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(LuJ Reprinted from the Archivee of Pathology April 1910, Vol. 09 Copyright 1970, by American Medical Auociolion i'lfOPtltari, ^ubUphtd JlLjf'f United Stotec of America 1/ r<Ferruginous Bodies*^ in Guinea Triigi s Fine Structure Produced Experiment&llyJProm Minerals Other Than Asbestos John M. C. 7>nr|i, PhPfCambridgr, England; and Paul Crou, MD, ----------- -- and Robert T, P. De Trtville, MD, Pittsburgh Electronmicroscope studies of ''ferruginous" bodies, produced in guinea pigs in response to fine glass fiber end ceramic aluminum silicate, have shown that the fine structure of these bodies is identical to that of asbestos bodies. All these bodies are produced intrecellulerly, mainly in giant cells. Their coating consists of small dense granules, approximately 60 Ang stroms in diameter, that probably represent ferritin or hemosiderin. This coating may be laid down as a single dense layer or as a series of variable layers. Usually all bodies are sepa rated from the giant cell cytoplasm by a dis tinct membrane, but in some of the older bodies this membrane is no longer present. ASBESTOS bodies were probably first observed by Fahr and Feigel in 1914,1 but they were not immediately associated with asbestos exposure. Fahr and Feigel called them strange crystals, and later in 1924, Cooke2 suggested that because of their beaded nature they might be fungi. Stew art and Haddow* realized that they were an integral part of the disease. They therefore coined the term "asbestosis body," but it was later understood that the number of bodies present was not directly related to the degree of lung fibrosis. For this reason the bodies became looked upon Accepted for publication Oct 17, 1069 From the Department of Pathology, Cambridge University, Cambridge, England (Dr. Davis), and the Industrial Hygiene Foundation of America, Inc.. Pittsburgh (Drv Cross and De Trevllle). Dr. Davis ts member of the British Asbestosis Re search Council. Reprint requests to Department of Pathology, Cambridge University, Tennis Court Rd, Cam bridge, England (Dr. Davis). solely as an indication of asbestos expo sure. It was felt that they were produced in response to this mineral alone, and the term asbestosis body was changed to as bestos body. Gloyne, in 1932,4 demonstrat ed that each asbestos body contained a central core of asbestos dust surrounded by a coating which he thought contained iron and some protein material. The iron protein nature of the capsule was con- finned by Beger2 and Sundius and Bygden. After the 1920's the industrial hazards of asbestos exposure were well known and resulted in most countries in strict hygienic regulations for factories and mines. It was believed, however, that the use of products containing asbestos was not dangerous to the general public and did not result in the liberation of harmful asbestos dust. This idea was repudiated by the find ings of Thomson et al in 1963,5 who re ported that structures very similar to asbestos bodies were present in the lungs of 30% of unselected autopsy cases from Cape Town. It seemed, therefore, that as bestos might be becoming a general urban hazard and studies were undertaken in other cities. These confirmed Thomson's results and bodies were found in 30% of autopsies from Miami, 43% from Pitts burgh, and 48% from Montreal.10 A more recent study in Pittsburgh 11 has found bodies in nearly 100% of autopsy cases. In all these reports, however, only a few bodies were found in each case and they appeared to result in no pathological changes in the lungs. ___________ Arch Path--Vol 89, April 1970 PI-AINTIFF'S EXHIBIT FD-14S2 SCF-FA-6400 S005 1448 PRODUCED BY FORD "FERRUGINOUS BODIES" IN GUINEA PIGS--DAVIS ET AL MS Thomson had assumed that all the bod ies found in his cases were caused by asbestos, but anthracosis bodies have been known for a long time from the lungs of coal miners. Gloyne, in 1949,'* reported bodies similar to asbestos bodies in the lungs of workers exposed to graphite. This indicated that the process of body forma tion was not limited to asbestos, although, since the coal and graphite bodies had dark central cores, it was thought that they could not be confused with genuine asbestos bodies. In 1968, however, Gross et al15 reported that structures indistin guishable from asbestos bodies could be formed in experimental animals by the injection of such materials as aluminum silicate needles, silicon carbide whiskers, and fine glass fiber. Because all the bodies contained iron, it was suggested that they and asbestos bodies should be given the general name of "ferruginous bodies." Since minerals of similar size range to those used in these experiments are ubiquitous, it now seems very likely that many of the bodies seen in the lungs of the