Pathogenesis of Experimental Pulmonary Alveolar Proteinosis
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Thorax: first published as 10.1136/thx.25.2.230 on 1 March 1970. Downloaded from Thorax (1970), 25, 230. Pathogenesis of experimental pulmonary alveolar proteinosis B. CORRIN and E. KING Department of Morbid Anatomy, St. Thomas's Hospital Medical School, London, S.E.I, and the Occupational Hygiene Service, Manchester University Rats exposed to various airborne dusts developed a condition identical to pulmonary alveolar proteinosis as seen in man. The experimental condition developed through a stage of endogenous lipid pneumonia, characterized by numerous large foamy macrophages widely distributed through- out the lung. These cells broke down to release a finely granular material which finally condensed to reproduce the appearances of alveolar proteinosis. Electron microscopy indicated that the alveolar material was produced by type II pneumocytes and may therefore represent puImonary surfactant. A study of the dust-handling mechanism showed that in affected animals macrophage mobility was seriously impaired. Recent experiments comparing the effect of similar groups exposed under identical conditions to various dusts on the rat lung have been compli- various quartz modifications including the same purecopyright. cated by a process which as it advances repro- quartz did not contract alveolar proteinosis. Two duces the histological appearances of pulmonary groups of 24 rats each served as controls and alveolar proteinosis (Rosen, Castleman, and remained free of disease. The animals were killed Liebow, 1958). This complication was first en- with coal gas at monthly intervals up to 27 months. Details of the dusts, dust exposure apparatus, dust http://thorax.bmj.com/ countered in rats exposed to an aluminium dust concentrations, and chemical methods are given else- cloud (Corrin, 1962). A preliminary report of its where (Corrin, 1962; Corrin and King, 1969; presence in further dust inhalation experiments Corrin, Flindt, and King, 1970). (Corrin land King, 1966) stimulated its retrospec- At death the lungs were gently distended in situ tive recognition by Gough (1967), and the condi- with 4% formaldehyde and the entire thoracic con- tion has since been reported by others (Hepple- tents were then placed in an excess of fixative. ston, 1967; Gross and deTreville, 1968a). This Paraffin sections of a coronal slice of each lung and paper considers the development of experimental of the hilar lymph nodes were stained with haema- the of the toxylin and eosin, Masson's trichrome, sudan black alveolar proteinosis, efficiency pulmon- on September 26, 2021 by guest. Protected ary clearance mechanism in this condition, and for bound lipid, by Gordon and Sweet's method for the nature of the alveolar material as revealed by reticulin and by the periodic acid-Schiff (PAS) electron method. Frozen sections were stained for neutral microscopy. lipid with sudan red and by the Schultz reaction for MATERIALS AND MEETHODS cholesterol. For electron microscopy small pieces of fresh lung were immersed in cold (40 C.) 4% para- Alveolar proteinosis has been observed in the final formaldehyde in cacodylate buffer (pH 7-4) for one stage of two sets of experiments, both designed to hour, followed by cold osmium tetroxide in veronal test the fibrogenicity of various airborne dusts in the buffer (pH 74) containing 1% sucrose for a further rat lung. In the first experiment, 10 standard labora- hour. The tissues were embedded in maraglas. Ultra- tory albino rats were exposed to Pyro aluminium microtomy proved impossible with glass and a powder for 600 hours over a five-month period and diamond knife was required. The difficulty in sec- were killed with ether at 6, 7, 10, and 13 months. tioning was encountered even in relatively dust-free In the second experiment, four groups, each com- areas of the lung and appeared to be due to the prising 30 specific pathogen,free Wistar strain rats, alveolar proteinosis material as much as to the dusts. were exposed to pure quartz, quartz of 60% and 5% Thin sections were stained with uranium acetate and cristobalite content, and a non-fibrogenic feldspar, lead citrate and examined in a Siemens Elmiskop I for 3,000 hours over an eight-month period. Four electron microscope. 