Br. 1. Exp. Path. (I989) 70, 589-596

Current Status Review: A comparison of secretory epithelioid cells and phagocytosing in experimental mycobacterial J.L. Turk Department ofPathology, Royal College ofSurgeons ofEngland, Lincoln's Inn Fields, London WC2A 3PN, UK

The different histological appearance of gra- The production of epithelioid cell and non- nulomas induced by the same micro-orga- epithelioid cell granulomas nism dependent on the immunological status ofthe host is no better shown than in . The intradermal injection of BCG vaccine or In tuberculoid leprosy, where there is a high cobalt-irradiated Mycobacterium leprae into resistance to proliferation of the organism the dorsum of the ear of the guinea-pig and the development of T-- induced the development of granulomas in mediated allergic reactions, granulomas are the post-auricular lymph node immediately typically of the epithelioid cell type. In draining the site ofinjection. A direct paralle- lepromatous leprosy, where there is a failure lism was observed between the area of of T-cell-mediated immune reactions and granulomatous infiltration and the increase widespread proliferation of the infective in weight of the lymph node followed over a organism, the granulomas consist of macro- I 2-week period after intradermal injection of phages containing large numbers of the mycobacteria. Animals injected with BCG mycobacteria. In , epithelioid showed peak development 2 cell granulomas are classically associated weeks after injection, whereas those injected with proliferation and increased with M. leprae developed maximum granulo- local collagen synthesis. This is also a strong mas at 5 weeks (Narayanan et al. I982). The feature of epithelioid cell granulomas histological appearance ofthe lesion induced induced by other agents, such as beryllium, by BCG was that of a typical epithelioid cell and in . The demonstration of granuloma containing giant cells. As the rough endoplasmic reticulum in epithelioid lesions resolved there was an increase in the cells in granulomas in man (Ridley et al. amount of collagen as shown by conventio- I980) suggested a secretory function for nal stains and ultrastructurally (Fig. 3). these cells and indicated a need for the These granulomatous areas were heavily development ofsuitable experimental models infiltrated with cells of the mononuclear of these granulomas, particularly to study series (MPS) showing no evidence the possible connection between this func- of but containing extensive tion and fibroblast activation. One possible areas of rough endoplasmic reticulum model ofsecretory epithelioid cells associated suggestive ofa secretory function (Figs i and with increased fibroblast activation is the 4). By contrast, nodes from guinea-pigs zirconium granuloma in the guinea-pig injected with M. leprae showed a more mixed (Turk et al. 1978). However, these granulo- infiltration and under electron mas occur irregularly and being in the skin microscopy were found to have phagocy- are difficult to dissect out and study with any tosed organisms as well as a large amount of quantitative detail. degraded material (Figs 2 and 4) (Narayanan 589 J.L. Turk

Fig. i. Secretory epithelioid cell BCG granuloma 2 weeks. x 88oo. et al. i98 ia). were not a striking mon to other cells of the MPS. Moreover, by feature ofthese granulomas. In both types of the use of a directed granulomas the cells of the MPS were ester- against guinea-pig MHC Class II antigen, it ase positive and showed the presence of was found that although this antigen was fibronectin. However, the majority did not well expressed on the phagocytosing macro- carry receptors for the Fc component of IgG phages of the M. leprae granulomas, it was or the C3 component ofcomplement, nor did poorly expressed on the secretory epithelioid they exhibit peroxidase activity. The macro- cells of the BCG granulomas (Mathew et al. phages from the M. Ieprae granulomas were I983). highly glass adherent, whereas 80% of the Although in human leprosy the two types cells ofthe MPS in the BCG granulomas were of granuloma develop as a result of differ- non-glass adherent (Narayanan et al. I982). ences in the host's T-cell immune response, By the use of a monoclonal antibody that so far, these granulomas could be reproduced reacted specifically with activated macro- only by using the two different forms of phages, it was possible to demonstrate that mycobacteria. To bring the models into line the secretory epithelioid cells in the BCG with the human condition it was important granulomas, as well as the phagocytosing to see if changes in granuloma could be macrophages in the M. leprae granuloma, induced by changing the host's immune showed surface membrane antigenicity com- reaction to the organism. It was found that if Epitihelial cells and macrophages in granulomas 59I

