Document LpZRz0R9dMo65g2nObZ5GEoJ7

, '> *~'*z S\w* I I %y k w W4 ** sj/ `*4 V** 4* #"i i v i %<< -. *^* V / *, !? .% j, t * r fk ^k 4 V a / k Si **> 1 k 4 !-. 4 ", * t -*.. i y ; m i iiE?r.CSTE!) PP.Oil THE `iMUTISU JOUKHAE OP iSSPERIMEXTAL Patjioi.ogv', Vol. XLVIII, :-To. 4, August 1937. v J psnrrso ros t H. K. LEWIS & .Co. Ltd., Vi 'v'. 136, COVYSQ STT.EST, LO>'00>',- 'V.C.l. . . 51T' . ' AP'L.vr.D c: sox. 2'_kt;:olom^ rsEss, dd^xixo. ASARCO ELP 000314-4 379 THE EFFECTS OF CIIItVSOTILK ASBESTOS DUST OX LUNG MACROPHAGES MAINTAINED IN ORGAN CULTURE AX ELECTKOX-MICROSCOPE STUDY J. M. G. DAVIS From the Department of Pathology, University of Cambridge Recoivcd for implication Fobruary US, 1007 lx a previous study (Davis. 19(17) a technique of organ cull lire was reported which allowed lung macrophages to he maintained for at least !l-lo 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. 19G3. 6; Holt, Mills and Young. 1 iHi-t) had. however, shown that very large amounts of dust could be taken up by individual lung macrophages which were able to combine to produce mulliuucleate giant cells in only a few days. Later many macrophages became converted to fibro blasts, the dust still remaining in the fibroblast cytoplasm. It was of great interest, therefore, to find that in the organ culture normal undusted macrophages could undergo conversion to fibroblasts. The system was obviously well suited to the stud}- of this process in dusted cells. MATERIALS AND METHODS The apparatus mid methods of culture used previously to study uoruml lung tissue (Davis. 1967) were also used iu this study of dusted macrophages. It was decided to use ehrysotilc asbestos dust us this was known to cause few problems of embedding uud svciioning for elcctrou-microscopr examination, and a sample of finely milled dust was nliiuiued from Dr. P. F. Holt of Kcudiiig University. This dust was similar to that usiil iu previous experiments (Holt it at.. 1964) and consisted mainly of fibres a it or less in length. Initially it was found diilicult to introduce the dust to the vultures iu u sutisfuetory maimer. If the dust was simply added to the culture medium none of it penetrated into the lung at all. Keen simple injeetion of a suspension of dust iu culture fluid was unsatisfactory ns most of the dust was very quickly squeezed out of the cut surface of the ltitiir- Finally it was found necessary to inject the dust into the tip of a small lung lobe and then tie a suture at the injection site to retain the dust. The final method adopted was as follows. Chrysotilc asbestos was suspended iu physiological saline to give a concentration of approximately 5 mg. per ml. This stts|ivusiou was sterilised by being brought quiekly to the boil and was then allowed to cool during which time the dust sediments to the bottom of the container. This allows 75 per cent of the saline to be drawn off and this is replaced hv an eipial volume of sterile culture fluid. The mixture which is kept at 37 is injected into the line points of small lung IoIk-s so us to distend the lung slightly at the injection site. A BUturo is then passed round the tip of the lobe and tightened, after which the sutured bag of tissue is severed from the rest of the hint; and. placed in a culture chamber (Fig. 1). At the end of each experimentthe lung tissue was divided into two lobes. One was fixed iu forinol saline for light micro scopic study, while the other was fixed in buffered osmium tetroxide for electron-microscope examination. This tissue was embedded iu Arulditc by the method of Davis (in.'ilt) except that total embedding time was reduced to 3 days before the tissue was dispensed into gelatin capsules to liardcn. After sectioning the tissue was stained on the grids with lead citrate (Reynold, 19G3) before being exumiued in the electron microscope. J ASARCO ELP 0003145 380 J. M. G. DAVIS RESULTS The appearance of guinea-pig lung containing asbestos clust differed frnormal lung slices maintained in organ culture in two respects. In the first ph it was found impossible to inject dust without distending the lung slightly and t made it dillicult to keep the diameter of the cultures down to the -2 mm. throe which oxygen can adequately diffuse. As a result central necrosis was oft found in the dusted cultures with a hand of living tissue about I nun. thick the outside, fit addition to this injection of dust containing fluid into thealvt caused them to collapse rather more quickly than a