Document 9B2j9bJwQdK3ZBkmMo867Ngp

Induction of early alveolar injury by inhaled asbestos and silica1 ARNOLD R. BRODY, LILA H. HILL. AND DAVID B, WARHEIT Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park. North Carolina 27709 i' A P A T CO t e t t I,inhalation of toxic particulates such as asbestos and silica causes interstitial fibrotic lung disease in humans and animals (4, 17, 23,28). Little is known about the biochemical and cellular mechanisms through which the in haled particulates cause disease (4, 9, 15). Ongoing studies on rats and mice exposed briefly to aerosols of asbestos or silica have expanded our knowl edge of the initial cellular events that occur consequent to particle deposi tion on alveolar surfaces. However, the basic mechanisms by which such particles stimulate interstitial fibro blasts to replicate and/or produce increased amounts of connective re main undefined. In this paper we shall summarize the results of exper iments that illustrate the following: ABSTRACT Inhaled asbestos fibers and silica crystals are known to cause interstitial fibrotic lung disease in animals and humans. The initial cellular events and biochemical mechanisms that lead to development of disease are poorly understood. In ongoing studies reviewed here it has been shown that inhaled particulates small enough to pass through the conducting airways are deposited initially at the bifurcations of alveolar ducts. Within hours after brief exposure, alveolar epithelial cells phagocylose inhaled particles that subsequently are translocated to interstitial matrix and fibroblasts. Within 48 h after exposure, inhaled asbestos on alveolar surfaces activates a complement-dependent chemotactic factor for macrophages that accumulate at duct bifurcations. Epithelial cells, macrophages, fibroblasts, and the interstitial matrix are significantly altered by brief (1- 5-h) exposure to chrysolite asbestos. The basic mechanisms that mediate these initial events remain to be defined.--Brody, A. R.; Hill, L. H.j Warhest, D. B. Induction of early alveolar injury by inhaled asbestos and silica. Federation Proc. 44: 2596-2601; 1985. /) Initial deposition pattern of in haled inorganic particles 2) Response of epithelial cells, fibro blasts, and macrophages to dust depjosition 3) Elaboration of a complement-de pendent chemotactic factor for macrophages induced by asbestos on alveolar surfaces 4) Development of an early asbestosinduced interstitial lesion INHALED PARTICLES ARE DEPOSITED AT ALVEOLAR DUCT BIFURCATIONS Inhaled particles small enough to pass through the conducting airways reach the alveolar (gas exchange) regions of the lung (9, II, 19). For many years, investigators have assumed that inhaled particles deposit randomly along alveolar duct surfaces and within individual alveolar spiaces (6, 19). This misconception has persisted because of the knowledge that airflow velocity is very low distal to the bron chial tree (6, 19). The large number of terminal bronchioles normally found in the lung creates a large cumulative cross section for flow, which suggests that particle dep>osition should be controlled by sedimentation or diffusion (6, 19). Recently, using scanning electron microscopy and specialized dissection techniques, we quantified the deposition pattern of five aerosolized dusts: chrysotile and crocidolite asbestos, fiber glass, alphaquartz, and ash from the Mt. St. Helens volcano (11). The results showed that regardless of size, min eral nature, or concentration, inhaled particulates small enough- to...reach the alveolar zopc-ate.drpnsitrd.maialy at alveolar duct bifurcations. The al veolar deposition pattern at duct bi furcations most likely is a result of airflow characteristics that cause en hanced deposition of particles at bi furcations intersecting the flow. This is similar to deposition piatterns oc- < curring at bifurcations of conducting airways (19), although we recognize j that the mechanisms controlling de- : position may be different. Figure 1 illustrates the deposition pattern of chrysotile asbestos immediately after , a 1-h exposure. The significance of this finding is that progression of asbestos-induced lung disease is initTT ~ated at the alveolar diiCLbifurcatioos, pierhaps as a result of the enhanced fiber deposition at these sites. This hypothesis is addressed further in the following sections. ` From Response to Injury Thematic Mini symposium Response to Acute Lung Injury pre sented by the American Association of Pathol ogists at the 68th Annual Meeting of the Federation of American Societies for Experi mental Biology, St. Louis. Missouri, April 4. 1984. Acepted for publication September IS, 1984. 