Thorax (1973), 28, 559.

Electron microscopy of chlorphentermine lung

PAUL SMITH, DONALD HEATH, and P. S. HASLETON

The Department of Pathology, University of Liverpool

Smith, P., Heath, D., and Hasleton, P. S. (1973). Thorax, 28, 559-566. Electron microscopyof chlorphentermine lung. Electron microscopy of lung tissue from rats treated with chlor-phentermine shows an intense hyperplasia in the alveolar walls of granular pneumocyteswhich remain attached and do not migrate into the alveolar spaces. Numerous 'foam cells'are found free in the alveoli and are histiocytes which have ingested osmiophilic lamellarbodies. Similar laminated inclusions are found in granular pneumocytes, membranouspneumocytes, bronchiolar Clara cells, pulmonary macrophages, and endothelial cells ofpulmonary capillaries. The intra-alveolar debris is rich in osmiophilic lamellar bodies withassociated phospholipid lattices. The appearances are those of a toxic alveolitis. Furtherstudies of chronic toxicity are suggested to ascertain if this acute lesion progresses to fibrosingalveolitis.

In the preceding paper we showed that admini-stration of chlorphentermine for 50 days to ratsdoes not give rise to hypertensive pulmonaryvascular disease but instead leads to pronouncedchanges in the lung parenchyma (Heath, Smith,and Hasleton, 1973). These consist of a cellularhyperplasia of the alveolar walls and an accumu-lation of 'foam cells' in the alveolar spaces. In thepresent study we identified these cells in thealveolar walls and spaces by electron microscopyand studied the ultrastructure of the associatedintra-alveolar debris.

METHODS

A piece of lung tissue for electron microscopy wastaken from the lateral third of the superior lobe of theright lung in each rat. This was placed on a piece ofcard in a pool of glutaraldehyde and cut into smallfragments measuring approximately 1 mm across.These were transferred to bottles containing ice-coldbuffered glutaraldehyde and allowed to fix for 24hours. The blocks of tissue were washed in isotonicsucrose solution and post-fixed in 1 % buffered osmiumtetroxide for 1 hour before being embedded in Araldite.Thin sections (1 ,um) were cut and stained withtoluidine blue for the selection of suitable areas forelectron microscopic examination. The blocks werethen trimmed and fine sections were cut with an LKBUltrotome III ultramicrotome to obtain silver-colouredsections. The sections were floated onto copper grids,stained with uranyl acetate and lead citrate, andexamined with an AEI EM6B electron microscopeusing an acceleration voltage of 60 kV.

RESULTS

There was an intense hyperplasia of cells attachedto the alveolar walls. These had the ultrastructuralcharacteristics of granular (type II) pneumocyteswhich have been described elsewhere (Brewer,Heath, and Asquith, 1969). In particular, thecells contained many lamellar bodies (Fig. 1).These granular pneumocytes were shaped like thesegment of a circle, the chord of this segment beingrepresented by the straight surface of the cellwhich was attached to the alveolar wall. Thecurved surfaces of the cells projecting into thealveolar space were covered by short straightmicrovilli (Fig. 1). Such ultrastructural featureswere found only in cells still attached to thealveolar wall.Many large cells were lying free in the alveolar

spaces and they contained large and numerousosmiophilic lamellar inclusions (Fig. 2). At firstsight they appeared to be granular pneumocytesbut on closer examination they included lysosomaldark bodies, many of which surrounded, or werein close association with, lamellar material. Thesecells appeared to be macrophages which had in-gested material derived from the true lamellarbodies of granular pneumocytes (Figs 3 and 4).In this paper we shall restrict the term 'lamellarbody' to the osmiophilic laminated bodies foundonly in a recognizable granular pneumocyte.We shall refer to laminated material of similarstructure in other cells as osmiophilic lamellar

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Electron microscopy of chlorphentermine lung

inclusions. Virtually all cells lying free in alveolarspaces were macrophages of this type. All thecells that could be identified positively as granularpneumocytes were attached to alveolar walls.Many of the intra-alveolar cells showed centralpale areas in which the cytoplasm and its containedorganelles appeared to be in the process of dis-integration (Fig. 5). Such areas of pallor clearlycorresponded to the pale-staining areas in intra-alveolar cells that we had previously observed onlight microscopy (Heath et al., 1973).

