Br. J. exp. Path. (1978) 59, 395
THE MORPHOLOGY OF ALVEOLAR TISSUE DESTRUCTION P. GROSS From0 the Departmient of Pathology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29403 Receive(d for publication March 13, 1978
Summary.-Destruction of intra-alveolar stroma in regions of tuberculous pneumonia is described. This destruction is associated with phagocytosis of argentophilic granules and fibre fragments by macrophages. The specific features of stromal destruction consist of sequestration, fragmentation, lysis, and other, less obvious, changes in reticulin and collagen fibres found in the air spaces. These findings have important implications in chronic fibrosing pneumonitis where similar intra-alveolar stromal changes occur. Destructive stromal changes have also been noted within granulomatous nodules of sarcoidosis in the region of giant cells. Some of the latter also contain argentophilic fibre fragments.
DESTRUCTION of alveolar septa in a lung abscess, in acid-fast or mycotic cavities, and within invasive lung tumours is a familiar sight to pathologists. The endstage of a more benign, and subtle lytic destruction of alveolar stroma that occurs in emphysema is also commonly seen in necropsy material although the earlier developmental stages of this destruction generally escape recognition. Far less note has been made of the degeneration and destruction of alveolar stroma that occurs in experimentally produced chronic pneumonitis as well as the chronic fibrosing pneumonitis found in hospital patients. The destruction of intra-alveolar inflammatory reticulin in experimental chronic pneumonitis has been interpreted as a "type of resolution" with the notation that this resolution is generally incomplete because the alveolar walls commonly do not return to their normal status but retain a residual, often considerable, degree of thickening (Gross et al., 1962). On the basis of 52 cases of active chronic pneumonitis studied, the destruction of intraalveolar inflammatory stroma has recently been characterized with the following distinctive features: 1. the presence of 27
structureless eosinophilic intra-alveolar material containing karyolytic nuclear remnants, 2. the presence of degenerated reticulin fibres within the air spaces, and 3. the phagocytosis of reticulin fibre fragments by macrophages (Gross and Goodwin, 1976). Whereas the presence of intra-alveolar structureless material associated with nuclear remnants is undoubtedly indicative of necrosis, this does not exclude the possibility that new stroma is being formed therein as has been claimed for organizing pneumonia. Although few doubts will arise with regard to the significance of phagocytosis of reticulin fibre fragments, more rigorous proof may be desirable to establish that the reticulin fragments demonstrated within the necrotic intraalveolar material are indeed a product of degeneration rather than of neoformation. In order to differentiate the reticulin stroma that has been newly formed from that undergoing necrosis, a study was made of the changes in this stroma in tuberculous pneumonia as compared to the appearance of the reticulin fibres in active sarcoidosis. It is the purpose of this paper to report my findings and conclusions of this study.
396
P. GROSS MATERIALS AND METHODS
Cases of pulmonary tuberculosis were reviewed and slides demonstrating caseous pneumonia or a desquamative type of pneumonitis were selected for further study. Cases of pulmonary sarcoidosis were similarly reviewed and slides were selected which contained granulomnata with prominent giant cells. All slides had been stained with haematoxylin and eosin. Forty-nine fields on slides from 12 cases were photographed at magnifications ranging from 33 to 150. The coordinates of each field were recorded. Following completion of the photography, the coverslips were removed, the sections were decolourized and subjected to the Gordon and Sweets method of silver impregnation (Gordon and Sweets, 1936). Because the sections tend to become detached during this procedure, a special technique (Tolker and Gross, 1961) was instituted before the silver impregnation to prevent the detachment. The same fields were then rephotographed and an additional 44 fields wAere photographed. After study of the negatives, it appeared desirable to obtain better visualization of the macrophages in relation to the connective tissue fibres. The coverslips were therefore remnoved a second time and the slides were subjected to a very brief staining with eosin. An additional 105 fields wrere then photographed. RESULTS