normal lung and in so: NYLON 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 unaffecti by the presence of the asbestos dust and could be kept alive for at least ft day During this time the alveolar epithelial cells and the cells forming blood vessc and bronchioles showed exactly the same structures as the corresponding cc! in undusted cultures when examined by both light and electron mieroscop In fact the onlv difference to be seen between dusted and undusted cultures l light microscope methods was the presence of foamy macrophages in the tisst spaces of the former. Electron-microscope examination of the dusted tissue showed, however, tli. the dust is quite well distributed from the original injection site, aiul is very quick: taken up bv the lung macrophages. During this study no evidence was seen th: ASARCO ELP 0003146 KI-TIX'T tip ASIlpSTOS lH'ST OX l.L'Xt: .MACJUH'ItACK.S 3SI Ik* asbestos dust was taken U|> by any cells apart from tin* macrophages or that :lu* dust was able to penetrate the tissues by moehauioal aetion. Dust u as iireseut vithiu the tnaerophancs witiiitt as little as ISO mill, and almost all macrophages .-untamed dust by 2 hr. utter the culture was set up (Fig. 2). The macrophages in a lusted area develop elongated phagocytic processes on their surface membranes -vithin a few min. anil if those contact a dust particle they surround it and fold back into the cell surface to enclose it in a phagocytic vacuole or phagosome (l'ig. :5). The phagocytic processes have a fairly constant thickness of about loop A but rlioy vary considerably in length and some can be as long as 10 /t. Initially nhagosomes are dilated spherical structures which, in these experiments at least, jontain culture thud in which the dust particles float freely (Fig. 4). The longest period that dust particles can remain in phagosomes is impossible to determine as fresh phagocytosis occurs to some extent throughout the experiment-, but within 2 hr. of tile start of the experiment some asbestos dust has escaped from the phagosome and can be found free in the cytoplasm (Fig. II). It is not possible to be certain of the reason for phagosome rupture in all cases. Sometimes it may be due to chemical damage to the phagocytic meinhraiie. but in certain eases it is >bvious 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. A). 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 cylosome. but whose present terminology is rather confused. This point is dealt with in the discussion. Asbestos containing " eytosoines " are shown in Figs. 7. S. !i and 10. It can he *i 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 product- laminated or "myelin" structures. Dust containing " cylosome " structures were absent from macrophages during the first few days of etillttrc anil were most common after :l or 4 days indicating that it takes some time for the phagosome to contract fully. Daring 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 <:> a in diameler und surrounded by a single membrane (Fig. I A). They are believed to contain katabolio- enzymes wliiclt- 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 iu macrophages that has just been described, when* the dust is either finally liberated, into the cytoplasm or walled up iu " residual bodies", is by far the most usual otic. In sonic 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 iu 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 docs not seem likely that a foaming macrophage is simplv a dying cell. I I* i li i i ASARCO ELP 0003147 382 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 1 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 cell formation in culture is exactly similar that described by Davis (111036) for giant cell formation in live guinea-pig lun The macrophage phagocytic processes become entangled and contract to bind t cells together (l*'ig. 12). Eventually the dividing celtwalls between the individi macrophages break down and a truly multinueleate cell is produced. This proc-' can occur very quickly and some such cells were found after as little as 24 hr. EXPLANATION! 01" TLATES Fits. 2. An alveolar muerophngo from guiuea-pig him* maintained in organ culture for 2 hi*. In tins Jntrl. tuna the cell has phagocytosod a considerable amount of aslK\st<w dust much of which is still contained in tlio phagosomes hul somo of which (arrowed) is already lying freo in tho colt cytoplasm. X22.2UI). Fm. 3. --A plmgoeviic process from a guinea-pig lung maccuplmgo engulfing crystals ofchrvso* tilo nshcstoH dust. This cell hud been maintained in orpin culture for 2 hr. X60,0l)0. Fm. 4. - A hundlo of chrysotilo crystals routaiuod in a plmgosoum from a guiuea-pig lung macrophage. This coll had 1kh*u maintained in orpin culture for 1 hr. Fm. f*.