Thorax: first published as 10.1136/thx.29.1.115 on 1 January 1974. Downloaded from Thorax (1974), 29, 115.

Fine structure and histochemistry of epithelioid cells in

E. M. VALERIE JAMES and W. JONES WILLIAMS Pathology Department, Welsh National School of Medicine, The Heath, Cardiff

James, E. M. Valerie and Jones Williams, W. (1974). Thorax, 29, 115-120. Fine structure and histochemistry of epithelioid cells in sarcoidosis. In this fine structure study of epithelioid cells in the granulomata of sarcoidosis we confirm our previous findings that they appear to be synthesizing rather than phagocytosing cells. The numerous characteristic intracyto- plasmic vesicles, 0 5 ,u in size, are shown to contain mucoglycoprotein and only rarely acid phosphatase activity. Epithelioid cells show varying quantities of extravesicular acid phos- phatase activity which is sometimes on the outer surface of the protein-containing vesicles. The possible role of secretory products of epithelioid cells in the formation and persistence ofgranulomata is discussed. It is suggested that epithelioid cells may be forming lymphokines.

We have previously demonstrated that the fine that of Ericsson and Trump (1965) using the lead structure of granulomata in sarcoidosis and tuber- nitrate Gomori technique. Substrate-free and sodium culosis (Jones Williams, Erasmus, James, and fluoride control blocks were also prepared. After being Davies, 1970), in the Kveim test lesion (Jones washed in acetate buffer solutions the tissues were post-fixed in Millonig's osmium tetroxide, dehydrated, Williams, 1972), and in chronic beryllium disease and embedded in Araldite. Sections were cut on an (Jones Williams, Fry, and James, 1972a) are LKB ultramicrotome and 0.5 ,u sections were stained http://thorax.bmj.com/ similar. The constituent epithelioid and giant cells with toluidine blue for light microscopy identification show numerous intracytoplasmic vesicles, varying of epithelioid cell granulomata. Ultrathin sections were in size from 04 to 07 ,u in diameter, and contain examined, either unstained or stained with uranyl amorphous lightly granular material (Fig. 1). acetate only. Evidence of active or previous in In order to assess the methods used we examined epithelioid cells is uncommon, and we consider and demonstrated intravesicular acid phosphatase that the cells are essentially biosynthetic rather activity in from a number of tissues, than phagocytic. including inflammatory lymph nodes, hyperplastic

spleen, and biochemically proven lysosomal fractions on October 2, 2021 by guest. Protected copyright. Our present histochemical study demonstrates from another hyperplastic spleen. that the majority of the vesicles contain muco- glycoproteins but not acid phosphatase. The sig- MUCOGLYCOPROTEINS Tissue blocks from two medi- nificance of these findings and the possible role astinal sarcoid lymph nodes and one positive Kveim of secretory substances in formation biopsy were fixed in 3% glutaraldehyde in 0-1 M are discussed. phosphate buffer pH 7-4 for two to four hours. After being washed ovemight in 0-1 M phosphate buffer the tissue was dehydrated, without postfixation in MATERIAL AND METHODS osmium tetroxide, and embedded in Araldite. Ultrathin sections of granulomatous areas were ACID PHOSPHATASE Tissue blocks of three sarcoid mounted on uncoated gold grids and were stained lymph nodes and one sarcoid skin biopsy were fixed for the detection of 1:2 glycol groups using the in 3% glutaraldehyde in 0-067 M cacodylate buffer, periodic acid, chromic acid silver methenamine (PA- pH 7-4 for two to four hours. After being washed CrA-silver) technique of Rambourg (1967) and overnight in 01 M cacodylate buffer the tissues were Rambourg, Hernandez, and Leblond (1969). Control incubated for 15 to 30 minutes in the acid phosphatase grids included those (a) unoxidized, and (b) blocked medium as either minute tissue blocks or 50 a Sorvall for four hours with a 2: 3 solution of acetic anhydride sections. The acid phosphatase technique used was and pyridine before oxidation. 115 Thorax: first published as 10.1136/thx.29.1.115 on 1 January 1974. Downloaded from 116 E. M. Valerie James and W. Jones Williams

