Fine structure and histochemistry epithelioid cells in

Thorax (1974), 29, 115.

Fine structure and histochemistry of epithelioidcells in sarcoidosis

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 structureand histochemistry of epithelioid cells in sarcoidosis. In this fine structure study of epithelioidcells in the granulomata of sarcoidosis we confirm our previous findings that they appearto 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 acidphosphatase 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 persistenceof granulomata is discussed. It is suggested that epithelioid cells may be forming lymphokines.

We have previously demonstrated that the finestructure of granulomata in sarcoidosis and tuber-culosis (Jones Williams, Erasmus, James, andDavies, 1970), in the Kveim test lesion (JonesWilliams, 1972), and in chronic beryllium disease(Jones Williams, Fry, and James, 1972a) aresimilar. The constituent epithelioid and giant cellsshow numerous intracytoplasmic vesicles, varyingin size from 04 to 07 ,u in diameter, and containamorphous lightly granular material (Fig. 1).Evidence of active or previous phagocytosis inepithelioid cells is uncommon, and we considerthat the cells are essentially biosynthetic ratherthan phagocytic.Our present histochemical study demonstrates

that the majority of the vesicles contain muco-glycoproteins but not acid phosphatase. The sig-nificance of these findings and the possible roleof secretory substances in granuloma formationare discussed.

MATERIAL AND METHODS

ACID PHOSPHATASE Tissue blocks of three sarcoidlymph nodes and one sarcoid skin biopsy were fixedin 3% glutaraldehyde in 0-067 M cacodylate buffer,pH 7-4 for two to four hours. After being washedovernight in 01 M cacodylate buffer the tissues wereincubated for 15 to 30 minutes in the acid phosphatasemedium as either minute tissue blocks or 50 a Sorvallsections. The acid phosphatase technique used was

that of Ericsson and Trump (1965) using the leadnitrate Gomori technique. Substrate-free and sodiumfluoride control blocks were also prepared. After beingwashed in acetate buffer solutions the tissues werepost-fixed in Millonig's osmium tetroxide, dehydrated,and embedded in Araldite. Sections were cut on anLKB ultramicrotome and 0.5 ,u sections were stainedwith toluidine blue for light microscopy identificationof epithelioid cell granulomata. Ultrathin sections wereexamined, either unstained or stained with uranylacetate only.

In order to assess the methods used we examinedand demonstrated intravesicular acid phosphataseactivity in macrophages from a number of tissues,including inflammatory lymph nodes, hyperplasticspleen, and biochemically proven lysosomal fractionsfrom another hyperplastic spleen.

MUCOGLYCOPROTEINS Tissue blocks from two medi-astinal sarcoid lymph nodes and one positive Kveimbiopsy were fixed in 3% glutaraldehyde in 0-1 Mphosphate buffer pH 7-4 for two to four hours. Afterbeing washed ovemight in 0-1 M phosphate bufferthe tissue was dehydrated, without postfixation inosmium tetroxide, and embedded in Araldite.Ultrathin sections of granulomatous areas weremounted on uncoated gold grids and were stainedfor the detection of 1:2 glycol groups using theperiodic acid, chromic acid silver methenamine (PA-CrA-silver) technique of Rambourg (1967) andRambourg, Hernandez, and Leblond (1969). Controlgrids included those (a) unoxidized, and (b) blockedfor four hours with a 2: 3 solution of acetic anhydrideand pyridine before oxidation.

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116 E. M. Valerie James and W. Jones Williams

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FIG. 3. PA-CrA-silver stained vesicles illustrating the presence of 1: 2 glycol groups in an epithelioid cell. EMx 31,185.

RESULTS

ACID PHOSPHATASE The localization of acidphosphatase, sodium ,3-glycerophosphatase inepithelioid cells was similar when studied in 50 ,uSorvall sections or tiny blocks. The epithelioidcells showed slight to heavy granular lead depositsfree in the cytoplasm surrounding the mitochondriaand characteristic vesicles (Fig. 2). Those cells withheavy enzyme staining contained abundant, large,

pale vesicles which on light microscopy appearedfoamy. Those with less heavy enzyme stainingcontained fewer, smaller, and more granularvesicles which were inconspicuous on light micro-scopy.The enzyme staining was only rarely within the

characteristic vesicles but was sometimes attachedto their outer membrane. However, occasionalcells showed a few small isolated vesicles withheavy enzyme staining. Lymphocytes and non-epi-

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Fine structure and histochemistry of epithelioid cells in sarcoidosis

thelioid mononuclear cells also showed occasionalstained vesicles but no generalized cytoplasmicstaining.The majority of cell nuclei showed a varying

density of lead staining but this could not becorrelated with the cell type.