Lamellar material was not confined to granularpneumocytes and intra-alveolar macrophages.Osmiophilic lamellar inclusions were also foundabundantly within the bronchioles in Clara cellswhich are devoid of cilia and quite distinct instructure from the respiratory epithelial cells (Fig.6). They were also seen within the cytoplasmof membranous type I pneumocytes which mayhave engulfed this material from the debris lyingfree in alveolar spaces (Fig. 7). Alternatively, thepresence of osmiophilic lamellar bodies in thesecells may be an expression of cellular degenera-tion. The membranous pneumocyte could beidentified by its flattened, attenuated form and itsposition between the fused basement membranesof the alveolar wall and the cavity of the alveolus(Fig. 7). Endothelial cells, lining pulmonarycapillaries and lying within the fused basementmembranes of the alveolar wall, also containedlamellar inclusions (Fig. 8). Hence osmiophiliclamellar inclusions were found in no fewer thanfive classes of cell in the lung, namely the granularpneumocyte, the membranous pneumocyte, theClara cell, the intra-alveolar macrophage, and theendothelial cell. It is not surprising that lamellarmaterial of this type was so widespread since thealveolar spaces contained large numbers ofdisintegrating intra-alveolar cells and much debrisresulting from this disintegration. As a result thealveoli were packed with lamellar material andlattices of phospholipid (Fig. 9). The combinationof substances corresponds to the apparentlyamorphous debris staining a cherry-pink colourwith the periodic acid Schiff stain that was foundon light microscopy. The intra-alveolar cellsstaining a magenta colour with this staincorrespond to intra-alveolar macrophages.

Electron microscopy also revealed the earlystages of interstitial pulmonary oedema with theproduction of blebs and endothelial vesicles whichprotruded into the lumina of the pulmonarycapillaries (Fig. 10). There was no evidence ofearly interstitial pulmonary fibrosis.

DISCUSSION

Our study shows that in the lungs of rats receivingchlorphentermine there is an intense hyperplasiaof granular pneumocytes in the alveolar walls.Their cytoplasm becomes filled with lamellarbodies and it seems likely that many of these areliberated into the alveoli. Significantly we foundnone of these granular pneumocytes lying free inthe alveolar spaces; they always remained attachedto the alveolar walls. This suggests that the 'foamcells' found so profusely in the alveolar spaces onlight microscopy (Heath et al., 1973) are notgranular pneumocytes but another variety of cell.The ultrastructural features of the free intra-

alveolar cells, which gave a magenta colour withthe PAS stain on light microscopy, suggest thatthey are macrophages. They present difficulty inidentification because they ingest so much of theintra-alveolar debris rich in osmiophilic lamellarmaterial and phospholipid that they come toresemble granular pneumocytes. Their trueidentity is revealed by the presence of lysosomalbodies. Groups of these pulmonary histiocytescongregate together in alveoli and then undergodisintegration to give rise to the particulate intra-alveolar debris which on light microscopy staineda cherry-pink colour with the PAS stain. As wehave seen, this debris is packed with osmiophiliclamellar material and phospholipid lattices.

Chlorphentermine certainly produces a remark-able pathological picture in the lungs of rats withosmiophilic lamellar inclusions abounding notonly free in the alveolar spaces but in no fewerthan five classes of cell, namely the granular(type II) pneumocyte, the membranous (type I)pneumocyte, the bronchiolar Clara cell, the intra-alveolar macrophage, and the endothelial cellsof pulmonary capillaries. Nevertheless, this re-markable pulmonary histiocytosis and profusionof lamellar material is in no sense specific forchlorphentermine administration. Virtually identi-cal changes were noted by Vijeyaratnam andCorrin (1972a) in the lungs of rats receiving oraliprindole. This imino dibenzyl derivative is usedclinically as an antidepressant drug and whengiven to rats gives rise to an accumulation of cellsin the alveolar spaces similar in appearance to thosewhich we have seen in desquamative interstitialpneumonia in man (Brewer et al., 1969). Vijeya-ratnam and Corrin (1972a) found that the freeintra-alveolar cells showed high acid phosphataseand f3 glucuronidase activity, suggesting that theywere macrophages. This was confirmed byelectron microscopy and by the avid ingestion of