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RESULTS pale vesicles which on light microscopy appeared foamy. Those with less heavy enzyme staining ACID PHOSPHATASE The localization of acid contained fewer, smaller, and more granular phosphatase, sodium ,3-glycerophosphatase in vesicles which were inconspicuous on light micro- epithelioid cells was similar when studied in 50 ,u scopy. Sorvall sections or tiny blocks. The epithelioid The enzyme staining was only rarely within the cells showed slight to heavy granular lead deposits characteristic vesicles but was sometimes attached free in the cytoplasm surrounding the mitochondria to their outer membrane. However, occasional and characteristic vesicles (Fig. 2). Those cells with cells showed a few small isolated vesicles with heavy enzyme staining contained abundant, large, heavy enzyme staining. and non-epi- Thorax: first published as 10.1136/thx.29.1.115 on 1 January 1974. Downloaded from Fine structure and histochemistry of epithelioid cells in sarcoidosis 119 thelioid mononuclear cells also showed occasional phagocytic ( type) cells. They rarely stained vesicles but no generalized cytoplasmic contain foreign material, residual bodies, phago- staining. cytic vesicles or acid phosphatase containing The majority of cell nuclei showed a varying vesicles. Our results also show that they do contain density of lead staining but this could not be numerous nmucoglycoprotein-containing vesicles, correlated with the cell type. Golgi complexes, and mitochondria. We cannot Both the substrate-free and sodium fluoride accept that the so-called 'epithelioid cells' pro- control technique resulted in completely negative duced under experimental conditions and develop- acid phosphatase staining in all epithelioid cells. ing from /macrophages (Papadimitriou and Spector, 1971 and 1972) are comparable to MUCOGLYCOPROTEINS The epithelioid and giant human epithelioid cells. These experimental cells cell vesicles gave a strong positive reaction with are rarely in closely packed foci, as in the human PA-CrA-silver technique (Fig. 3). The larger situation, are often described as containing lyso- vesicles gave a stronger reaction than the smaller somes, lipid droplets, and sometimes phagocytosed vesicles and the reaction product was most con- debris, and do not contain the characteristic 0-5 ,u spicuous on the inner aspect of the vesicle mem- diameter vesicles of human epithelioid cells. brane. Many of the vesicles showed a central On light microscopy we have previously demon- globular area of negative staining. Unoxidized strated (Williams, Jones Williams, and Williams, and blocking controls resulted in negative staining. 1969) with the Gomori technique the presence of These results demonstrate the presence of 1: 2 acid phosphatase in epithelioid cells and interpreted glycol-groups in the vesicles. the findings as indicating the presence of lyso- Both epithelioid and giant cells also show vary- somes. However, our present ul(trastructural ing numbers of electron dense, 222-296 A results do not confirm the above findings as the diameter, particles. These are randomly distributed enzyme activity when present was predominantly throughout the cytoplasm and are occasionally extravesicular and sometimes attached to the outer aggregated within membrane-bound vesicles. These membrane of the vesicles. We cannot explain the particles are considered to be glycogen as they difference merely on technique as within the same do not stain with the silver methenamine technique tissue we could show intravesicular staining in after periodic oxidation alone but are stained non-epithelioid cells and in macrophages in inflam- after treatment with both periodic and chronic matory lymph nodes. Further, we think it unlikely acids. This result is similar to that obtained in that intravesicular enzyme has leaked into the staining glycogen in liver. The particles are not cytoplasm because the dense activity which would http://thorax.bmj.com/ ribosomes, as ribosomes are difficult to visualize in have been expected on the inner surface of the unstained sections and measure only 148-178 A in vesicles was inconspicuous. We, therefore, con- diameter. clude that we may have misinterpreted the In addition, the carbohydrate cell coat, occa- granular staining on light microscopy as mem- sional small Golgi vesicles, and collagen were also brane-bound when in fact it was extravesicular stained and considered to be 1:2 glycol groups, and on the outside of the membrane. as the reaction was abolished in unoxidized and The occasional close apposition of acid phos- blocking controls. Ribosomes and nuclear phatase activity around secretory granules parallels on October 2, 2021 by guest. Protected copyright. chromatin remained positive in the controls and that found by Smith and Farquhar (1966), who therefore were regarded as being non-specifically in a study of hormone secretory granules in stained. pituitary glands showed an increase of enzyme activity around secretory vesicles commensurate with their increase in number. They interpreted DISCUSSION