Both the substrate-free and sodium fluoridecontrol technique resulted in completely negativeacid phosphatase staining in all epithelioid cells.

MUCOGLYCOPROTEINS The epithelioid and giantcell vesicles gave a strong positive reaction withPA-CrA-silver technique (Fig. 3). The largervesicles gave a stronger reaction than the smallervesicles and the reaction product was most con-spicuous on the inner aspect of the vesicle mem-brane. Many of the vesicles showed a centralglobular area of negative staining. Unoxidizedand blocking controls resulted in negative staining.These results demonstrate the presence of 1: 2glycol-groups in the vesicles.Both epithelioid and giant cells also show vary-

ing numbers of electron dense, 222-296 Adiameter, particles. These are randomly distributedthroughout the cytoplasm and are occasionallyaggregated within membrane-bound vesicles. Theseparticles are considered to be glycogen as theydo not stain with the silver methenamine techniqueafter periodic oxidation alone but are stainedafter treatment with both periodic and chronicacids. This result is similar to that obtained instaining glycogen in liver. The particles are notribosomes, as ribosomes are difficult to visualize inunstained sections and measure only 148-178 A indiameter.

In addition, the carbohydrate cell coat, occa-sional small Golgi vesicles, and collagen were alsostained and considered to be 1:2 glycol groups,as the reaction was abolished in unoxidized andblocking controls. Ribosomes and nuclearchromatin remained positive in the controls andtherefore were regarded as being non-specificallystained.

DISCUSSION

Our present report highlights the problem as tothe exact nature and role of epithelioid cells insarcoid&type granulomata; in particular, whetherthey are primarily phagocytic (macrophages) orsynthesizing cells. The problem is complicated inthat the two functions are not necessarily mutuallyexclusive and may change with time.We consider that epithelioid cells in mature

human granulomata do not show the features of

phagocytic (macrophage type) cells. They rarelycontain foreign material, residual bodies, phago-cytic vesicles or acid phosphatase containingvesicles. Our results also show that they do containnumerous nmucoglycoprotein-containing vesicles,Golgi complexes, and mitochondria. We cannotaccept that the so-called 'epithelioid cells' pro-duced under experimental conditions and develop-ing from monocytes/macrophages (Papadimitriouand Spector, 1971 and 1972) are comparable tohuman epithelioid cells. These experimental cellsare rarely in closely packed foci, as in the humansituation, are often described as containing lyso-somes, lipid droplets, and sometimes phagocytoseddebris, and do not contain the characteristic 0-5 ,udiameter vesicles of human epithelioid cells.On light microscopy we have previously demon-

strated (Williams, Jones Williams, and Williams,1969) with the Gomori technique the presence ofacid phosphatase in epithelioid cells and interpretedthe findings as indicating the presence of lyso-somes. However, our present ul(trastructuralresults do not confirm the above findings as theenzyme activity when present was predominantlyextravesicular and sometimes attached to the outermembrane of the vesicles. We cannot explain thedifference merely on technique as within the sametissue we could show intravesicular staining innon-epithelioid cells and in macrophages in inflam-matory lymph nodes. Further, we think it unlikelythat intravesicular enzyme has leaked into thecytoplasm because the dense activity which wouldhave been expected on the inner surface of thevesicles was inconspicuous. We, therefore, con-clude that we may have misinterpreted thegranular staining on light microscopy as mem-brane-bound when in fact it was extravesicularand on the outside of the membrane.The occasional close apposition of acid phos-

phatase activity around secretory granules parallelsthat found by Smith and Farquhar (1966), whoin a study of hormone secretory granules inpituitary glands showed an increase of enzymeactivity around secretory vesicles commensuratewith their increase in number. They interpretedthese findings as indicating a protective feed-backprocess in cells with excessive hormone produc-tion. A similar explanation may apply to excessivesecretion in epithelioid cells.Our description of the fine structure of

epithelioid cells agrees with that of other workers(Wanstrup and Christensen, 1966; Epstein, 1971)and we have now confirmed Gusek's (1965)suggestion that epithelioid cells might containmucoglycoproteins. This latter feature, togetherwith the prominent Golgi complexes, suggests

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E. M. Valerie James and W. Jones Williams

that the cells are actively engaged in biosynthesis.It is interesting to speculate on the possible role

of secretory products of epithelioid cells. It hasbeen suggested that they form para-amyloid(Wanstrup and Christensen, 1966), amyloid(Gusek, 1968), and a granuloma-inciting factor(Epstein, 1971). Jones Williams et al. (1970)suggested that the product may stimulate antigeniccommitted lymphocytes to develop into epithelioidcells. It has recently been shown (Becker, Krull,Deicher, and Kalden, 1972; Jones Williams, Pioli,Jones, and Dighero, 1972b) that lymphocytes fromsarcoid patients, when stimulated in vitro byKveim antigen, produce a macrophage migrationinhibition factor which Remold and David (1971)on chemical analysis consider to be a glycoprotein.