One of the anticipated rewards of the of the Gordon and Sweets technique is the excellent delineation of the alveolar stromal architecture even in regions of caseous necrosis (Fig. 1 and 2). However, in keeping with the concept of a fibrocaseous inflammation, the alveolar architecture is completely obliterated in some areas of caseous necrosis by proliferated reticulin, as well as collagen fibres. In other regions, the alveolar walls are markedly thickened by collagen and reticulin fibres. The most interesting and pertinent finding is the presence of reticulin and, much less frequently, of collagen fibres within the air spaces in association with degenerated macrophages or caseous material (Fig. 3). The reticulin fibres within the air spaces form irregular masses or solitary fragments. They are usually sequestered, i.e., they are separated from, and are not connected with the septal stroma. They differ from normal reticulin fibres by their irregular calibre caused by condensations and use
attenuations. Another difference is that many of the fibres fade off into invisibility (Fig. 3). Whereas in a normal reticulin network projecting naked ends of fibres are unusual, these are common in the sequestered reticulin masses and isolated fragments. Such naked ends are also common in the septal stroma near the alveolar surface where they are often oriented perpendicular to the surface. In many regions macrophages, though necrotic, are still recognizable. Occasionally a macrophage is seen in which the cell outline appears partially impregnated with argentophilic material (Fig. 3). In other regions, macrophages are filled with coarse argentophilic granules and occasional irregular, short, linear argentophilic fragments (Fig. 4). The presence of occasional coarse fibre fragments in the air spaces which have taken on the gold toning of collagen indicates that the destruction of stroma is not limited to reticulin. However, a more subtle indication of collagen destruction is noted in regions where the interstices within the basket-weave stain grey as though there has been a diffusion of argentophilia from the fibre bundles analogous to the diffusion of water-soluble paint upon a wet surface. The fact that fibres adjacent to the grey interstices stain grey in contrast to the black fibres elsewhere supports this interpretation (Fig. 5). A study of the granulomatous nodules in both tuberculosis and sarcoidosis disclosed that the reticulin fibre network is seriously disrupted in the region of the giant cells. The latter are nestled in spaces devoid of reticulin fibres (Fig. 6 and 7). Small fragments of reticulin fibres devoid of branches are found near the giant cells and many of these naked ends have beaded terminals. An occasional giant cell is found to contain fragments of reticulin fibres. No evidence of stromal destruction is found in the sections of sarcoidosis other than in the vicinity of the giant cells. In the peripheral portions of the granulomatous nodules or where there are no giant
*:. i ,w-
MORPHOLOGY OF ALVEOLAR TISSUE DESTRUCTION
4~~~~~~
FIG. 1. Early caseation in tuberculous pneumonia. Some of the air spaces are outlined by clefts caused by shrinkage of the intra-alveolar exudate. H. and E. x 110. FIG. 2. The same field as in Fig. 1 after silver impregnation showing the stromal architecture. Many septal walls are considerably thickened. The air spaces contain irregular masses of reticulin stroma that show varying degrees of degeneration and destruction. Gordon and Sweets. x 110. FIG. 3.-Intra-alveolar stroma in tuberculous pneumonia showing a highly irregular arrangement of the fibres with some naked ends demonstrable. Other fibres appear to fade off into invisibility. Many fibres are non-uniform in calibre and staining intensity. The cell walls of several macrophages appear partially argentophilic. Gordon and Sweets with light eosin counterstain. x 445. FIG. 4. The contents of another air space in tuberculous pneumonia showing macrophages filled with irregular coarse argentophilic granules and larger fragments. Argentophilic fibre fragments of various sizes are present among the macrophages. Gordon and Sweets with light eosin counterstain. x 445.
