Document Ra3kNZwD1pDDvgb6o2VqGy9N8

379 the EFFECTS OF CiriiVSOTILH ASBESTOS DUST OX LUXO XAOROPIUUE.S UAIXTAIXED IX ORGAN CULTURE ` AX ELECTKOX-JflCROSCOPE STUDY J. M. G. DAVIS From the Department of Pathology, University of Cambridge Recoivcct for publication February 15, 1907 t lx a previous study (Davis. 19(17) a technique of organ culture was reported which allowed lung macrophages to he maintained for at. least. 9-1 (> days. This technique was developed in order to study in detail the uptake of asbestos dust by these macrophages, a thing that had been difficult to accomplish in vivo. Experiments with guinea-pigs (Davis, l9G3a. 6; Holt, Mills and Young. 1994) had. however, shown that very large amounts of dust could be taken up by individual ' lung macrophages which were able to combine to produce multiuucleate giantcells in only a few days. Later many macrophages became converted to libroblasts, the dust still remaining in the fibroblast cytoplasm. It was of great interest, therefore, to iind that in the organ culture normal undusted macrophages could undergo conversion to fibroblasts. The system was obviously well suited to the study of this process in dusted cells. MATERIALS .VXD METHODS The apparatus and methods of culture used previously to study normal lime tissue (Davis. 1967) were also used in this study of dusted macrophages. it was derided to use chrysotilc asbestos dust as this was known to cause few problems of embedding mid sect inning for electron-microscope examination, anti a sample of finely milled dust was oliliiiued from Dr. P. F. Holt of Reading University. This dust was similar to that used iu pievjous experiments (Holt rt <tl.. 19(14) and consisted mainly of fibres 5 jt or less in length. Initially it was found diflicult to introduce the dust to the cultures in a satisfactory maimer. If the dust was simply added to the culture medium none of it penetrated into the lung at all. Even simple injection of a suspension of dust iu culture lluid was unsatisfactory as most of the dust was very quickly squeezed out of the cut surface of the hint;. Finally it was found necessary to inject the dust into the tip of n small lung lobe and then tiu a suture itt the injection site to retain the dust. The final method adopted was as follows. Chrysotilc asbestos was suspended in physiological saline to give a concentration of approximately 5 mg. per ml. Tin's suspension was sterilised liy being brought quickly to the boil and was thru allowed to cool during which time the dust sediments to the bottom of the continuer. This allows 75 per cent of the saline to be drawn off and this is replaced by an equal volume of sterile culture fluid. The mixture which is kept nt 37 is injected into the line points of .-mall lung lobes so us to distend the lung slightly at the injection site. A suture is then passed round the tip of the lobe anti tightened, after which the sutured bag of tissue is severed from the rest of the lung and placed in a culture chamber (Fig. 1). At the cud of each experiment the tiing tissue was divided into two lobes. One was fixed in formol saline for light micro scopic study, while the other was fixed in buffered osmium tetroxide for electron-microscope examination. This tissue was embedded in Aruldite by the method of Davis (lOaO) except that total embedding time was reduced to 3 days before the tissue was dispensed into gelatin capsules to harden. After sectioning the tissue was stained on the grids with lead citrate {Reynold, 19G3) before being examined in the electron microscope. ASARCO 3S0 J. M. G. DAVIS RESULTS The appearance of guinea-pig lung containing asbestos dust differed fr. normal lung slices maintained in organ culture in two respects. Tn the first pi. it was found impossible to inject dust without distending the lung slightly and t made it difficult to keep the diameter of the cultures down to tiled mm. throu which oxygen can adequately diffuse. As a result central necrosis was of; found in the dusted cultures with a hand of living tissue about l mm. thick the outside. Tn addition to this injection of dust containing fluid into the alvc caused them to collapse rather more quickly than a normal lung and in so: NYLON Fig. !. A diuprurnumtir roprosontatinn of rhi* method used to inject nshosUis dust into the lunp pieces to ho maintained in nrpati culture. cases the original outline of the alveoli was difficult to follow after as little as 3 da; of culture. Apart from this, however, the lung tissue as a whole seemed unaffects by the presence of the asbestos dust and could be kept alive for at least 0 day During this time the alveolar epithelial cells and the cells forming blood vess; and bronchioles showed exactly the same structures as the corresponding ce: in undustecl cultures when examined by both light and electron mieroseop In fact the only difference to he seen between dusted and undusted cultures 1 light microscope methods was the presence of foamy macrophages in the tis.