230 Thorax: first published as 10.1136/thx.25.2.230 on 1 March 1970. Downloaded from Pathogenesis of experimental pulmonary alveolar proteinosis '231 RESULTS ill-defined areas were affected throughout the lungs, which to the naked eye showed large chalky In the final stages of the experiments, areas of the grey or yellow patches on the pleural and cut lung were observed in which the alveoli were surfaces. The appearances at this stage were those filled with a finely granular eosinophilic deposit of endogenous lipid pneumonia (Gross, Brown, (Fig. 1). This contained occasional cholesterol and Hatch, 1952; Corrin and King, 1969) which crystals, dust particles and phagocytes with an has also been termed pulmonary lipidosis abundant eosinophilic cytoplasm similar in ap- (Jennings, Gresham, and Howard, 1965) and pearance to the surrounding alveolar material. multifocal histiocytosis (Yang, Yang, and Grice, The alveolar deposit was strongly PAS-positive 1966). Next, the macrophages broke down, releas- (diastase-resistant). Apart from the presence of ing a loose faintly eosinophilic floccular material dust the appearances were identical to those into the alveoli (Fig. 3). The cell margins of the described in man by Rosen et al. (1958). macrophages often appeared indistinct and there The development of this alveolar proteinosis was a marked resemblance of the extra-cellular could be traced from its earliest stages in those material to the swollen macrophage cytoplasm. experiments in which the animals were killed suc- Cholesterol crystals were found in the free cessively from the beginning of the dust expo- material, and occasionally cholesterol pneumonitis sures. In all dusted animals there was an increase with focal interstitial fibrosis was observed. More in alveolar macrophages, but this was more frequently, the cholesterol excited no inflamma- marked in those groups which later developed tory reaction and the alveolar walls remained thin alveolar proteinosis. Early animals from these with no evidence of fibrosis or cellular infiltration. groups showed particularly large numbers of Sudan red stains were weakly positive. Sudan macrophages, which had an unusually swollen black gave a stronger reaction and petroleum cytoplasm. These cells maintained a fairly diffuse ether extraction reduced the dry weight of the distribution in the lungs. They progressively in- lungs by 30% (controls 0 2 %). The faintly eosino- creased in number and size, assuming a large philic material was finely granular, weakly posi- copyright. vacuolated or foamy appearance (Fig. 2). Wide tive with PAS and light green with Masson's tri- http://thorax.bmj.com/ on September 26, 2021 by guest. Protected b~~~~~~~~~~~~~~~~~~~~~~~o FIG. 1. A strongly PAS positive deposit containing cholesterol crystal clefts fills the aveoli, reproducing the appearances of alveolar proteinosis. Rat lung 23 months after the commencement of feldspar exposure. PAS x 480. Thorax: first published as 10.1136/thx.25.2.230 on 1 March 1970. Downloaded from 232 B. Corrin and E. King .p .4 0_. a i: :w 4? _ ::: df ,j' ^ _ --: W_ *SIt.* x' a,,s* *^e . ._#i. 9,: ...'5,.t .:4.+ *itF.t :.a " ;' o * £ rSr , ..........8w> t,i s eFs . * .f :S FIG. 2. The alveoli contain many large foamy macrophages...:::',/ 'Rat . Xf_tlung z after 5 months' exposure to 60% cristobalite content quartz. H. and E. x 480. copyright. http://thorax.bmj.com/ f.*..*#.S. ; «2_ ...............~~A" ~* V *.: -,-e.d .i *'-, on September 26, 2021 by guest. Protected 43.. *:: 4tBe :. A o.'_. .} - <_ * _ J j: .,. t * u :: fiz: tw xs tt M _SPy tX .:a -t IA Si: ^s ° i L. w ..z _ w E; o i} ::. .: _e. *qr ws. ..:3.:y;,.,'. : ... *: :FIG. 3. A loose faintly staining deposit similar in appearance to the cytoplasm of neigh- bouring foam cells is seen in the alveolar space. Rat lung 23 months after the commence- ,nent of cristobalite and quartz exposure.eFFews w:^R\:: e,.:,-P .?H. ,and w - v '.*E. ::4sB. ......... .x¢,;.480. t. .... ' _w. _'' ;t:t Thorax: first published as 10.1136/thx.25.2.230 on 1 March 1970. Downloaded from Pathogenesis of experimental pulmonary alveolar proteinosis 233 .j, R: .>. 4.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.;:. FIG. 4. The finely granular but still loose comes to the alveoli. Condensation deposit fill copyright. of this material leads to the appearances seen in Fig. 1, 20 months after the commencement ofquartz exposure. H. and E. x 480. chrome stain. It gradually increased in amount an inverse relationship. The reason for this ap- http://thorax.bmj.com/ (Fig. 4) and became more compact. Concomitant peared to be a failure of the foamy macrophages with this it assumed a more deeply eosinophilic to achieve a high local concentration of silica. appearance, stained bright green with Masson's Even where the two conditions were closely asso- trichrome and became strongly PAS positive. By ciated, dust cells in the silicotic nodules differed this time the appearances of alveolar proteinosis from neighbouring foam cells in being smaller, already described were reproduced (Fig. 1). non-vacuolated and containing more dust. Trans- Whereas affected rats maintained a diffuse distri- port of dust to the hilar lymph nodes and subse- bution of swollen macrophages, in unaffected quent fibrosis were facilitated by the presence of animals relatively fewer phagocytes were able to lipid pneumonia, but in the lymph nodes the on September 26, 2021 by guest. Protected concentrate the inhaled dust at the centres of the characteristic foam cells seen in the lung were lung lobules (Macklin's dust sumps). Moreover, absent. Small compact macrophages were involved these macrophages were unaltered in appearance in the transport of dust to the lymph nodes and apart from containing dust and did not have an the larger foamy macrophages appeared to be abundant foamy cytoplasm.