i i t ' f , ,t. a i F~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.p

Fig. 2. Phagocytic macrophage M. leprae granuloma 5 weeks. x i8 000. the immune response was reduced by treat- on M. leprae. It was decided to add an extra ment with hydrocortisone or cyclosporine, strong antigenic determinant to M. leprae by the granulomas that developed in response the addition of the hapten fluorescein, by to BCG did not contain secretory epithelioid treating the organism with fluorescein isoth- cells. The cells of the MPS did not contain iocyanate. Such treatment caused the M. rough endoplasmic reticulum in their cyto- Ieprae to induce a secretory epithelioid cell plasm. In the case of cyclosporine-treated granuloma in which the cells of the MPS animals the cells contained phagocytosed were non-phagocytosing and contain rough material similar to that found in the M. leprae endoplasmic reticulum. At the same time granulomas (Gupta et al. I985b). Thus, the there was a marked increase in cell-mediated presence of secretory epithelioid cells in BCG immunity (Verghese et al. I987). Thus, there granuloma appeared to depend upon the appears to be a strong association between presence of a strong cell-mediated immune epithelioid cell formation and T-cell- response. mediated immunity in this model. The next question to be asked was, whether a secretory epithelioid cell granu- The association between epithelioid loma could be induced by M. leprae if the formation and increased collagen synthesis antigenicity of this organism was enhanced. It has been suggested that BCG contained a One of the ways to assay collagen synthesis number of antigens that were either not in tissues is that described by Peterkofsky and present or not as strongly expressed as those Diegelmann (I97I). In this, the tissue is 592 J.L. Turk

Fig. 3. Fibroblasts embedded in collagen BCG granuloma i week. X 1200. incubated with '4C-proline for 24 h. After genase used in these experiments contained homogenization the tissue is divided into two less than i% non-specific protease activity equal aliquots. One is then hydrolysed with when tested on '4C-tryptophan-labelled pro- the enzyme collagenase. The radioactivity in tein prepared from guinea-pig skin fibro- this fraction (H) is then subtracted from the blasts cultured in vitro. The specificity of the radioactivity in the unhydrolysed fraction collagenase was further assessed by its effect (T). The difference T-H then gives the '4C- on '4C-glycine labelled collagen prepared proline incorporated into collagen. The colla- from guinea-pig skin. There was a release of Epithelial cells and macrophages in granulomas 593 Antigen

Lymphokine

T-Lymphocyte Activated macrophage

Epithelioid cell

Fig. 4. Maturation of cells of the mononuclear phagocyte system.