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Paul Smith, Donald Heath, and P. S. Hasleton

thorium particles introduced via the trachea.These authors were convinced that the iprindolelesion represents a pulmonary histiocytosis ratherthan an alveolar epithelial desquamation and thismirrors our own opinion of the chlorphenterminelesion.The origin of the free pulmonary histiocytes

was demonstrated by Vijeyaratnam and Corrin(1972b), who studied mitotic counts and the ultra-structure of the lungs of iprindole-treated rats.They found that the pulmonary histiocytosis wasaccompanied by proliferation of cells in theinterstitial tissues of the lung which they regardedas the immediate precurors of the alveolar macro-phages. They point out that such findings arenot incompatible with cells derived from extra-pulmonary tissues, such as bone marrow, under-going further division and maturation within theinterstitial tissues of the lung before entering theair-spaces as alveolar macrophages.The interstitial oedema that we have found in

the lungs of rats treated by chlorphentermine(Fig. 10) was also noted by Vijeyaratnam andCorrin (1973) in their studies on iprindole. Theyfound a leakage of thorium particles which hadbeen injected intravenously and they noted-aswe have in the case of chlorphentermine-degenerative changes in the endothelial cells ofpulmonary capillaries. This suggested to them thatthe pulmonary oedema was due to increasedcapillary permeability rather than to raised intra-vascular pressure. Just as in the case of chlor-phentermine Vijeyaratnam and Corrin foundlamellar osmiophilic inclusions in all five classesof pulmonary cell that we list above.The present study reveals only the ultra-

structural details of the acute stage of the pul-monary disease induced by the administration ofchlorphentermine; it does not demonstrate thecourse and nature of the chronic lesions that mustfollow those which we have described here. Theprofuse hyperplasia of granular pneumocytesfollowing the administration of a drug is somewhatreminiscent of busulphan lung, a diseasecharacterized by the subsequent developmentof fibrosing alveolitis (Littler, Kay, Hasleton, andHeath, 1969). In view of this we think it desirableto carry out chronic studies with chlorphenter-

mine to see if the acute lesions described here willprogress to fibrosing alveolitis. In other words,it is important to ascertain if the predominantaction of the drug is on the lung parenchyma. Inview of the continuing controversy surroundingthe suspected relation between anorexigens andpulmonary hypertension it seems equally im-portant to exclude the possibility that chronicadministration of the drug may lead to involve-ment of the pulmonary vasculature withhypertensive pulmonary vascular disease.

Vijeyaratnam and Corrin (1972a) note that pul-monary histiocytosis is common in rats andconsidered that the absence of this cellularexudation in other species justified the use ofiprindole in clinical trials. On the other hand,we feel that chlorphentermine is only one ofmany anorexigens available to the medical pro-fession and doubt the wisdom of prescribing adrug that can lead to such dramatic changes inthe lung parenchyma of a laboratory animal. Itshould be noted here that similar changes are notproduced by the anorexigen aminorex fumarateat present suspected of causing pulmonaryhypertension. In our previous studies on thisdrug (Kay, Smith and Heath, 1971) we have notfound the remarkable proliferation of pulmonaryhistiocytes such as is produced by chlorphen-termine.

REFERENCESBrewer, D. B., Heath, D., and Asquith, P. (1969). Electron

microscopy of desquamative interstitial pneumonia.Journal of Pathology, 97, 317.

Heath, D., Smith, P., and Hasleton, P. S. (1973). Effectsof chlorphentermine on the rat lung. Thorax, 28, 551.

Kay, J. M., Smith, P., and Heath, D. (1971). Aminorex andthe pulmonary circulation. Thorax, 26, 262.

Littler, W. A., Kay, J. M., Hasleton, P. S., and Heath, D.(1969). Busulphan lung. Thorax, 24, 639.

Vijeyaratnam, G. S., and Corrin, B. (1972a). Pulmonaryhistiocytosis simulating desquamative interstitial pneu-monia in rats receiving oral iprindole. Journal ofPathology, 108, 105.

- and (1972b). Origin of the pulmonary alveolarmacrophage studied in the iprindole-treated rat.Journal ofPathology, 108, 115.

- and (1973). Fine structural alterations in the lungsof iprindole-treated rats. Journal ofPathology (In press).

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