- A phagosome from a guiueu-pig lung nmrrophugc in thn process of rupturing. The particle of nsltcsto* dust (arrowed) appears to Ik> escaping into tho cell cytoplasm. At tho same time cytoplasmic material can In: seen (lowing into tho phagosome. This celt had been maintained in orpin culture for 24 hr. >;42.<I0U. Fie. <. - A punctured phagosome from n guinea-pig lung macrophage. Much of tho contained chrysotilo aslicstos dust has remained within tho phngosume and the. phagosome membrane is collapsing and contracting around it. This macrophage was maintained in organ culture for 4 S hr. xf:U>M>. Fins. 7* 8. A and in.- These 4 tigs, show stages in the contraction of phagosomes containing aslicstos dust. Tho photographs were tuken from guinea-pig lung macrophages maintained in organ culture for 3 days. Most of these contracting phagosomes contain cell debris* especially mombmnos, in addition to the asbestos dust. >C 54,000 in each ease. Fig. 11.--An area of cytoplasm from a guinea-pig lung macrophage maintained in orpiu culture for 3 days. Crystals of chrysotilo asbestos are shown lying completely freo in the cell cytoplasm, x 54.000. Fie. 1:2.--Guinea-pig lung macrophages combining in tho early stages of giant cell formation. The phagocytic processes of theso cells have intordigitutcd and the cells arc effectively tied together. Theso macrophages had lieen maintained in organ culture for *4 hr. xlS.hOO. Fie. 13.--A multimiclcato giant coll found in guinea-pig lung after only -4 hr. of organ culture. In this short timo the junction lino between tho individual macrophages had disappeared and th coll membranes havo fused. Phagocytic processes am, however, still present ou the surface of this giant cell (arrowed) and it therefore seems likely that the cell could stilt pick up aslicstos dust. X 0,300. Fig. 14.--A lung macrophago from guinea-pig lung maintained in organ culture for 24 hr. Th cell is still actively phagocytic but considerable numbers of new A-cynmicmhraues have recontly been produced (arrowed). Fig. 15.--An area of cell cytoplasm from a guinea-pig lung macrophage maintained m organ culture for 4 days. Xew a-cytomembrancs are present as well as crystals of chrvsotde asbestos dust lying freo in tho cytoplasm. This photograph shows 2 typical primary lysosomes whicli ar arrowed. x 42,000. Fig. 16.--A cell from guinea-pig lung maintained in organ culture for C days. The cell contains a particle of asbestos dust (arrowed) and numbers of long .vcytonn'mhranes. This cell was found surrounded by collagen or rcttculin fibres aiul had no ph;ig*eytie processes on its surface membrane. The a*cytomcmbnutcs arc not short or dilated enough tor the cell to be considered a typical fibroblast and it probably represents an intermediate stage in tho conversion of dust containing macrophage to dust containing fibroblast. \2l.uluK Fia. 17.--A fibroblast from guinea-pig lung maintained in orzan culture for 6 days. The cell contains crystals of chrysotilo aslM**tus dust (arrowed) mid the A-eytotuetuhraiic* have becomo dilated to form sacs which npj>cnr to lie tilled with amorphous material. Handed collagen or reticulin fibres can ho seen outside the cell. * 4l,tUi>. ASARCO ELP 0003148 UlUTIMl Jl>L U.NAL Ur* ICM'KIUMUSTAI. I'ATIIOLUl. V Vul. XLYHI. Xu. 4. ASARCO ELP 0003149 .Iui uvai. or i:\ri:i;tMi:\r\i. l\vn:oi,i...v Vol. Xl.VUl, X,.. 4. ASARCO ELP 0003150 UlUTISll JoCU.VAt. Of ICXnUtlMKNTAI. 1'A'lttlH.llliV Vl. XLVX1I, -Yu. 1. Davis. ASARCO ELP 0003151 iiitimi Joi'un'ai. if E\i`i:i:imi:n tai. I'.iTitnMniv. Vo!. XLVIlt, Xo. 4, I ASARCO ELP 0003152 J ASARCO ELP 0003153 CiiiTiMi Joi'unai. <>i- K\n:i;nn:\r.u. I,athiiijii:v. Vi.l. XIjVIII, Xu. 4. 8I Davis. ASARCO ELP 0003154 Bitrrtstt .IiicitVAi. or Km:i!i)insTAi, r.vriini.onv Voi, xi,vm, Xo. 4. J s Davis. ASARCO ELP 0003155 lluirtsu -lot i:nai. V..I, XI.V1U. N`... 4. 