Epithelioid granuloma induced by muramyl dipeptide in immunologically deficient rats.

When WKA rats were either neonatally thymectomized or injected with anti-rat thymocyte sera, their T-cell functions were effectively suppressed. When neonatally thymectomized plus anti-rat thymocyte serum-treated rats were injected with non-immunogenic muramyl dipeptide in water-in-oil emulsion, they produced massive epithelioid granulomas. Essentially, no morphological difference was noticed between granulomas induced in untreated rats and in thymectomized plus anti-rat thymocyte serum-treated rats. These findings strongly suggest that muramyl dipeptide-induced epithelioid granulomas required no T cells for their formation. In contrast, the induction of adjuvant arthritis appeared to depend on the presence of T cells.     

Epithelioid granuloma formation by a synthetic bacterial cell wall component, muramyl dipeptide (MDP).

A synthetic muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-D-isoglutamine) is a minimal essential structure that is contained generally in bacterial cell walls and is responsible for their many biologic activities such as adjuvant activity, pyrogenicity, and a capacity to confer resistance against bacterial and viral infections. We found that this MDP evoked dose-dependently massive organized epithelioid granulomas in guinea pigs, when injected in the form of Freund-type water-in-oil emulsion. Granuloma formation reached a peak at 3 weeks. A minimal effective dose of MDP was 0.1 microgram. Essentially, no difference was observed qualitatively among granulomas evoked by MDP, MDP plus antigen, and killed tubercle bacilli incorporated in the emulsion. Quantitatively, however, MDP was stronger in its granulomagenic capacity than tubercle bacilli. Antigenicity of MDP was not detectable. These findings support our proposal that MDP may be a chemical structure in tubercle bacilli essential for epithelioid granuloma formation and that the MDP-induced epithelioid granuloma may be of a nonallergic nature.     

Comparison of mycobacterial granulomas guinea-pig lymph nodes

A study was made of mycobacterial-induced granulomas in guinea-pig lymph nodes. Live BCG (Pasteur) induced a granuloma containing epithelioid cells while Cobalt irradiated Mycobacterium leprae induced a granuloma comprised of phagocytic macrophages. The granulomas were quantitated by measurement of lymph node weight and the areas of infiltration in histological sections. The time course of granuloma formation induced by Co-irradiated M. leprae was very different from the time course of the granuloma formation induced by BCG. Collagen synthesis assessed by incorporation of ¹⁴C-proline into collagenase sensitive protein was greater in lymph nodes draining the site of injection of Co-irradiated BCG than those draining the site of injection of Co-irradiated M. leprae during the first 10 weeks. Collagen synthesis was delayed in the nodes from animals injected with live BCG for at least 10 weeks. Single cell suspensions of draining lymph nodes containing granulomas consisted of lymphocytes and large cells (epithelioid cells and macrophages). A high proportion of the large cells were found to be non-adherent in the live BCG-induced epithelioid cell granuloma. In contrast, M. leprae-induced granulomas contained a high percentage of adherent large cells. In both the granulomas, the majority of large cells were esterase positive and showed the presence of fibronectin. Most of the large cells in the granulomas did not carry receptors for the Fc component of IgG or the C3 component of complement and did not exhibit peroxidase activity.     

Isolated epithelioid cells from disaggregated BCG granulomas—an ultrastructural study

Non-caseating epithelioid granulomas have been induced in rats by the subcutaneous injection of BCG vaccine and their cellular disaggregation to yield isolated epithelioid cells has been achieved using collagenase. Ultrastructurally epithelioid cells in both intact granulomas and disaggregated cell suspensions showed a spectrum of appearances ranging from cells with conspicuous rough surfaced endoplasmic reticulum (“plasmacytoid epithelioid cells”) to cells containing numerous cytoplasmic membrane-bound vesicles (“vesicular epithelioid cells”). Numerous intermediate forms were present. Endocytosed material was inconspicuous. Investigation of disaggregated 4, 10 and 28 day lesions showed that the proportion of vesicular epithelioid cells increased with maturation of the granulomas. Nevertheless a full spectrum of epithelioid cell morphology was present as early as 4 days. It is suggested that mononuclear phagocyte cells entering the granuloma transform into vesicular epithelioid cells via an intermediate plasmacytoid stage. The successful isolation of viable epithelioid cells from granulomas may allow th function of these cells to be evaluated further.     