-i spaces of the former. Electron-microscope examination of the dusted tissue showed, however, th. the dust is quite well distributed from the original injection site, and is very quick taken up by (lie lung macrophages. During this study no evidence was seen tin ASARCO ALV 0005276 c KFKKCT OF ASliFsTOS IH'sT OX IXXO JlACKOl'IIACK.S :j.si ;he asbestos dust was taken U|> by any cells apart from the* macrophages or that :ho (hist was able to penetrate the tissues by mechanical action. Dust was present within the macrophages within as little as :ii> min. and almost all macrophages -untamed dust by 2 hr. after theculture was set up (Fig. d). The macrophages in a lusted area develop elongated phagocytic processes on their surface membranes -vithiu a few min. and if these contact a dust particle they surround it and fold back jmto the cell surface to enclose it in a phagocytic vacuole or phagosome (L'ig. :>). The phagoevtie processes have a fairly constant thickness of about UMii.) A but they vary considerably in length and some can be as long as ID//. Initially nlmgosomes are dilated spherical structures which, in these experiments at least, contain culture fluid in which the dust particles float freely (Fig. 4). The longest period that dust particles can remain in phagosomes is impossible to determine as :resh phagocytosis.occurs to some extent throughout the experiment, but within 2 hr. of the start of the experiment some asbestos dust has escaped from the phagosome and can be found free in the cytoplasm (Fig, 11). Tt is not possible to be certain of the reason for phagosome rupture in all cast's. .Sometimes it may be due to chemical damage to the phagocytic membrane, but in certain cases it is jbvious that lung dust "fibres have punctured the phagosome wall by purely mechanical action. Whatever the cause of the membrane rupture, however, cytoplasmic material Hows into a punctured phagosome, the membrane of which remains intact for much of its circumference (Fig. .">). This membrane, however, eventually collapses (Fig. 0) and contracts enclosing any remaining dust to form a dense structure that in the past would have been called a eylosoinc. but whose present terminology is rather confused. This point is dealt with in the discussion. Asbestos containing " cytosomes " are shown in Figs. 7. S. !l and !(>. ft can be l that in addition to the dust, these bodies contain a considerable amount of .ris including portions of membranes that often have been folded and packed to produce laminated or " myelin " structures. Dust containing " eytosome " structures were absent from macrophages during the first few days of culture and were most common after :! or 4 days indicating that it takes some time for the phagosome to contract fully. During the process of dust phagocytosis it was noticeable that the number of primary lysosomes in the cells increased consider ably. These structures are spherical granular masses about i>.'{// in diameter and surrounded by a single membrane (Fig. l.">). They are believed to contain katabolie enzymes which can be liberated into the phagosomes to undertake digestion. A few of these structures are present in normal lung macrophages, but it was found in this study that they become much more common from about 4S hr. onwards. This probably means that new ones are produced if the macro phage enters a prolonged period of phagocytic activity. The fate of asbestos dust in macrophages that has just been described, where the dust is either finally liberated into the cytoplasm or walled up in " residual bodies ", is by far the most usual one. In some cells, however, the phagosome seems neither to rupture or contract and as new phagosomes are formed the cell takes on the highly vacuolated appearance of a " foamy macrophage ". In these cells most of the dust is retained in phagosomes for several days and very little of it is ever found free in the cytoplasm. How these cells differ from normal macro phages is uncertain but most of the cell structures including mitochondria are quite normal so that it does not seem