normal urban population result from minerals other than asbestos. The deposition of a coating containing iron around a variety of different min eral fibers indicates that a very general process is involved, and it is of great im portance, therefore, to elucidate the bio chemical conditions required for body formation. As a first step, an electron microscope study of a variety of experi mentally produced ferruginous bodies was undertaken to see if all these bodies have the same ultrastructure, and whether this is exactly the same as asbestos bodies. Asbestos bodies from guinea pigs and one human had previously been examined by Davis in 1965," ,s and it was reported that these structures were always formed in tracellularly within macrophages or giant cells. The body coating consisted of small granules approximately 60 A in diameter and it was suggested that these might be ferritin. The body coating was sometimes deposited as a single layer but could con sist of a number of layers of variable thickness and density. Sometimes the outermost layer consisted of radially ar ranged fine filaments about 60 A in di ameter, and it was suggested that these represented some form of calcium deposit. It has now been shown that the fine struc ture of apatite crystals is very similar to these asbestos body filaments, and it now seems likely that the outer layer of some asbestos bodies is made up of this material. In the experimental studies of ferrugi nous bodies, the following minerals were used: ceramic aluminum silicate, silicon carbide whiskers, and fine glass fiber. In addition to this, a commercial prepara tion of elastin was also tested to check on the structural similarity between elastosis bodies and other ferruginous bodies. Materials and Methods In the first experiments, mineral samples were Injected intratracheaUy into hamsters as reported by Gross et a) " in 19C8. It was found, however, that although the resulting ferruginous bodies were common enough to be easily found with the light microscope, they were too dispersed to be found with the electron microscope except on rare occasions. It was known, however (Davis, unpublished data), that the intrapleural injec tions of asbestos dust into guinea pigs resulted in the production of large granulomas which contained many asbestos bodies after as little as two weeks. For this reason it was decided to use the intrapleural technique with other minerals. For these experiments. 25 mg of test material was suspended in 1 ml of physiological saline and injected into the right lower thoracic cavity of guinea pigs. The animals were killed between two and six weeks after injection and the result ing granulomas were divided into two segments. One was fixed in formol saline for light micro scope examination and the other was fixed in buffered osmium tetroxide for electron micro scope study. The light microscope sections were stained by Perl's method for iron. This stains the coating material of the ferruginous bodies a dense blue, making them easily visible against the back ground of a pale, neutral red counterstain. The material for electron microscopy was embedded in epoxy resin and stained with lead citrate. The injected materials were as follows: 1. Glass fiber, a commercial sample with an average fiber diameter of 0.05m to 0.99m. Initially Arch Path--Vol 89, April 1910 8005 1449 PRODUCED BY FORD *M "FERRUGINOUS BODIES" IN GUINEA FIGS--DAVIS ET AL supplied u a felt, this was finely ground before injection. 2. Aluminum silicate, an uncoated ceramic fiber with a median diameter of 2m. Fifty percent of the fibers were under 75m in length and many of the fibers were beginning to stain blue. In this case, however, the eiastin fibers were much larger than asbestos bodies and could not be confused with them. filaments were shorter than 15m. No free silica was detected in the fibers. S. Silicon carbide whiskers, 995% silicon car* bide. Fiber diameter ranged from 0.5m to 3m, and fiber length ranged from 100m to 750m. 4. Eiastin, a commercial sample consisting of relatively short thick fibers with diameters of 5m Electron miscroscope examination re vealed that the cellular response to fine glass fiber and aluminum silicate was ex actly similar to that induced by asbestos dust. Although the glass fiber used had an average diameter of 0.05p to 0.99y, much to 15m and lengths from 50m to 200m. of the dust was thinner than this and some Observations of it was as small as 250 A in diameter. These particles were, therefore, almost as When the animals were examined six weeks after injection, it was found that all test materials had caused the formation of large granulomas. These were fairly evenly distributed over both visceral and parietal pleura and the diaphragm. The histological patterns of these granulomas varied, however, with