90% of counts from the labelled collagen. It association between epithelioid cell forma- was clear that the nodes from BCG injected tion and increased fibroblast activity. As the animals synthesized high levels of collagen epithelioid cells were particularly character- when compared with nodes draining the site ized by the presence of a rough endoplasmic of application with the chemical sensitizer reticulum, it seemed reasonable to look for 2,4-dinitrofluorobenzene (DNFB) to the skin. the production of soluble mediators pro- Only nine out of 82 lymph nodes from duced by those cells that might affect fibro- animals injected with M. leprae showed blast function (Narayanan et al. i98ib). positive incorporation of '4C into collagen, Supernatants from the culture of lymph whereas 23 out of 84 nodes from the animals nodes containing BCG and M. leprae granulo- injected with the same dose of BCG showed mas were studied for their effect on protein positive incorporation (Narayanan et al. and DNA synthesis in homologous fibroblast 1982). Maximum collagen synthesis in BCG cultures. Supernatants from both types of nodes was between the fourth and fifteenth granulomas stimulated amino acid incorpor- weeks after injection. A similar increased ation and depressed DNA synthesis. A simi- incorporation of '4C-proline into collagen lar activity was released from lymph nodes has been described in the livers of mice containing activated by DNFB. infected with Schistosoma mansoni (Dunn et This suggested that the activity was derived al. I977). The finding of a significant in- from activated T-lymphocytes rather than crease in collagen synthesis by lymph nodes from cells of the MPS. However, dissociation containing epithelioid cell granulomas over from migration inhibitory factor suggested those containing non-epithelioid cell granu- an activity separate from more classical lomas suggested that there might be an lymphokines. 594 J.L. Turk Production of secretory products derived on epithelioid cells from BCG granulomas as directly from cells of the MPS opposed to M. Ieprae macrophages. Thus the stimulation of PGF2cx production could be The availability of a monoclonal antibody related to the poor expression of Class II specific for cells of the mononuclear phago- antigen. As PGE and PGE2 suppressed Class II cyte system of the guinea-pig (Mathew et al. expression in schistosome granulomas, it is I983) allowed the separation of these cells not surprising that no increase in the pro- from granulomas and their preparation with duction of these mediators was discovered in relative purity. This had not been previously the supernatants from mycobacterial granu- possible for epithelioid cells as these cells lomas. which were non-phagocytosing did not Two enzymes secreted by cells of the adhere to plastic or glass (Narayanan et al. mononuclear phagocyte system are lyso- I982). Epithelioid cells from BCG granulo- zyme and angiotensin-converting enzyme mas and macrophages from M. leprae granu- (ACE). High levels of circulating ACE are a lomas could then be stimulated with lipoly- particular feature of epithelioid cell granulo- saccharide (LPS) in vitro and the culture mas, particularly in sarcoidosis (Leiberman supernatants examined for their content of 1975). Increases in both lysozyme and ACE interleukin-i (IL-i). LPS-stimulated epithe- were found in lymph nodes carrying both lioid cells showed significantly higher IL-i BCG and M. leprae granulomas, but the levels activity, whereas LPS failed to enhance IL-i detected were no greater than in lymph production from M. leprae macrophages. nodes without granulomas which increased There have been a number of reports of the in size as part of an immune response. The association of IL-i activity and fibroblast increase in enzyme content paralleled the proliferation activity (Schmidt et al. I982; increase in weight of the node. Increased Wahl et al. 1978; Wahl & Wahl I98I; serum levels of these enzymes were also Leibovich & Ross 1976; Postlethwaite et al. found following the development of these I 983). The increased IL-i activity in epithe- granulomas. It