2596 0014-9446/8S/0044-2SM/S01.2S. FASEfl ST0272782 71cpirHl-IAL CELLS. teRSTITIAL FIBROBLASTS. , macrophages ^AGOCYTOZE inhaled chestos and silica s,udy the earliest cellular response foaled particles, n is necessary to 10 bi,sh the initial deposition sites of 'he dust (as described above) and `hen focus on these regions at appro- riaie postexposure periods. In the |-h of exposure to chrvsolile as- ^tos or 3 h of exposure to crvstallinc silica is sufficient to evoke a variety 0f measurable biological responses (9. |2), We found that within the first hour of exposure to asbestos and after 3 h of exposure to silica, these particles are taken up by alveolar epithel ial cells covering the alveolar duct bifurcations (9. 10. 12). Figure 2 illustrates this finding During the first 3-4 h after exposure, the partijcs are translocated to he alveolar Figure 2. Transmission electron micrograph of aiveobr<apilbry membranes from the lung of a rai 48 h after a 1 -h exposure to chrysotile asbestos. The gas exchange region of the lung is comprised of a thin type ! epithelium (Ep) that lines alveolar spaces (AS), basement membrane (arrowheads), and an endothelium (En) that lines the capillaries (Ca). A thick cross-sectioned aibestos fiber (arrows) and a single asbestos fibril (arrow) are seen within the cytopbsm of a type 1 epithelial cell. Underlying collagenous connective tissue (CT) is obvious. figure I. Scanning electron micrograph of terminal bronchiole (TB) and us branching alveolar duCU (AO) from the lung of a rat exposed to chrvsotile asbestos for l h. The first alveolar duct bifurcation besond the terminal bronchiole is demarcated by the rectangle and is magnified in 8 The higher magnification (B) illustrates numerous chrysotile fibers (arrowheads) that have deposited on the epithelial surfaces (EP) of the duct bifurcation. From ref I I. $o interstitium by as yet undefined mechanisms. We have presented pre liminary evidence (10) suggesting that the intraepithelial translocation is mediated by actin-comaining micro filaments that bind to asbestos fibers. This mechanism would be similar to that described for intracellular trans port of mucin granules (1) or chro mosomes (2). Whatever the transport system may be. it is clear that large numbers of inhaled particles accu mulate in the lung interstitium (3, 8). At duel bifurcations, interstitial fi broblasts phagoevtose asbestos fibers; as early as I month after a 1-h ex posure to chrysotile. the fibers have induced the formation of intracellular microcalcifications (3, 8). Figure 3 illustrates an asbestos-containing mi crocalcification in an interstitital fi broblast. The pathogenic significance and sequelae of intracellular calcifi cation have not yet been established, although we have postulated that the lesion develops as a result of asbestosinduced membrane injury that allows abnormal ion flux. Calcification con sequent to membrane injury is a wellknown pathological phenomenon (14), and we have demonstrated as bestos-induced alteration of ion flux in red blood cells under conditions where positively charged fibers bind to and cause the redistribution of sialic acid groups (7). Whether similar membrane events occur in pulmonary RESPONSE TO ACUTE LUNG INJURY 2597 ST0272783 Figure S. One month after a !*h exposure to chryjotile asbestos, numerous microcalcifications (M) are found in the lung mcerstitium at alveolar duct bifurcations (8). This calcification is typical because it contains a central asbestos fiber (delineated and magnified in the inset); it is in the cytoplasm of an interstitial fibroblast (F) and is comprised of calcium and phosphorus (8). AS, alveolar space. fibroblasts and macrophages remains to be determined. During the first 4-5 h after asbes tos and silica inhalation, local popu lations of pulmonary macrophages are attracted to sites of the initial particle deposition (25) (i.e , duct bifurcations as shown above). The mechanisms that mediate migration of macro phages to alveolar duct bifurcations could be central to the development of the initial lesions of asbestosis and will be elaborated on in the next section. Here we illustrate that brief inhalation of potentially toxic asbestos or silica particles results in rapid phagocytic uptake by macrophages. After 3 h of silica exposure, approx imately 25-35% of the pulmonary macrophages contain particles (12). The 25% figure was achieved by an alyzing the silica content of lavaged cells in vitro and the 35% figure was obtained by counting silica-containing macrophages in situ on alveolar sur faces. This approach, using electron microscopy in concert with X-ray en ergy spectrometry (12), has validated the use of cells lavaged from exposed animals to study particle content. The populations of macrophages lavaged from the lung and populations re maining attached to alveolar surfaces exhibited similar percentages of par ticle-containing cells. The percentages increased from 36% immediately af ter exposure to 66% during the 24 h after exposure. It was fascinating to learn that this high percentage of macrophage participation was main tained for more than 3 wk after ex posure and then returned to 25% 42 days after the original 3-h exposure (12). As described above, the per centages of silica-containing macro phages recovered by lavage were sim ilar to those studied in situ. Even though the percentages of these cells remained steady, the amount of silica per cell decreased during the 12-h to 24-day period. Interestingly enough. 