Soderholm S, Cox C, Baggs R, Finkelstein J: Inhaled ultrafine particles: evidence of their increased pulmonary toxicity. Am Rev Resp Dis 1991, 143:A700 16. Ferin J, Feldstein ML: Pulmonary clearance and hilar lymph node content in rats after particle exposure. Environ Res 1978,16:342-348 17. Bowden DH, Adamson IYR: Role of monocytes and interstitial cells in the generation of alveolar macrophages: Kinetic studies of normal mice. Lab Invest 1980, 42:511-517 18. Sorokin SP, Hoyt RF, Jr.: Pure population of non-monocyte derived macrophages arising in organ cultures of embryonic rat lungs. Anat Rec 1987, 217:35-52 19. Sweeney TD, Brown JD, Tryka AF, Godleski JJ: Retention of inhaled particles in hamsters with pulmonary fibrosis. Am Rev Resp Dis 1983,128:138-143 20. Slauson DO, Lay JC, Castleman WL, Neilsen NR: Acute inflammatory lung injury retards pulmonary particle clearance. Inflammation 1989, 13:185-199 21. Harris JA, Hyde DM, Wang Q, Stovall MY, Giri SN: Repeated episodes of C5a-induced neutrophil influx do not result in pulmonary fibrosis. Inflammation 1991, 15:233-250 22. Klotz 0: Pulmonary anthracosis: A community disease. Am J Publ Health 1914, 4:887-916