6 ! ASARCO ELP 0003156 UiariMi .Ioi-un.u. m- Km-hiumuntai. l\vrm>i..i<;v Voi. xr,via, Xu. 4. t ASARCO ELP 0003157 EFFECT OF ASBESTOS DOST ON LL'NU MACUOt'U.UiKiS :jtsu culture {Fig. 13). Once formed giant colls did not appear to change for the whole 9-10 days of the experiment. In normal guinea-pig lungs the macrophages contaiu many ribosomes but few if any a-cytomembrancs. Within 24 hr. of culturing, however, many macro phages produced sheaves of long a-cytomembranes and this often occurs while the cell is still actively phagocytic {Fig. 14). Eventually this type of macrophage loses its phagocytic processes (Fig. 1C) and the long a-cytomembranes 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 chrysotilc. DISCUSSION 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 anti known to occur in human lungs. Dust is taken up only by the lung macrophages and these may cither 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 cytological 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 uormat lung tissue. This, however, does 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. 19036) that giant cell formation does not necessarily result from the presence of foreign material too large to be phagocytosod by a single macrophage. (Jiant cells formed in the dusted cultures within 24 hr. and none of them was found to contaiu any dust particles more than 5/t 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 arc actively phagocytic but remain evenly distributed throughout the tissue and giant cells do not form. It seems likely that hi these cultures the stimulus to phngocyto.se 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 iuto a phagocytic vacuole or " phagosome " aud 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 ELP J as-i ' J. M. G. DAVIS neetion with the phagocytosis of asbestos were given the general name of cytosomc (Curasao and Fuvard. 105S; Gicseking, 1U5S; Karrer, 1*.)58), 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 plntgocytoscd materials and the role of the Ivsosome. It is now believed that the digestion of phagocytoscd materials is carried on inside tire phagosome by katabolic enzymes that are secreted into the vacuoles by structures called Ivsosomes. 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 lysosomc 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 Ivsosome except for the debris it contains and some workers have used the word lysosomc to describe these structures. Both de Duve (1!)03) and poldfiseher (UoUUisehcr. Jissner and Xovikoff, 1!HS4) consider that the primary lysosomc, the phagosome and its residual body should all be included under the general term " lysosomc As far us the phagosome is concerned this seems undesirable. If the primary lysosomc is to be considered as a bag of enzymes ready for use. th'en the phagosome is an entirely separate structure which is formed from the cell surface during phagocytosis. The fact that the Ivsosome enzymes may lie 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 lysosomc apart from containing a certain amount of debris. This fact suggests the idea that the Ivsosome enzymes may not be used up or inactivated during digestion in the phagosome and are retained in the residual body in a form iu which they could be reinjected into another phagosome. If this is the ease then the residual body could well be called a " secondary lysosomc ", but until this is definitely proved it would be best to retain the term " residual body To call sucli a structure a lysosomc unless it can be proved .still capable of acting as a source of usable enzymes would obviously be incorrect. SUMMAltV Chrysotiie asbestos dust was injected into small pieces of adult guiuea-pig lung maintained in organ culture for 10-14 days. This dust was taken up by the lung macrophages iu a matter of hours, but dust was not found in any of the other ceils 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 body. In the cultured lung it was found that dust containing macrophages could ASARCO ELP 0003159 Kt-'FKCT OK ASUliSTOS DUST OX I-UXC! MACUOl'UAGKS 3S.1 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 fibroblast cytoplasm. REFERENCES . Carasso. N. and Favard. 1'.--(1950) C.r. Acad. Set. (Paris), 246, 1594. Davis, J. 31. G.--(1959) Xnittre, Land.. 1S3. 200.--(19G3) Er. J. exp. Path., 44, 454.-- (19036) Er. J. exp. Path., 44, 50$.--(1907) Er. J. exp. Path., 48, 371. de Dove, C.--(1903) Scientific American, 64, 200. Giesekixg, R.--(193S) Peril. ditch. path. Gcs., 42, 344. Goldfisciier, s>.. Essxek, E. axi> Novikoff, A. B.--(1904) J. Ilhtochem. Cytochcm., 12, 72. Holt, P. F.. Mills, J. 31. and Young, D. K.--(1904) J. Path. Bad., S7,15. Tvarp.eu. H. E.--(19,1$) J. biophys. hinrhem. CyloL, 4, 093. Reynolds, S.--(1903) J. Cell Biol., 17,20S. ASARCO ELP 0003160