likely that a foaming macrophage is simply a dying cell. I ASARCO ALV 0005277 3S2 J. M. G. DAVIS In the previous paper on the structure of normal lung maintained in or;, culture it was reported that the lung macrophages were actively phagocytic I they did not combine to form giant cells. In dusted cultures, however, d: containing macrophages quickly combine and quite large giant cells can be form within 24 hr. The process of giant coll formation in culture is exactly similar that described by Davis (IUG36) for giant cell formation in live guinea-pig Iuu The macrophage phagocytic processes become entangled and contract to bind t cells together (Ifig. 12). Eventually the dividing cell walls between the individi macrophages break down and a truly multinuclcate cell is produced. This proccan occur very quickly and some such cells were found after as little as 24 hr. EXPLANATION OP TLATES Fits. 2. An tilvfolnr macrophage from guinea-pig him: maintained in input cttHuro for 2 hr. In this hnr(. turn? lho coll 1ms phagoeytosod a couMilomhln amount of usbostns dust much of which is still contained in tho phagosomes 1ml sumo of which (urruwed) is already lying fcco in the roll cytoplasm. X22.2U0. Fn:. 3. --A phagocytic process from a guinea-pig lung umcrophngo engulfing crystals of chryso- tilo asbestos dust. This coll hud boon maintained hi orpm culture for 2 hr. X 60,000. Fit:. 4.- A httmllo of ehrysotilo crystals contained in a phagosome from a guinea-pig lung macrophage. This cell had been maintained hi orpui culture for l hr. Fl. n.- A phagosome from a guinea-pig lung macrophage in thn process of rupturing. The purtielo of asbestos dust (arrowed) appears to bo escaping into the cell cytoplasm. At tho Mime time cytoplasmic material eau ho seen (lowing into tho phagosome. This cell had been maintained in organ culture for 24 hr. X42.0U0. Fits. f>. - A punctured phagosome from a guinea-pig lung macrophage. Much of tho contained ehrysotilo asbestos dust has remained within tho phagosome and tho phngosomo membrano is collapsing and contracting around it. This nmcrophugo was maintained in organ culturo for 4S hr. x53.(H)l>. Fins. 7, 8. ft and 10.-- Thcso 4 (igs. show stages in the contraction of phagosomes containing asliestos dust. Tho photographs were taken from guinea-pig lung macrophages maintained in organ culturo for 3 days. Most of these contracting phagosomes contain cell debris, especially tnomhruncs, in addition to the asbestos dust. Xo4,UU0 iu each case. > Fic. 11.--An area of cytoplasm from a guinea-pig lung macrophage maintained in organ culture for 3 days. Crystals of ehrysotilo asbestos uro shown lying completely frco in the cell cytoplasm. X 54,000. Fig. 12.--Uuiiica-pig lung macrophages combining in tho early stages of giant cell formation. Tho phagocytic processes of thcso cells have iuterdigitated and tho cells arc effectively tied together. Thcso macrophages had been maintained in organ culturo for 24 hr. XlS.OOO. Fig* 13.--A multiuuclcato giant coll found in guinea-pig lung after only 24 hr. of organ culture. In this short titno the junction lino between tho individual macrophages had disappeared and tho coll membranes have fused. Phagocytic processes arc, however, still present on the surface of this giant cell (arrowed) and it therefore seems likely that the cell could still pick up asbestos dust. Xp,300. - Fig. 14.--A lung macrophago from guinea-pig lung maintained in organ culture for 24 hr. The cell is still actively phagocytic but considerable numbers of new A-cytomcmluunes have recently bcon produced (arrowed). Fie. 15.--An area of cell cytoplasm from a guinea-pig lung macrophage maintained in organ culture for 4 days. Xew a-cytomembranes are present as well as crystals of ehrysotilo asbestos dust lying frco in tho cytoplasm. This photograph shows 2 typical primary lysosomes which are arrowed. X 42,000. Fig. 1G.--A cell from guinea-pig lung maintained in organ culture for G days. The cell contains a particle of asbestos dust (arrowed) and numbers of long A-cytomembraties. This cell was found surrounded by'collagen or rcticulin fibres atul had no phagocytic processes on its surface membrane. Tho a-cytomembranes arc not short or dilated enough tor the cell to be considered a typical fibroblast and it probably represents an intermediate stage in the conversion of dust containing macrophage to dust containing fibroblast. ^ >. 2l.0u0. Fig. 17.