would appear, therefore, that lioid cell supernatants was demonstrated in a lymph nodes containing BCG and M. leprae- fibroblast proliferative assay as well as in a induced granulomas show an increased syn- thymocyte comitogenic proliferative assay thesis of lysozyme and ACE. However, this is (Montreewasawat et al. I987). As no such not specific to the granulomatous changes fibroblast-stimulating activity was detected and could be partly the result ofproliferating in the culture supernatants from LPS-stimu- T-lymphocytes (Rea et al. I983). lated M. leprae granuloma macrophages, it could well be that IL-i secreted by epithelioid cells from BCG-induced granulomas plays a Accessory cell function of MPS cells in role in the resolution ofthese granulomas by granulomas fibrosis. There was no detectable amount of The demonstration of a difference in Class II E2 (PGE2) in the culture super- histocompatibility antigen expression on natants from either unstimulated or LPS- cells of the MPS derived from granulomas stimulated BCG and M. leprae-induced gra- prompted an investigation into the accessory nuloma cells. However, BCG granuloma cells cell function of these cells. Class II antigen secreted PGF2x spontaneously at higher expression is classically associated with anti- levels than macrophages from M. leprae gen presentation by accessory cells. An granulomas (Montreewasuwat et al. I987). accessory cell may be defined as a cell of the PGF2a has been reported to augment MHC MPS necessary for antigen presentation and Class II antigen expression in schistosome T-lymphocyte proliferation. Initially in these granulomas (Chensue et a). I983; Kunkel et experiments, cells were separated on the al. I984). Class II antigen is poorly expressed fluorescent-activated cell sorter using a Epithelial cells and macrophages in granulomas 595 monoclonal antimacrophage antibody. The References epithelioid cells from the BCG granulomas CHENSUE S.W., KUNKEL S.L., WARD P.A. & HIGASHI which expressed Class II antigen poorly were G.I. (I983) Exogenously administered prostag- able to support lymphocyte proliferation in landins modulate pulmonary granulomas the presence of the non-specific mitogen induced by Schistosoma mansoni eggs. Am. J. concanavalin-A (Con-A) but not with tuber- Path. III, 78-87. culin-PPD. Preparations of Class II positive COWLEY S., BuTTER C., VERGHESE S., CURTIS J. & from M. leprae granulomas did TuRuC J.L. (I988) Nerve damage induced by macrophages mycobacterial granulomas in guinea pig sciatic not show responsiveness to Con-A or PPD nerves. Int. 1. Leprosy 56, 2. (Gupta et al. i985a). Peripheral macro- DASTUR D.K. (1978) Leprosy (an infectious and phages from peritoneal of animals immunological disorder ofthe nervous system). carrying both BCG and M. leprae granulomas In Handbook of Clinical Neurology Volume 33, in the lymph nodes were able to act as Eds P.J. Vinken & G.W. Bruyn Amsterdam: accessory cells for both Con-A and antigen North Holland. pp. 421-468. proliferation in the presence of tuberculin- DUNN M.A., ROJKIND M., WARREN K.S., HAIT P.K., RIFAs L. & SEIFTER S. (I9 77) Liver collagen PPD. Although specific macrophage antigen synthesis in murine schistosomiasis. 1. Clin. positive cells from M. leprae granulomas were Invest. 59, 666-6 74. not able to act as accessory cells, there was a GuPTA S., CURTIS J. & TuRK J.L. (I 98 5a) Accessory population of Class II antigen positive cells cell function ofcells ofthe mononuclear phago- from these granulomas containing lymph cyte system isolated from mycobacterial granu- nodes that were able to perform this function lomas. Cell Immunol. 91, 425-433. GuPrA S.K., CURTIS J. & Tuiu J.L. (I985b) The (Montreewasuwat et al. I986). These cells effect of hydrocortisone and cyclosporin A on could be identified as dendritic cells using a bacillus Calmette-Guerin epithelioid cell granu- specific monoclonal antibody that reacted lomas. Cell Immunol. 