397
398
P. GROSS
MORPHOLOGY OF ALVEOLAR TISSUE DESTRUCTION
cells, the reticulin fibres forming a very loose network are thin, of uniform calibre and interweave without showing naked projecting ends. DISC USSION
W!hether or not pulmonary stroma has been destroyed is often not ascertainable from sections stained to demonstrate cellular details. Silver impregnation is essential for this determination. W"Iith the use of this technique it has been demonstrated in experimental animals that cellular necrosis and degeneration of alveolar stroma may occur independently of one another. It has been observed that cellular necrosis may be advanced and complete in the presence of persisting reticulin stroma sharply outlining alveolar and bronchiolar walls. Contrariwise, it has been noted that alveolar reticulin stroma may undergo degeneration and lysis without overt evidence of cellular necrosis (Gross, 1961). The literature contains brief references to the destruction of alveolar stroma in various diseases. For instance, "changes" in reticulin fibres as well as in elastic fibres have been noted within several hours of exposure to aerosolized papain (Johanson, 1972). Following exposure of rabbits to ozone, alveolar septa were found shortened, polypoid or mushroom-shaped, and the expanded peripheral portion generally contained a button-shaped mass of condensed reticulin (Gross, Scheel and Stokinger, 1965). In a study of radiation pneumonitis produced by x-rays, punctate and linear argentophilic fragments were observed within alveolar macrophages. This was considered suggestive of lysis of reticulin. A photomicrograph (Fig. 7) in this paper illustrates severe disruption and loss of alveolar reticulin (DeVilliers and Gross, 1967). The present observations on the character of the reticulin in regions of caseous necrosis confirm that previously published criteria of degeneration of reticulin fibres as encountered in chronic
399
fibrosing pneumonitis are indeed indicative of stromal destruction. The features characteristic of stromal destruction are the following: 1. The presence of many projecting naked ends of fibres. 2. The presence of isolated short fibre fragments. 3. The non-uniform calibre of fibres with foci of condensation and attenuation. 4. The sequestration of irregular stromal masses within the air spaces. 5. The fading of some fibres into invisibility. 6. The presence of coarse, irregular, argentophilic granules and argentophilic fibre fragments within macrophages. 7. The focal loss of gold toning of collagen. 8. The diminution of staining intensity of collagen fibre bundles with concomitant diffusion of argentophilic staining into the interstitiurn. With the exception of the first two of these features, which were also found in the region of giant cells in sarcoidosis, these changes have not been observed in the stroma of the latter disease. In our previous studies of granulomatous nodules containing giant cells, it has been a commonplace observation that the reticulin network in the region of giant cells is defective, but this has not been more closely examined until the present time. The occurrence of reticulin fibre fragments in the region of giant cells and the occasional finding of fibre fragments apparently within their cytoplasm suggests that these cells are capable of causing dissolution of the granulomatous nodule. This capability should not be surprising, since the macrophages that fused to form giant cells are known to possess proteolytic and phagocytic capabilities. Inasmuch as it is now believed that the end-stage of chronic fibrosing pneumonitis is the honeycomb lung (Spencer, 1968), it would appear reasonable that in addition to the collagenous fibrosis there must have been also considerable loss (by
400
P. GROSS
dissolution) of parenchymatous tissue to account for the large holes lined by bronchial or squamous epithelium that are characteristic of this disease. The hallmark of dissolution is to be found in those cases of fibrosing alveolitis in which the stroma is still largely composed of reticulin rather than collagen and in which intraalveolar structureless necrotic material is present (Gross and Goodwin, 1967). This material will generally contain remnants of reticulin stroma that has many of the features found in tuberculous pneumonia and which have been here listed. REFERENCES DEVILLIERS, A. J. & GROSS, P. (1967) Radiation Pneumonitis. X-ray induced Lesions in Hamsters and Rats. Arch. Environ. Health, 15, 649.
GORDON, H. & SWEETS, H. H., JR (1936) A Simple Method for Silver Impregnation of Reticulum. Am. J. Path., 12, 545. GROSS, P. (1961) The Morphology of Pulmonary Alveolar Reticulin Tissue. Arch. Pathol., 72, 438. GROSS, P. & GOODWIN, M. N. (1976) Tissue Destruction in Chronic Pneumonitis. Arch. Pathol., 100, 613. GROSS, P., MCNERNEY, J. M., WESTRICK, M. L. & BABYAK, M. A. (1962) Resolution of Chronic Interstitial Pneumonitis. Experimental Observations. Arch. Pathol., 74, 81. GRoss, P., SCHEEL, L. D. & STOKINGER, H. E. (1965) Ozone Toxicity Studies: Destruction of Alveolar Septa-a Precursor of Emphysema. Med. Thorac., 22, 376. JOHANSON, W. (1972) In Pulmonary Emphysema and Proteolysis. Charles Mittman, Ed. New York: Academic Press. p. 409. SPENCER, H. (1968) Pathology of the Lung, 2nd Edn. New York: Pergamon Press. p. 739. TOLKER, E. B. & GRoss, P. (1961) Detachment of Sections of Tissue: Its Prevention. Am. J. clin. Path., 36, 188.