--A fibroblast from guinea-pig lung maintained in organ culture for 6 days. The cell contains crystals of ehrysotilo asbestos dust (arrowed) and the A-eytomembrancs have becomo dilated to form sacs which npjtcftr to ho filled with amorphous material. Handed collagen or roticuliu fibres can bo seen outside the cell. 4l.tUU. ASARCO ALV 0005278 UlUTlMl JulliXAL UK KxrKKlMUJiTAI. i'.U'UULUt.V Vul. XLVXII. No. 4. ASARCO ALV 0005279 ASARCO ALV 0005280 f UlUTISlC JoLUN'AL Of Km'Kulmkntal I'atiuuaicv i Vol. XLV11I, Xu. 4. ( ASARCO ALV 0005281 I'Iiitimi .lufus'Ai. or li\n:i:iMi:.vfAi. l'.\'ni<iun:v. Vul. XLVIIt, Xo. 4. I ASARCO ALV 0005282  BulTlSlI JlllMlVAI. <>K liSl' UIMKN'TAl. I'.Vrill'I.OfiV Vnl. X1.VUI, N". 4. | a! i /I ASARCO ALV 0005283 IlltlTIMt ! Of UN All Of KnI'UIUMUNTAI. I'ATIIOl.OU V Vul. XI.VIII, Xo. 4. !i Davis. ASARCO ALV 0005284 Davis. I ASARCO ALV 0005285 lir.insu .loi iiNAt. of IMII.STAI. I'ATUUI.UCV V..I, M.VIll. Xm. 1. ti 1 ! / ASARCO ALV 0005286 Bkitisii Jui'iiNAi. ui' I'.vrmn.uisv Vul. XLVIII, Xu. 4, Davisi. ASARCO ALV 0005287 EFFECT OF ASUESTUS OUST ON LL'NU MACKOHIACfK.S 383 culture (Fig. 13). Once formed giant ceils did not appear to change for the whole 9--10 days of the experiment. In normal guinea-pig lungs the macrophages contain many ribosomes but few if any a-cytomembrancs. Within 24 hr. of culturing, however, mauy macro phages produced sheaves of long a-evtomembranes and this often occurs while the cell is still actively phagocytic (Fig. 14). Eventually this type of macrophage loses its phagocytic processes (Fig. 16) and the long a-cytomembrancs break up into short dilated lengths (Fig. 17). The cells finally exhibit the structure that is normally considered typical of fibroblasts, and are usually found surrounded by masses of collagen on reticulin fibres. From these observations it will be seen that the pathological changes previously reported for dusted guinea-pigs in vivo can all be produced in pieces of lung maintained in organ culture. This system should therefore be useful for studying the effect of various chemicals on asbestos uptake and it is hoped in the near future to study the effect of P 204 on cultures dusted with chrysotile. DISCCSSIOX In this study it was reassuring to find that the fate of asbestos dust in organ culture is exactly the same as that previously demonstrated in dusted animals anil known to occur in human lungs. Dust is taken up only by the lung macrophages and these may either combine to form giant cells or at a rather later stage become converted into fibroblasts. In this respect the system of organ culture is ideally suited for a cvtological study of the changes produced by asbestos. It would'bo less satisfactory for examining the pathological stimulus of collagen production by asbestos because, as was shown in the preceding paper, fibrosis can occur in the later stages of organ culture of normal lung tissue. This, however, docs not invalidate the suggestion that asbestos dust causes fibrosis by direct action on the macrophage cytoplasm, but merely emphasises the fact that fibrosis is a normal pathological process that can be initiated by a number of different stimuli. This study has confirmed the previous suggestion (Davis, l!)i;:i6) that giant cell formation docs not necessarily result from the presence of foreign material too large to be phagoevtosed by a single macrophage, (iiant cells formed in the dusted cultures within 24 hr. and none of them was found to contain any dust particles more than 5 ji in length. The reasons for giant cell formation are probably seen by comparing normal and- dusted organ cultures of lung tissue. In normal cultures the macrophages are actively phagocytic but remain evenly distributed throughout the tissue and giant cells do not form. It seems likely that in these cultures the stimulus to phagocytosc is widespread and the macrophages do not aggregate in any particular area. In dusted cultures, however, the dust fibres tend to occur in clumps and many macrophages become attracted to small areas where they aggregate. When this occurs their phagocytic processes inter twine and giant cells are formed. From this evidence it would appear that giant cell formation results from a localisation of foreign material and the size of this material is not important. By the use of organ culture it has been possible to follow the uptake of asbestos dust into a phagocytic vacuole or " phagosome " and to study the way in which these vacuoles collapse to form residual bodies". This, however,*raises the problem of terminology. Originally, all the structures mentioned here in con- 35 ASARCO ALV 0005288 384 