In conclusion, we consider that epithelioid cellsshow both morphological and histochemical evi-dence of a synthesizing cell. The product appearsto be a mucoglycoprotein which, if epithelioidcells develop from lymphocytes, may be a lympho-kine (Dumonde, 1970) and thus play a part inboth the formation and persistence of granulo-mata.

E. M. V. J. was supported by the Clinical ResearchFund of the Welsh Regional Hospital Board.

REFERENCES

Becker, F. W., Krull, P., Deicher, H., and Kalden, J. R.(1972). The leucocyte-migration test in sarcoidosis.Lancet, 1, 120.

Dumonde, D. C. (1970). 'Lymphokines': molecular mediatorsof cellular immune responses in animals and man.Proceedings of the Royal Society of Medicine, 63, 899.

Epstein, W. L. (1971). Metal-induced granulomatoushypersensitivity in man. In Advances in Biology of Skin.Vol XI: Immunology and the Skin, Ed. W. Montagnaand R. E. Billingham, p. 313. Appleton-Century-Crofts,New York.

Ericsson, J. L. E. and Trump, B. F. (1965). Observationson the application to electron microscopy of the leadphosphate technique for the demonstration of acidphosphatase. Histochemie, 4, 470.

Gusek, W. (1965). Histologie und elektronenmikroskopischekomparative Zytologie tuberkuloser und epitheloid-zelliger Granulome. Fortschritte der Tuiberkuilose-Forschung, 14, 97.(1968). Formale Pathogenese der hyalinen Transforma-tion des Sarkoidosegranuloms. Virchovs Arlchilt A.Pathologische Anatomie, 345, 264.

Jones Williams, W. (1972). La Sarcoidose. Rappotrts diuSymposium Europ&en de la Sarcoilose, Genmhe, 1971,edited by Y. Gallopin, p. 29., Erasmus, D. A., James, E. M. Valerie, and Davies, T.(1970). The fine structure of sarcoid and tuberculousgranulomas. Postgraduate Medical Journal, 46, 496.Fry, Elizabeth, and James, E. M. Valerie (1972a).

The fine structure of Beryllium granulomas. .4ctaPathologica et Microbiologica Scandanavica, Section A,80, Suppl. 233, 195., Pioli E., Jones, D. J., and Dighero, M. (1972b). TheKmif (Kveim-induced macrophage migration inhibitionfactor) test in sarcoidosis. Journal of Clinical Pathology,25, 951.

Papadimitriou, J. M. and Spector, W. G. (1971).. The origin,properties and fate of epithelioid cells. Journsial ofPathology, 105, 187.

(1972). The ultrastructure of high- and low-turnover inflammatory granulomata. Journal oJ Path-ology, 106, 37.

Rambourg, A. (1967). An improved silver methenaminetechnique for the detection of periodic acid-reactivecomplex carbohydrates with the electron microscope.Journal of Histochemistry and Cytochemistiy, 15, 409.

- Hernandez, W., and Leblond, C. P. (1969). Detectionof complex carbohydrates in the Golgi apparatus of ratcells. Journal of Cell Biology, 40, 395.

Remold, H. G. and David, J. R. (1971). Further studies onmigration inhibitory factor (MIF): evidence for itsglycoprotein nature. Journal of Immunology, 107, 1090.

Smith, R. E. and Farquhar, Marilyn G. (1966). Lysosomefunction in the regulation of the secretory process incells of the anterior pituitary gland. Journal of CellBiology, 31, 319.

Wanstrup, J. and Christensen, H. E. (1966). Sarcoidosis.1. Ultrastructural investigations on epithelioid cellgranulomas. Acta Pathologica et Microbiologica Scanda-navica, 66, 169.

Williams, D., Jones Williams, W., and Williams, J. E. (1969).Enzyme histochemistry of epithelioid cells in sarcoidosisand sarcoid-like granulomas. Journal of Pathology andBacteriology, 97, 705.

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