Granuloma formation by synthetic bacterial cell wall fragment: muramyl dipeptide.

A synthetic muramyl dipeptide, N-acetylmuramyl-L-alanyl-D-isoglutamine, which possesses the same structure as that of a part of the peptidoglycan monomer of wax D of tubercle bacilli or bacterial cell walls was found to induce, when injected in water-in-oil emulsion, massive granulomas often accompanying abscesses in the site of injection and draining lymph nodes of guinea pigs and rats. The granulomas were composed mainly of epithelioid cells 2 weeks after injection and were indistinguishable from those induced by tubercle bacilli. The granulomas induced in rats were less mature than those induced in guinea pigs. Allergic reaction appeared to play no important role in the development of the muarmyl dipeptide-induced granuloma.     

L3T4+ T cells induce hepatic lesions resembling primary biliary cirrhosis in mice with graft-versus-host reactions due to major histocompatibility complex class II disparity

We had shown that the appearance of hepatic lesions such as epithelioid granulomas and chronic nonsuppurative destructive cholangitis (CNSDC)-like bile duct changes characteristic of primary biliary cirrhosis (PBC) in mice undergoing MHC class II-disparate graft-versus-host reactions (GVHR). To further examine the pathogenesis of the disease, we examined in the present study which T cell subset, i.e., L3T4+ or Lyt-2+ T lymphocytes, had an ability to induce such hepatic lesions. (B6 x bm12)F1 recipients were injected with unseparated T cells, L3T4+, or Lyt2+ T cells of B6 mice and on various days postinjection liver specimens were obtained. At Day 14 postinjection, livers of mice injected with whole T cells or L3T4+ T cells showed PBC-like histological changes, but none of the lesions were induced by Lyt-2+ T cells. Immunohistochemical studies revealed that Lyt-2+ as well as L3T4+ T cells were detected around bile ducts and some of them were infiltrating among bile duct epithelial cells. Kinetic studies showed that shortly after injection of L3T4+ T cells, L3T4+ T cells appeared around bile ducts and then Mac-1+ cells emerged. Lyt-2+ T cells and surface IgM+ B cells were detected on Day 5 and increased thereafter. Hepatic granulomas consisted of both L3T4+ and Lyt-2+ T cells with a few B cells. The aberrant expression of MHC class II (Ia) antigen was detected mainly at the lateral surface of bile duct epithelial cells by Day 14 postinjection. Based on these findings, the developmental mechanism of PBC-like hepatic lesions induced in mice with GVHR was discussed.     

Plasmacytoid Monocytes in Epithelioid Cell Granulomas: Ultrastructural and Immunoelectron Microscopic Study

Plasmacytoid monocytes, the so-called plasmacytoid T cells, were originally described in rare cases of lymphadenitis. Recent immunohistochemical studies have demonstrated their monocytic origin. Plasmacytoid monocytes have in common with epithelioid cells and multinucleated giant cells the expression of several antigens; they also occur in close topographic association with epithelioid and multinucleated giant cells in epithelioid cell granulomas. On the basis of these data it has been suggested that plasmacytoid monocytes may transform into epithelioid cells. The present ultrastructural and immunoelectron microscopic study of epithelioid cell granulomas provides furthei arguments in favor of this hypothesis. Moreover, the existence of a transitional cell type with characteristics of plasmacytoid monocytes and epithelioid cells is documented. Subplasmalemmal linear densities present on focal areas of the plasma membrane of the main cell components of granulomas are also discussed.     

The role of Kupffer cells in glucan-induced granuloma formation in the liver of mice depleted of blood monocytes by administration of strontium-89

In order to elucidate the role of Kupffer cells in granuloma formation in the liver of mice under a condition of severe monocytopenia induced by administration of strontium-89, granulomas were produced by particulate glucan injection and examined histopathologically, immunohistochemically, by [3H]thymidine autoradiography, and in culture experiments. Hepatic granulomas were smaller, less numerous, and more irregularly shaped in the monocytopenic mice than in the control mice. The granulomas were composed of multinuclear giant cells, epithelioid cells, Kupffer cells, and T lymphocytes, but not monocytes or granulocytes. Kupffer cells were heavily labeled with [3H]thymidine in the monocytopenic mice, particularly just before the stage of granuloma formation, and then clustered in the liver sinusoids. At 8 days, they formed granulomas, transformed into epithelioid cells, and transformed further into multinuclear giant cells. Although the culture of liver cell suspensions prepared from the livers of monocytopenic mice sustained diffuse proliferation of macrophages on a monolayer of mouse stromal cell line (ST2), no monocyte/macrophage colonies were formed. From these results, it is reasonable to conclude that Kupffer cells alone are activated in a condition without a supply of monocytes from peripheral blood; proliferate and cluster in the hepatic sinusoids; transform into peroxidase-negative macrophages, epithelioid cells, and multinuclear giant cells; and participate in granuloma formation in loco together with T lymphocytes.     