J. M. a. DAVIS ucct ion with the phagocytosis of asbestos were given the general name of cytosomc (Carasso and Favard. IO.'iS; Gicseking, 1058; Karrer, 1058), but now the impor tance of the different types is recognised. In order to discuss their terminology, however, it will be necessary to summarise the present concepts of the enzymatic digestion of phagocytosed materials and the role of the Iysosome. It is now believed that the digestion of phagocytosed materials is carried on inside the phagosome by katabolic enzymes that are secreted into the vacuoles by structures called lysosomes. These lysosomes are dense granular bodies bounded by a single membrane. The enzymes they carry are either inactive or unable to damage the cell because they are separated from the cytoplasmic structures by a membrane. Once in the phagosome the enzymes are activated and digest any organic material present. It is in the subsequent fate of the phagosome and the Iysosome enzymes it contains that the confusion arises.. When the phagosome contracts to form a residual body it ofton contains the remains of membrane structures that become packed together to give a myelinated " appearance. The residual body is very much smaller than the original phagosome. It contains dense granular material and is surrounded by a single membrane. In fact the residual body is very much like a Iysosome except for the debris it contains and some workers have used the word Iysosome to describe these structures. Both de Duvc (1%!!) and vGoldfischer (Uoldfiseher. Essncr and Xovikolf, l!)fi4) consider that the primary Iysosome, the phagosome and its residual body should all be included under the general term " Iysosome ". As far as the phagosome is concerned this seems undesirable. If the primary Iysosome is to be considered as a bag of enzymes ready for use, then the phagosome is an entirely separate structure which is formed from the ceil surface during phagocytosis. The fact that the Iysosome enzymes may be injected into the phagosome does not alter this fundamental difference. When the phagosome has contracted to form a residual body, however, it does show a similar structure to the primary Iysosome apart from containing a certain amount of debris. 'Phis fact suggests the idea that the Iysosome enzymes may not be used up or inactivated during digestion in the phagosome and are retained in the residual body in a form iti which they could be reinjected into another phagosome. If this is (he case then the residual body could well be called a " secondary Iysosome ", but until this is definitely proved it would be best to retain the term " residual body ". To call such a structure a Iysosome unless it can be proved still capable of acting as a source of usable enzymes would obviously be incorrect. SUMMARY Cluysotile asbestos dust was injected into small pieces of adult gumea-pig lung maintained in organ culture for 10-14 days. This dust was taken up by the lung macrophages in a matter of hours, but dust was not found in any of the other cells during the course of these experiments. The macrophages in contact with dust produce elongated phagocytic processes on the cell surface and these encircle the dust- and fold back onto the cell to form phagocytic vacuoles or phagosomes. The dust may remain in the phagosomes for some time or it can quickly escape into the cytoplasm perhaps by mechanical action. If the dust remains in a phagosome this eventually contracts to form a small dense structure usually called a residual bod}-. In the cultured lung it was found that dust containing macrophages could ASARCO ALV 0005289 ta KL'iT.CT OF ASUIibTOS UL'.ST OX U_'XO MAt'lIOFllAlilvS asr* quickly combine to form giant cells, and quite large specimens were often found within 24 hr. Some macrophages eventually become converted into fibroblasts with asbestos dust still retained in the tibroblast cytoplasm. HEFEREXCES . Carasso. X. and Favac d. L\--(1958) C.r. Acad. Sci. (Pam), 246, 1594. Davis, J. M. G.--(1959) Suture, Laud.. 1S3. 200.--(1903c) Dr. J. exp. Path., 44, 454.-- (19036) Dr..}. exp. Path., 44, 508.--(1907) Dr. J. exp. Path., 48, 371. de Dove, C.--(1903) Scientific Americun, 64, 208. Giesekixo, R.--(195S) Peril, dt-sch. path. Gee., 42, 344. Goldfiscuer, 8.. Essxek, E. and Xovikoff, A. 13.--(1904) J. Ilklochem. Cytochcm., 12. 72. Holt, P. F.. Mills. J. M. and Young, D. Iv.--(1904)./. Path. Duel., S7, 13. Karrek. H. E.--(1958) J. biophys. binrhem. CytoL, 4, 093. Reynolds, S.--(1903) J. Ceil Dial., 17, 208. i } J I ASARCO ALV 0005290