THE MORPHOLOGY OF ALVEOLAR TISSUE DESTRUCTION P. GROSS From0 the Departmient of Pathology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29403 Receive(d for publication March 13, 1978
Summary.-Destruction of intra-alveolar stroma in regions of tuberculous pneumonia is described. This destruction is associated with phagocytosis of argentophilic granules and fibre fragments by macrophages. The specific features of stromal destruction consist of sequestration, fragmentation, lysis, and other, less obvious, changes in reticulin and collagen fibres found in the air spaces. These findings have important implications in chronic fibrosing pneumonitis where similar intra-alveolar stromal changes occur. Destructive stromal changes have also been noted within granulomatous nodules of sarcoidosis in the region of giant cells. Some of the latter also contain argentophilic fibre fragments.
DESTRUCTION of alveolar septa in a lung abscess, in acid-fast or mycotic cavities, and within invasive lung tumours is a familiar sight to pathologists. The endstage of a more benign, and subtle lytic destruction of alveolar stroma that occurs in emphysema is also commonly seen in necropsy material although the earlier developmental stages of this destruction generally escape recognition. Far less note has been made of the degeneration and destruction of alveolar stroma that occurs in experimentally produced chronic pneumonitis as well as the chronic fibrosing pneumonitis found in hospital patients. The destruction of intra-alveolar inflammatory reticulin in experimental chronic pneumonitis has been interpreted as a "type of resolution" with the notation that this resolution is generally incomplete because the alveolar walls commonly do not return to their normal status but retain a residual, often considerable, degree of thickening (Gross et al., 1962). On the basis of 52 cases of active chronic pneumonitis studied, the destruction of intraalveolar inflammatory stroma has recently been characterized with the following distinctive features: 1. the presence of 27
structureless eosinophilic intra-alveolar material containing karyolytic nuclear remnants, 2. the presence of degenerated reticulin fibres within the air spaces, and 3. the phagocytosis of reticulin fibre fragments by macrophages (Gross and Goodwin, 1976). Whereas the presence of intra-alveolar structureless material associated with nuclear remnants is undoubtedly indicative of necrosis, this does not exclude the possibility that new stroma is being formed therein as has been claimed for organizing pneumonia. Although few doubts will arise with regard to the significance of phagocytosis of reticulin fibre fragments, more rigorous proof may be desirable to establish that the reticulin fragments demonstrated within the necrotic intraalveolar material are indeed a product of degeneration rather than of neoformation. In order to differentiate the reticulin stroma that has been newly formed from that undergoing necrosis, a study was made of the changes in this stroma in tuberculous pneumonia as compared to the appearance of the reticulin fibres in active sarcoidosis. It is the purpose of this paper to report my findings and conclusions of this study.
396
P. GROSS MATERIALS AND METHODS
Cases of pulmonary tuberculosis were reviewed and slides demonstrating caseous pneumonia or a desquamative type of pneumonitis were selected for further study. Cases of pulmonary sarcoidosis were similarly reviewed and slides were selected which contained granulomnata with prominent giant cells. All slides had been stained with haematoxylin and eosin. Forty-nine fields on slides from 12 cases were photographed at magnifications ranging from 33 to 150. The coordinates of each field were recorded. Following completion of the photography, the coverslips were removed, the sections were decolourized and subjected to the Gordon and Sweets method of silver impregnation (Gordon and Sweets, 1936). Because the sections tend to become detached during this procedure, a special technique (Tolker and Gross, 1961) was instituted before the silver impregnation to prevent the detachment. The same fields were then rephotographed and an additional 44 fields wAere photographed. After study of the negatives, it appeared desirable to obtain better visualization of the macrophages in relation to the connective tissue fibres. The coverslips were therefore remnoved a second time and the slides were subjected to a very brief staining with eosin. An additional 105 fields wrere then photographed. RESULTS