93, I89-I98. with dendritic cells, but not with macro- KUNKEL S.L., CHENSUE S.W., PLEWA M. & HIGASHI phages (Verghese et al. I988). G.I. (I984) Macrophage function in the Schis- tomsoma mansoni egg-induced pulmonary gra- nuloma. Role ofarachidonic acid metabolites in Granuloma formation in peripheral nerves macrophage Ta antigen expression. Am. J. An important feature of both tuberculoid Pathol. 114, 240-249. and lepromatous leprosy in man is peripheral LIEBERMAN J. (I 9 75) Elevation of serum angioten- nerve damage (Dastur 1978). The intra- sin-converting enzyme level in sarcoidosis. Am. J. Med. 59, 365. neural injection into the sciatic nerve of the LEIBOVICH S.J. & Ross R. (I976) A macrophage guinea-pig of BCG vaccine or M. leprae dependent factor that stimulates the prolifer- induces an epithelioid cell granuloma or a ation of fibroblast in vitro. Am. J. Pathol. 84, macrophage granuloma respectively. These 50I-5I4. are similar to those described previously in MATHEW R.C., KATAYAMA I., GUPTA S.K., CURTIS J. lymph nodes ofguinea-pigs, draining the site & TURK J.L. (I983) Analysis of cells of the mononuclear phagocyte series in experimental of intradermal injection of the respective mycobacterial granulomas by monoclonal mycobacteria. Nerve damage was observed antibodies. Infect. Immun. 39, 344-352. as a result of both types of granuloma. This MONTREEWASUWAT N., CURTIS J. & TURK J.L. (I 986) was in both cases associated with demyelina- Accessory cell function of cells isolated from tion and some degree of Schwann cell pro- Mycobacterium leprae-induced granulomas. Cell liferation. Electrophysiological studies of Immunol. 102, 346-354. affected nerves showed a functional deficit in MONTREEWASUWAT N.. CURTIS J. & TURK J.L. (I 98 7) as a result ofboth of Interleukin i and prostaglandin production by nerve transmission types cells of the mononuclear phagocyte system granuloma (Cowley et al. I988). This is isolated from mycobacterial granulomas. Cell consistent with the observation that nerve Immunol. 104, I2-23. damage can occur in both clinical forms of NARAYANAN R.B., BADENOCH-JONES P. & TURK J.L. leprosy. (198 i a) Experimental mycobacterial granulo- 596 J.L. Turk mas in guinea pig lymph nodes: ultrastructural lear-phagocyte series (MPS) across the leprosy observations. J. Pathol. I34, 253-265. spectrum. 1. Pathol. 130, 223-227. NARAYANAN R.B., CURTIS J. & TURK J.L. (I98Ib) SCHMIDr J.A., MIZEL S.B., COHEN D. & GREEN I. Release of soluble factors from lymph nodes (I982) Interleukin-i, a potential regulator of containing mycobacterial granulomas and fibroblast proliferation.J. Immunol. I28, 2I77- their effect on fibroblast function in vitro. Cell 2I82. Immunol. 65, 93-IO2. TURK J.L., BADENOCH-JONES P. & PARKER D. (I978) NARAYANAN R.B., BADENOCH-JONES P., CURTIS J. & Ultrastructural observations on epithelioid cell TuRK J.L. (I982) Comparison of mycobacterial granulomas induced by zirconium in the gui- granulomas in guinea-pig lymph nodes. J. nea-pig. J. Pathol. 124, 45-49. Pathol. 138, 219-233. VERGHESE S., CuRTIs J. & TuRK J.L. (I987) Epithe- PETERKOFSKY B. & DIEGELMANN K. (I 9 7 I) Use of a lioid cell granuloma induction in the guinea pig mixture of proteinase-free collagenases for spe- by haptenated Mycobacterium leprae. Cell Immu- cific assay of radioactive collagen in the pres- nol. 107, 307-3 I 6. ence of other proteins. Biochemistry io, 988- VERGHESE S., HEALEY D.G., CURTIS J. & TURK J.L. 994. (1988) Accessory cell function ofdendritic cells Postlethwaite A.E., Lachman L.B., Mainardi C.L. from lymph nodes containing Mycobacterium & Kang A.H. (1983) Interleukin-i stimulation leprae induced granulomas. Int. Arch. Allergy of collagenase production by cultured fibro- Appl. Immunol. 87, 392-399. blasts. J. Exp. Med. 157, 8oi-8o6. WAHL S.M., WAHL L.M. & MCCARTHY J.B. (1978) REA T.H., NARAYANAN R.B. & TURK J.L. (I983) Lymphocyte-mediated activation of fibroblast Lysozyme and angiotensin converting enzyme proliferation and collagen production. J. Immu- levels in experimental mycobacterial granulo- nol. 121, 942-946. mas. 1. Pathol. 139, 207; 2I6. WAHL S.M. & WAHL L.M. (I98I) Modulation of RIDLEY M.J., BADENOCH-JONES P. & TuRK J.L. fibroblast growth and function by monokines (I980) Ultrastructure of cells of the mononuc- and lymphokines. Lymphokines, 2, I79-201.