the different ma terials. Granulomas produced in response to glass or aluminum silicate were very cellular and consisted mainly of giant cells mixed with free macrophages and some fibroblasts. There was some deposition of reticulin fibers in these lesions from about two weeks after injection, but collagen was limited to a capsule around each granuloma. In the case of eiastin, however, although the foreign material was quickly encased in b thin collagenous capsule, the cellular response around the eiastin fibers was poor. In some areas, macrophages and small as crystals of chrysotile asbestos. The aluminum silicate dust was much coarser and very little of it was below 0.5y in diameter. AU sizes of dust were taken up into macrophage or giant cell phagosomes, and these quickly contracted either to sur round the dust with a closely apposed membrane or to form dust containing sec ondary lysosomes, as previously reported for asbestos dust by one of us (J.M.G.D.. 1967). As with asbestos dust, it was found that the coating of dust to form ferrugi nous bodies was the exception rather than the rule, and it would appear that well below 1% of dust was ever involved in this process. Ferruginous bodies were al ways intracellular, usually in giant cells, and the deposition of their coating ap peared in all respects similar to the forma tion of asbestos bodies. fibroblasts infiltrated between the indi The body coating consisted of dense vidual eiastin fibers, but other areas re granules approximately 60 A in diameter mained quite acellular. which probably represent ferritin (Fig 4 Perl's stain revealed that with both to 6). Most frequently the granules were glass fiber and aluminum silicate, some of deposited in a single dense layer up to 5ii the fibers were coated with iron-contain thick (Fig 7), but occasionally they were ing material as little as two weeks after arranged in layers of uneven thickness injection. By six weeks many ferruginous and density. In many cases there was bodies, some of them beaded, were pres a distinct membrane between the ferrugi ent in the granuloma, and they appeared nous body Bnd the surrounding cell cyto in all respects similar to asbestos bodies plasm (Fig 6), but this was not always produced in guinea pigs by the intra present (Fig 4). It can be seen in Fig 4 pleural injection of chrysotile asbestos that the good preservation of many cyto (Fig 1 to 3). In the lesions produced by plasmic membrane structures in close eiastin, Perl's staining appeared negative proximity to the ferruginous body pre after two weeks, but by six weeks some cludes the possibility that a surrounding Arch Poth-Vol 89, April 1910 8005 1450 PRODUCED BY FORD membrane has been destroyed during fix* ation. In the case of the elastin lesions the ferruginous material still consisted of small ferritin-like granules approximately 60 A in diameter, but in this case the granules were not deposited as a coat around the elastin fiber but penetrated into the matrix of the fiber itself. This impregnation was found most frequently in areas where the masses of elastin fibers were well infiltrated with macrophages and fibroblasts, but giant cells were not found, and only rarely were the elastin fibers small enough to be phagocytosed by a single macrophage. The formation of elastosis bodies would appear, therefore, to be an extracellular process in contrast to other types of ferruginous body. The earliest sites of granule deposition were usually found in areas where the elastin fibers were closely opposed to one an other. and the areas of impregnation ap peared to extend inwards from adjacent points on the surfaces of both fibers to form hemispherical masses. The greatest density of granular deposition was usually Fig l^ksbestos bodies produced in guinea pig pieu* rat cavity six weeks after injection of chrysotiie asbes tos dust. This Illustration is presented for comparison with Fig 2 end 9 (Peri s stein, reduced from x 000). Fig 2.--Ferruginous bodies produced in guinea pig pleural cavity six weeks after injection of glass liber (Perl's stain, reduced from x 000). Fig 3.--Ferruginous body produced in guinea pig pleural cavity six weeks sMer injection of ceramic aluminum silicate fibers (Perl's stain, reduced from x 000). 1 I j II i 8005 1451 i i I i i PRODUCED BY FORD Fig 4--Surface of ferruginous body formed around glass fiber. Body Is lying in cytoplasm of giant cell at point where two macrophages have not com pletely fused and where interdlgltad phagocytic pro cesses are still visible. Although surface membranes of these processes are excellently preserved, there is no clear membrene between body and rest of cy toplasm. It can be seen that body coating consists largely of small dense granules approximately 60 A in diameter (arrows) (x 110.000). Fig Ferruginous body formed in response to glass fiber in cytoplasm of giant cell. This body is surrounded by very distinct membrane (arrows) lx 7!.000). 