One of the anticipated rewards of the of the Gordon and Sweets technique is the excellent delineation of the alveolar stromal architecture even in regions of caseous necrosis (Fig. 1 and 2). However, in keeping with the concept of a fibrocaseous inflammation, the alveolar architecture is completely obliterated in some areas of caseous necrosis by proliferated reticulin, as well as collagen fibres. In other regions, the alveolar walls are markedly thickened by collagen and reticulin fibres. The most interesting and pertinent finding is the presence of reticulin and, much less frequently, of collagen fibres within the air spaces in association with degenerated macrophages or caseous material (Fig. 3). The reticulin fibres within the air spaces form irregular masses or solitary fragments. They are usually sequestered, i.e., they are separated from, and are not connected with the septal stroma. They differ from normal reticulin fibres by their irregular calibre caused by condensations and use
attenuations. Another difference is that many of the fibres fade off into invisibility (Fig. 3). Whereas in a normal reticulin network projecting naked ends of fibres are unusual, these are common in the sequestered reticulin masses and isolated fragments. Such naked ends are also common in the septal stroma near the alveolar surface where they are often oriented perpendicular to the surface. In many regions macrophages, though necrotic, are still recognizable. Occasionally a macrophage is seen in which the cell outline appears partially impregnated with argentophilic material (Fig. 3). In other regions, macrophages are filled with coarse argentophilic granules and occasional irregular, short, linear argentophilic fragments (Fig. 4). The presence of occasional coarse fibre fragments in the air spaces which have taken on the gold toning of collagen indicates that the destruction of stroma is not limited to reticulin. However, a more subtle indication of collagen destruction is noted in regions where the interstices within the basket-weave stain grey as though there has been a diffusion of argentophilia from the fibre bundles analogous to the diffusion of water-soluble paint upon a wet surface. The fact that fibres adjacent to the grey interstices stain grey in contrast to the black fibres elsewhere supports this interpretation (Fig. 5). A study of the granulomatous nodules in both tuberculosis and sarcoidosis disclosed that the reticulin fibre network is seriously disrupted in the region of the giant cells. The latter are nestled in spaces devoid of reticulin fibres (Fig. 6 and 7). Small fragments of reticulin fibres devoid of branches are found near the giant cells and many of these naked ends have beaded terminals. An occasional giant cell is found to contain fragments of reticulin fibres. No evidence of stromal destruction is found in the sections of sarcoidosis other than in the vicinity of the giant cells. In the peripheral portions of the granulomatous nodules or where there are no giant
*:. i ,w-
MORPHOLOGY OF ALVEOLAR TISSUE DESTRUCTION
4~~~~~~
FIG. 1. Early caseation in tuberculous pneumonia. Some of the air spaces are outlined by clefts caused by shrinkage of the intra-alveolar exudate. H. and E. x 110. FIG. 2. The same field as in Fig. 1 after silver impregnation showing the stromal architecture. Many septal walls are considerably thickened. The air spaces contain irregular masses of reticulin stroma that show varying degrees of degeneration and destruction. Gordon and Sweets. x 110. FIG. 3.-Intra-alveolar stroma in tuberculous pneumonia showing a highly irregular arrangement of the fibres with some naked ends demonstrable. Other fibres appear to fade off into invisibility. Many fibres are non-uniform in calibre and staining intensity. The cell walls of several macrophages appear partially argentophilic. Gordon and Sweets with light eosin counterstain. x 445. FIG. 4. The contents of another air space in tuberculous pneumonia showing macrophages filled with irregular coarse argentophilic granules and larger fragments. Argentophilic fibre fragments of various sizes are present among the macrophages. Gordon and Sweets with light eosin counterstain. x 445.