these findings as indicating a protective feed-back process in cells with excessive hormone produc- Our present report highlights the problem as to tion. A similar explanation may apply to excessive the exact nature and role of epithelioid cells in in epithelioid cells. sarcoid&type granulomata; in particular, whether Our description of the fine structure of they are primarily phagocytic (macrophages) or epithelioid cells agrees with that of other workers synthesizing cells. The problem is complicated in (Wanstrup and Christensen, 1966; Epstein, 1971) that the two functions are not necessarily mutually and we have now confirmed Gusek's (1965) exclusive and may change with time. suggestion that epithelioid cells might contain We consider that epithelioid cells in mature mucoglycoproteins. This latter feature, together human granulomata do not show the features of with the prominent Golgi complexes, suggests Thorax: first published as 10.1136/thx.29.1.115 on 1 January 1974. Downloaded from 120 E. M. Valerie James and W. Jones Williams that the cells are actively engaged in biosynthesis. Gusek, W. (1965). Histologie und elektronenmikroskopische speculate on the possible role komparative Zytologie tuberkuloser und epitheloid- It is interesting to zelliger Granulome. Fortschritte der Tuiberkuilose- of secretory products of epithelioid cells. It has Forschung, 14, 97. been suggested that they form para-amyloid (1968). Formale Pathogenese der hyalinen Transforma- (Wanstrup and Christensen, 1966), amyloid tion des Sarkoidosegranuloms. Virchovs Arlchilt A. (Gusek, 1968), and a granuloma-inciting factor Pathologische Anatomie, 345, 264. (Epstein, 1971). Jones Williams et al. (1970) Jones Williams, W. (1972). La Sarcoidose. Rappotrts diu suggested that the product may stimulate antigenic Symposium Europ&en de la Sarcoilose, Genmhe, 1971, committed lymphocytes to develop into epithelioid edited by Y. Gallopin, p. 29. , Erasmus, D. A., James, E. M. Valerie, and Davies, T. cells. It has recently been shown (Becker, Krull, (1970). The fine structure of sarcoid and tuberculous Deicher, and Kalden, 1972; Jones Williams, Pioli, . Postgraduate Medical Journal, 46, 496. Jones, and Dighero, 1972b) that lymphocytes from Fry, Elizabeth, and James, E. M. Valerie (1972a). sarcoid patients, when stimulated in vitro by The fine structure of Beryllium granulomas. .4cta Kveim antigen, produce a macrophage migration Pathologica et Microbiologica Scandanavica, Section A, inhibition factor which Remold and David (1971) 80, Suppl. 233, 195. chemical analysis consider to be a glycoprotein. , Pioli E., Jones, D. J., and Dighero, M. (1972b). The on Kmif (Kveim-induced macrophage migration inhibition In conclusion, we consider that epithelioid cells factor) test in sarcoidosis. Journal of Clinical Pathology, show both morphological and histochemical evi- 25, 951. dence of a synthesizing cell. The product appears Papadimitriou, J. M. and Spector, W. G. (1971).. The origin, to be a mucoglycoprotein which, if epithelioid properties and fate of epithelioid cells. Journsial of cells develop from lymphocytes, may be a lympho- Pathology, 105, 187. kine (Dumonde, 1970) and thus play a part in (1972). The ultrastructure of high- and low- both the formation and persistence of granulo- turnover inflammatory granulomata. Journal oJ Path- mata. ology, 106, 37. Rambourg, A. (1967). An improved silver methenamine E. M. V. J. was supported by the Clinical Research technique for the detection of periodic acid-reactive Fund of the Welsh Regional Hospital Board. complex carbohydrates with the electron microscope. Journal of Histochemistry and Cytochemistiy, 15, 409. - Hernandez, W., and Leblond, C. P. (1969). Detection of complex carbohydrates in the Golgi apparatus of rat REFERENCES cells. Journal of , 40, 395. Remold, H. G. and David, J. R. (1971). Further studies on Becker, F. W., Krull, P., Deicher, H., and Kalden, J. R. migration inhibitory factor (MIF): evidence for its http://thorax.bmj.com/ (1972). The leucocyte-migration test in sarcoidosis. glycoprotein nature. Journal of , 107, 1090. Lancet, 1, 120. Dumonde, D. C. (1970). 'Lymphokines': molecular mediators Smith, R. E. and Farquhar, Marilyn G. (1966). Lysosome of cellular immune responses in animals and man. function in the regulation of the secretory process in the Medicine, 63, 899. cells of the anterior pituitary gland. Journal of Cell Proceedings of Royal Society of Biology, 31, 319. Epstein, W. L. (1971). Metal-induced granulomatous hypersensitivity in man. In Advances in Biology of Skin. Wanstrup, J. and Christensen, H. E. 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New York. on October 2, 2021 by guest. Protected copyright. Ericsson, J. L. E. and Trump, B. F. (1965). Observations Williams, D., Jones Williams, W., and Williams, J. E. (1969). on the application to electron microscopy of the lead Enzyme histochemistry of epithelioid cells in sarcoidosis phosphate technique for the demonstration of acid and sarcoid-like granulomas. Journal of Pathology and phosphatase. Histochemie, 4, 470. Bacteriology, 97, 705.