8005 1452 PRODUCED BY FORD 'FERRUGINOUS BODIES" IN GUINEA FIGS--DAVIS ET AL StS found near the surface of the elastin fiber and became progressively less further in. In many cases the innermost layers of the area of deposition consisted not of gran* ules, but a radially arranged array of fine filaments about 60 A in diameter (Fig 6). The size of impregnated areas found in elastin fibers varied considerably, and in some cases the whole fiber was involved (Fig 8). It therefore seems logical to sug gest that the impregnation is a gradual process which proceeds until the whole elastin fiber is saturated with the ferrugi nous materials. Where the impregnation has involved a complete fiber, the 60-A granules extend throughout and no layer of fine filaments is present. It must be emphasized that by no means all elastin fibers in any area are impregnated within the duration of this experiment, and com pletely unchanged fibers can often be found adjacent to others in which impreg nation is complete (Fig 8). After it was discovered that the formation of elastosis bodies was by a process of impregnation, a control experiment was undertaken to Fig 6.--Area of impregnation in elastin fiber. Center of impregnated area consists of small dense granules approximately go A in diameter, while outer layer consists of fine . filaments of similar diameter that probably represent apatite crystals (x 74.000). I 8005 1453 PRODUCED BY FORD *70 `FERRUGINOUS BODIES" IN GUINEA PIGS--DAVIS ET AL make sure the initial sample of elastin was not impregnated in any way. For this purpose, elastin fibers from the commer cial samples were embedded directly in epoxy resin and examined in the electron microscope. No areas of impregnation were found. In a few cases the elastin fibers used in this experiment were small enough to be phagocytosed, and when this occurred it was sometimes found that the fiber was separated from the majority of the cyto plasmic organelles by a pale area showing a fine granulation (Fig 9). It is possible Fig 7.--Ferruginous body produced in response to gloss fiber lying in cytoplasm ol giant cell. The cytoplasm around dense "ferritin" of body Is devoid of cytoplasmic organelles and probably corresponds to pseudopodial ectoplasm. There ere hints of limiting membrane between body coaling end this pseudopodial material, but this is not very distinct (X 75.000). Arch Poth-Vol 89, April WO 8005 1454 PRODUCED BY FORD "FERRUGINOUS BODIES" IN GUINEA PIGS--DAVIS ET AL 371 that a membrane exists at the point of contact between the elastin fiber and this granular layer, but if so it must be very closely apposed to the fiber. It seems cer tain, however, that no membrane exists between the pale granular layer and the rest of the cell cytoplasm. Some of the ferruginous bodies from glass and alumi num silicate were also separated from the cytoplasmic organelles by a pale granu lar area (Fig 7). In this case a membrane was sometimes present between the body and the granular area, bat never betwen this area and the rest of the cytoplasm. These pale granular areas are very similar to the structure of pseudopodia of ameboid cells and were reported in lung macro phages by Collet and Reuet-Normand in 1967." To find that such areas occasionally remain for some time around large parti cles is perhaps not surprising. Comment The finding that the fine structure of ferruginous bodies formed from a number of different mineral fibers is identical to that of genuine asbestos bodies reempha sized the suggestion of Gross et al in 1967 that it is extremely difficult to distinguish Fig Area from center of meu of elastin fiben In guinea pig pleural cavity. There It alight Infiltration of fibroblatlt between elattin fibers end tome depotltion of collagen or retlculin. The variability of Impregnation of elastin fiben 1s ehown by feet thet while three fiben ere elmoit completely Impregnated end appear black in photograph, one large fiber (F) shows no signs of Impregnation at all (x 9,000). ArcH Poth-Vol 89, April 1970 8005 1455 PRODUCED BY FORD an "FERRUGINOUS BODIES IN guinea PIGS--DAVIS et al Fit t__Small claitln fiber that fid been prtgocyIpsed by a macro phage. Cell cytoplasm Immediately turrounding fiber it quite devoid ol cell organellet and probably corresponds to pseudopodial ectoplasm (x 40.0001. between any of these structures with the light microscope. Where a mineral fiber is large or pigmented, it may be possible to rule out asbestos as the core, but in many cases this cannot be done. This is especially true of the finest glass fiber which has similar dimensions to asbestos. For this reason electron microscope ex amination is the only