67% of the macrophages lavaged from the lungs of asbestos-exposed rats contained fibers 48 h after a 1-h exposure (25). This is a percentage remarkably similar to that reported after silica exposure (12). We specu late that the results are similar be cause of a common response of the macrophage populations to dust de position at alveolar duct bifurcations. Studies of asbestos-exposed animals show that significant numbers of macrophages migrate to these sites of dust deposition, phagocytoze fibers, and accumulate there (25). Figure 4 illustrates an accumulation of macloptiages, some containing asbcsioy at an alveolar duct bifurcation. Quantitating this response, we found that more than 90% of first alveolar duct bifurcations in rats exposed to asbestos exhibit macrophage accu mulation, sometimes as many as JO15 cells in a cluster. Sham-exposed animals rarely have even a single macrophage on duct bifurcation sur faces (25). Two-thirds of the macro phages that had accumulated could be removed from bifurcation surfaces by lavage (25). These cells were stud ied in vitro (25) and had significant changes in morphology (increased numbers of smooth-surfaced cells), phagocytic potential (decreased par ticle uptake), and chemotactic capac ity (fewer cells migrating in blindwell chambers). The significance of these findings in regard to the patho genetic mechanisms of asbestos-in duced disease have yet to be deter mined. PRODUCTION OF AN ASBESTOS-INDUCED COMPLEMENT-DEPENDENT CHEMOTACTIC FACTOR FOR MACROPHAGES A number of investigators have pre sented evidence that macrophages play a central role in mediating par ticle-induced lung disease (5, 16). Therefore, we have tried to elucidate a mechanism through which these cells could be attracted initially to sites of asbestos deposition. We hy pothesize that absestos fibers that are deposited on duct bifurcations rapidly activate complement proteins on al veolar surfaces, consequently produc ing C5a, a known chemotactic factor for macrophages (24). The comple ment component C5 reportedly is found in the lungs (18, 20, 21), prob ably as a result of normal transudation of serum proteins from the vascula ture to air spaces. It has been shown that chrysolite asbestos fibers activate serum complement (C5) in vitro to produce C5a through the alternative pathway (22, 27). We postulate that such activation takes place in vivo on alveolar surfaces after fiber inhala tion (26). The results of four separate sets of experiments support our hypoth esis. I) Rats were exposed to aerosol ized chrysotile asbestos or sham-cx- 2598 FEDERATION PROCEEDINGS VOL *4. NO 10 JULY 1965 ST027278L to air for 1 or 5 h. The animals' lu-it lavaged 'tn'i'c-rliJU'U after fXposure. and then at 3. 24. and 48 (, and 8 days after exposure. Proteins in the lavaged fluid were concentrated and tested for the presence of a che- ^otactic factor that causes pulmonary macrophages to migrate in blind-well chambers (24). At all time periods except 8 days after exposure, there vns significantly greater chemoiactic response induced by proteins from the animals exposed to asbestos. The presence of chemoiactic factor im mediately after exposure preceded the accumulation of macrophages at alveolar duct bifurcations, although significant chemoiactic activity re mained through the 48-h period after exposure (26). 2) To demonstrate the presence of chemotactic potential in cell-free lavaged fluids, proteins con centrated from the lungs of rats were treated in vitro with chrysotile asbes tos or zymosan (a known activator of complement proteins). Both materials induced the production of a chemo- tacnc factor for macrophages. This factor had characteristics of a com plement-derived protein because che motactic activity was stimulated by zymosan and could be diminished by Ca2* and Mg2* chelators as well as by heating to 56 C. 3) To further evaluate our hypothesis that the che motactic factor is derived from com plement proteins, we exposed com plement-deficient rats and mice to aerosolized chrysotile asbestos. Rats were treated with cobra venom factor to deplete complement proteins, and the mice were genetically deficient in C5 (normal congemc mice were ex posed as controls). Quantitative re sults showed that both complement- Figure 4. Scanning electron micrograph of a terminal bronchiole (TB) and alveolar ducts (AD) divided by a duct bifurcation (Bf) in the lung of a rat 48 h after a I*h exposure to chrysotile asbestos. Deposition of asbestos fibers at the bifurcation region (9. II) has attracted pulmonary macrophages (25), and four separate clusters of macrophages can be seen (arrows). The cluster delineated by a rectangle, magnified in B. exhibits several macrophages (> that contain asbestos fibers (arrowheads). deficient rats and mice exhibited significantly fewer macrophages mi grating to duct bifurcations after as serum and lavaged protein. The ac PULMONARY MACROPHAGES bestos exposure. However, pulmo nary macrophages lavaged from these animals showed normal chemotactic activity in vitro (26). 