397
398
P. GROSS
MORPHOLOGY OF ALVEOLAR TISSUE DESTRUCTION
cells, the reticulin fibres forming a very loose network are thin, of uniform calibre and interweave without showing naked projecting ends. DISC USSION
W!hether or not pulmonary stroma has been destroyed is often not ascertainable from sections stained to demonstrate cellular details. Silver impregnation is essential for this determination. W"Iith the use of this technique it has been demonstrated in experimental animals that cellular necrosis and degeneration of alveolar stroma may occur independently of one another. It has been observed that cellular necrosis may be advanced and complete in the presence of persisting reticulin stroma sharply outlining alveolar and bronchiolar walls. Contrariwise, it has been noted that alveolar reticulin stroma may undergo degeneration and lysis without overt evidence of cellular necrosis (Gross, 1961). The literature contains brief references to the destruction of alveolar stroma in various diseases. For instance, "changes" in reticulin fibres as well as in elastic fibres have been noted within several hours of exposure to aerosolized papain (Johanson, 1972). Following exposure of rabbits to ozone, alveolar septa were found shortened, polypoid or mushroom-shaped, and the expanded peripheral portion generally contained a button-shaped mass of condensed reticulin (Gross, Scheel and Stokinger, 1965). In a study of radiation pneumonitis produced by x-rays, punctate and linear argentophilic fragments were observed within alveolar macrophages. This was considered suggestive of lysis of reticulin. A photomicrograph (Fig. 7) in this paper illustrates severe disruption and loss of alveolar reticulin (DeVilliers and Gross, 1967). The present observations on the character of the reticulin in regions of caseous necrosis confirm that previously published criteria of degeneration of reticulin fibres as encountered in chronic
399
fibrosing pneumonitis are indeed indicative of stromal destruction. The features characteristic of stromal destruction are the following: 1. The presence of many projecting naked ends of fibres. 2. The presence of isolated short fibre fragments. 3. The non-uniform calibre of fibres with foci of condensation and attenuation. 4. The sequestration of irregular stromal masses within the air spaces. 5. The fading of some fibres into invisibility. 6. The presence of coarse, irregular, argentophilic granules and argentophilic fibre fragments within macrophages. 7. The focal loss of gold toning of collagen. 8. The diminution of staining intensity of collagen fibre bundles with concomitant diffusion of argentophilic staining into the interstitiurn. With the exception of the first two of these features, which were also found in the region of giant cells in sarcoidosis, these changes have not been observed in the stroma of the latter disease. In our previous studies of granulomatous nodules containing giant cells, it has been a commonplace observation that the reticulin network in the region of giant cells is defective, but this has not been more closely examined until the present time. The occurrence of reticulin fibre fragments in the region of giant cells and the occasional finding of fibre fragments apparently within their cytoplasm suggests that these cells are capable of causing dissolution of the granulomatous nodule. This capability should not be surprising, since the macrophages that fused to form giant cells are known to possess proteolytic and phagocytic capabilities. Inasmuch as it is now believed that the end-stage of chronic fibrosing pneumonitis is the honeycomb lung (Spencer, 1968), it would appear reasonable that in addition to the collagenous fibrosis there must have been also considerable loss (by
400
P. GROSS
dissolution) of parenchymatous tissue to account for the large holes lined by bronchial or squamous epithelium that are characteristic of this disease. The hallmark of dissolution is to be found in those cases of fibrosing alveolitis in which the stroma is still largely composed of reticulin rather than collagen and in which intraalveolar structureless necrotic material is present (Gross and Goodwin, 1967). This material will generally contain remnants of reticulin stroma that has many of the features found in tuberculous pneumonia and which have been here listed. REFERENCES DEVILLIERS, A. J. & GROSS, P. (1967) Radiation Pneumonitis. X-ray induced Lesions in Hamsters and Rats. Arch. Environ. Health, 15, 649.
GORDON, H. & SWEETS, H. H., JR (1936) A Simple Method for Silver Impregnation of Reticulum. Am. J. Path., 12, 545. GROSS, P. (1961) The Morphology of Pulmonary Alveolar Reticulin Tissue. Arch. Pathol., 72, 438. GROSS, P. & GOODWIN, M. N. (1976) Tissue Destruction in Chronic Pneumonitis. Arch. Pathol., 100, 613. GROSS, P., MCNERNEY, J. M., WESTRICK, M. L. & BABYAK, M. A. (1962) Resolution of Chronic Interstitial Pneumonitis. Experimental Observations. Arch. Pathol., 74, 81. GRoss, P., SCHEEL, L. D. & STOKINGER, H. E. (1965) Ozone Toxicity Studies: Destruction of Alveolar Septa-a Precursor of Emphysema. Med. Thorac., 22, 376. JOHANSON, W. (1972) In Pulmonary Emphysema and Proteolysis. Charles Mittman, Ed. New York: Academic Press. p. 409. SPENCER, H. (1968) Pathology of the Lung, 2nd Edn. New York: Pergamon Press. p. 739. TOLKER, E. B. & GRoss, P. (1961) Detachment of Sections of Tissue: Its Prevention. Am. J. clin. Path., 36, 188.