reliable method of distinguishing between the various types of mineral core in a sample of ferruginous bodies, and even then differentiation is often only possible if an electron diffrac tion pattern can be obtained from the mineral. Because of these difficulties, it is at present impossible to critically evaluate the finding of ferruginous bodies in mem bers of the normal urban population. It may be that many of these are genuine asbestos bodies, but on the other hand Arch Patfc--VoJ 89, April 1970 8005 K56 PRODUCED BY FORD 'FERRUGINOUS BODIES" IN GUINEA PIGS-DAVIS ET Ah 373 other minerals may predominate and very few of these bodies be due to asbestos. A number of studies are at present being undertaken to recognize the mineral core of bodies found in unselected autopsies, but the examination of even one body is tedious and no definite results are yet available. The finding that the formation of elastin bodies is a process of impregnation rather than coating raises some interesting queries regarding the chemical processes involved in the formation of all types of ferruginous body. The impregnation of basement membranes by ferritin is a wellknown phenomenon,1 e and colloidal iron is used as a stain for acid mucopolysac charides.10-*0 It may be, therefore, that some iron-containing molecules have an affinity for certain proteins, and perhaps some mucopolysaccharides, and will se lectively impregnate aggregates of these materials. It has been known for a long time that asbestos body coating contained both iron and protein, but the discovery that granules very similar to ferritin con stituted the bulk of the coating material suggested that this iron-containing protein might make up the coating exclusively. One of us (J.M.G.D., 1965), however, showed that some layers of asbestos body contained little ferritin, and it may be that body formation involves initially the coat ing of the dust with a layer of protein or mucopolysaccharide containing no iron. Later, ferritin may impregnate this layer in a number of patterns to produce either single- or multiple-layered bodies. References 1. Fahr, and Feigel: Aerztlicher Verein In Ham burg, Mvnchen Med Wschr 61:625-626 (March) 1914. 2. Cooke, W.: Fibrosis of the Lungs Due to the Inhalation of Asbestos Dust, Brit Med J 2:147 (July) 1921. 3. Stewart. M.J., and HBddow, A.C.: Demonstra tion of the Peculiar Bodies of Pulmonary Asbestosis (Asbestosis Bodies) in Material Obtained by Lung Puncture and in the Sputum, J Path Baet 32:172 (Jan) 1929. 4. Gloyne, S.R.: The Asbestosis Body, Lancet 219: 1351-1353 (June) 1932 5. Beger, PJ.: Ober die Asbestosiskorperchen, Virchow Arch 290:280-333 (March) 1933. 6. Sundius, N., and Bygden. A.: Der Staubinhalt einer Asbestosislunge und die Beshaffenheit der sogenannten Asbestosis korperchen. Arch Geuierbepoth Gewerbehyp 8:26-34 (March) 1937. 7. Thomson, J.G.; Kaschula, R.O.C.; and Mac donald. R.R.: Asbestos as a Modem Urban Hazard, 5 Air Med J 37:77-81 (Jan) 1963. 8. Thomson, J.G.; Path, F.C.; and Graves, W.M.: Asbestos as an Urban Air Contaminant, Arch Path 81:438-461 (May) 1966. 9. Cauna. D.; Totten, R.S.: and Gross, P.: Asbestos Bodies in Human Lungs at Autopsy. JAMA 192:371 373 (May 3) 1965. 10. Anjilvel, L., and Thurlbeck, W.M.: The Inci dence of Asbestos Bodies in the Lungs at Random Necropsies in Montreal, Canod Med Atioc J 95: 1179-1182 (Dec) 1966. 11. Utidjian, M.D.; Gross, P.; and de Treville, R.T.P.: Ferruginous Bodies in Human Lungs, Arch Environ Health 17:327-333 (Sept) 1968. 12. Gloyne, S.R.; Marshall, G.; and Hoyle, C.: Pneumoconiosis Due to Graphite Dust, Thorax 4: 31-38 (March) 1949. 13. Gross, P., el al: Pulmonary Ferruginous Bodies: Development in Response to Filamentous Dusts and a Method of Isolation and Concentration, Arch Path 85:539-346 (May) 1968. 14. Davis. J.M.G.: The Ultrastructure of Asbestos Bodies From Guinea Pig Lungs, Brit J Exp Path 45:634-641 (Dec) 1964. 15. Davis, JM.G.: The Ultrastructure of Asbestos Bodies From Human Lungs, Brit J Exp Path 45: 642-646 (Dec) 1964. 16. Gough, J.: Differential Diagnosis in the Pathology of Asbestosis, Ann NY Acad Set 132: 368-372 (Sept) 1965. 17. Collet A., end Reuet-Normand, C.: Aspects Infra-structuraux de la Traversee de la Paroi Alveolaire du Poumon par des Cellules Migratriees. Sem Hop Paris 43:1928-1937 (June) 1967. 18. Farquar, M.G., and Paladc, GJ2.: Glomerular Permeability: II. Ferritin Transfer Across the Glomerular Capillary Wall in Nephrotic Rats. J Exp Med 114:699-716 (Nov) 1961. 19. Curran. R.C.: Lovell, D.; and Clark, A.E.: Mucopolysaccharides in Peritoneal Granulomas in the Rat. J Path Bact 91:429-439 (April) 1966. 20. Roy, S.. and GhBdiBlIy, F.N.: Synthesis of Hyaluronic Acid by Synovia] Cells, J Path Bact 93: 555-557 (April) 1967. Arch Path--Vol 89, April 1970 8005 1457 PRODUCED BY FORD