4) Biochemical characterization was carried out on tivity was localized in the 14,000- to 18,000-dalton range, i.e., the mol wt of C5a. Taken together, the work reviewed above supports our hypothesis that AT ALVEOLAR DUCT BIFURCATIONS PARTICIPATE IN THE FORMATION OF AN ASBESTOS-INDUCED LESION the chemotactic factor in serum acti inhaled asbestos fibers activate com As a result of macrophage accumu vated with asbestos in vitro and in plement proteins on alveolar surfaces, lation at alveolar duct bifurcations (as lavaged fluids from rats exposed to consequently attracting local popula reviewed above (25)), the tissue area asbestos. The various molecular tions of macrophages to sites of as in this region more than doubled weight (mol wt) fractions chromato bestos deposition. A variety of mate only 48 h after a 1-h exposure to graphed from a Sephadex G-100 col rials activate serum complement; chrysotile asbestos. Macrophages cov umn were tested for chemotactic ac whether other inhaled particulates ered I b% of the surfaces of asbestos- tivity in blind-well chambers. Che activate complement and induce mac exposed bifurcations, whereas the motactic activity was clearly rophage accumulation in vivo is a macrophages on bifurcations in sham- demonstrated in both the activated topic of ongoing study. exposed rats were so few that they Response to acute lung injury 2599 ST0272785 5- TB 9 r 4 A' * i sisted for at least I month (13). Bv iiiis tunc, tlifie luti been .. normal ization of the epithelial cells and al veolar macrophages. In contrast, there was a dramatic threefold in crease in the numbers of interstitial macrophages and fibroblasts (13). Figure 5 illustrates the ultrastructural features of this lesion. This informa tion shows that a single, l-h exposure to chrysotile asbestos induces an an atomical lesion in which epithelial cells, macrophages, and fibroblasts participate. We anticipate that chronic exposure will cause this lesion to progress, forming first localized and then diffuse fibrotic scarring, and that an increase in asbestos dose will am plify the cellular responses illustrated above. Such ideas are being studied. SUMMARY AND CONCLUSIONS Figure 5. Light and transmission electron micrographs of an alveolar duct bifurcation in asbestos-exposed and sham-exposed rats. The light micrographs (A) illustrates a terminal bronchiole (TB) and two alveolar duct (AD) bifurcations (arrow) in the lung of an animal 48 h after a l-h exposure to chrysotile asbestos. The area delineated by a rectangle, magnified by transmission electron microscopy (B). shows macrophages (), prominent interstitial cells (1C), and connective tissue (CT). The duct bifurcation from a control animal (C) shows a prominent capillary bed (Ca). no macrophages, and comparatively little interstitial tissue (1). From ref 25. could not be measured by ultrastruc- exposed bifurcations were also signif tural morphometry. Interstitial and icantly increased in area (25). Some epithelial components of the asbestos- of these anatomical alterations per- In animals inhaling chrysotile asbestos and silica, we have shown that the particles are deposited initially on al veolar duct bifurcations and induce an almost immediate reaction of the alveolar epithelium. Subsequently, macrophages are recruited to sites of asbestos deposition by complement- derived chemotactic factors. In the interstitium of the duct bifurcation, fibers are phagocytized by fibroblasts, and intracellular microcalcifications commonly occur. In addition, a single l-h exposure to chrysotile asbestos leads to the development of a mea surable anatomical lesion that includes alterations of epithelial cells, macro phages, and fibroblasts at alveolar duct bifurcations. The biochemical and cellular mechanisms that mediate the early pathogenic events of silica- and asbes tos-induced disease have yet to be defined. We are pursuing the alter native, but not mutually exclusive, hypotheses that I) particle-induced interstitital lung disease is mediated by the effects of macrophage secre tions on interstitial fibroblasts, and/ or 2) fibrolic disease is the result of direct effects of toxic particles on a sensitive population of interstitial fi broblasts. REFERENCES I. Adler, K. B.; Brody, A. R.; Craig head, J. E. Studies on the mecha nism of mucin secretion by cells of 2600 the porcine tracheal epithelium. Proc. Soc. Exp. Btoi. Med 166: 3744: 1981. 2. Barak, L. S.; Nothnagel, E. A.; DeMarco, E. F. Differential stain ing of actin in metaphasc spindles .FEDERATION PROCEEDINGS VOL 44 NO. 10 JULY 1905 ST0272786 r v ,,,th 7-nitrobeti/-o\a-1 3.-<lij/iile- I I Brody, A. R.; Roe. M. W. Deposition 20 Reynolds. H. Y.; Newball, H. 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