Sarcoidosis--1977.

Sarcoidosis is defined as a multisystem disorder characterised by the finding of epithelioid cell granulomas in more than one system. Diagnosis is aided by the use of the Kveim Siltzbach skin test and the development of an "in vitro" Kmif test is discussed. Despite extensive researches the causative agent(s) remains unknown. The granulomas, morphologically, on light and electron microscopy and histochemistry may be indistinguishable from those caused by known agents. Inclusion bodies are also non specific. Central necrosis is rare, and can be usually distinguished from caseation. The close relationship between the monocyte derived, epithelioid cells and lymphocytes is emphasised. Evidence is accumulating that epithelioid cells in sarcoid type granulomas are primarily synthesising rather than phagocytic cells. The products are considered to be mucoglycoproteins and may have both local and systemic actions. Locally it is suggested that the products may be lymphokines which react with associated thymic derived (T) lymphocytes and mononuclear cells and thus play a role in perpetuating the granulomas. Epithelioid cells may also be a source of circulating T lymphocyte function depressants. It has further been suggested that epitheloid cells are the source of the raised angiotensin converting enzyme found in sarcoid sera. Study of epithelioid cell granulomas in sarcoidosis, despite the disappointing lack of evidence of a causative sarcoid agent(s), is thus of considerable interest in furthering knowledge of many diseases characterised by these curious cellular foci.     

Muramyl dipeptide and mononuclear cell supernatant induce Langhans-type cells from human monocytes

Muramyl dipeptide (MDP) in bacterial cell walls reportedly evokes epithelioid cell granulomas. We examined its effects on multinucleated-giant-cell (MGC) formation from monocytes. Supernatant of concanavalin A-stimulated peripheral blood mononuclear cells (conditioned medium) generated MGCs from monocytes. MDP significantly increased the fusion index of Langhans-type MGCs (LGCs) but did not affect total MGCs. N-Acetylmuramyl-L-alanyl-L-isoglutamine, an MDP analogue, had no effect on MGC formation. MGCs were produced by conditioned medium from CD14(++)/CD16(-) monocytes. MDP enhanced the LGC fusion index from CD14(++)/CD16(-) monocytes. MGCs were not produced from CD14(+)/CD16(+) monocytes or immature dendritic cells induced by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL) 4 and only weakly produced from macrophage (M)-CSF- or GM-CSF-induced macrophages. Added MDP did not generate MGCs from CD14(+)/CD16(+) monocytes or dendritic cells but enhanced LGC formation from macrophages. Because IFN-gamma, IL-3, and GM-CSF reportedly are important in LGC induction, we added anti-IFN-gamma, anti-IL-3, or anti-GM-CSF monoclonal antibody (mAb) concomitantly to the monocyte culture treated with conditioned medium alone or plus MDP. Anti-IFN-gamma mAb completely abrogated MGC generation, whereas anti-GM-CSF and anti-IL-3 mAbs significantly inhibited LGCs. These findings suggest that CD14(++)/CD16(-) monocytes are fused to form LGCs by MDP derived from granulomatous-disease-causing pathogens with inflammatory mediators such as IFN-gamma, IL-3, and GM-CSF.     

Immunohistochemical observation of lysozyme in macrophages and giant cells in human granulomas.

Lysozyme activity of macrophages and giant cells in various human granulomas were examined with immunoperoxidase bridge method in tissue sections. Various numbers of epithelioid cells and giant cells of epithelioid cell granulomas of tuberculosis, sarcoidosis and Crohn's disease exhibited intense granular cytoplasmic lysozyme activity. Foreign body granulomas induced with various substances showed negative or faintly positive lysozyme stain. Macrophages and giant cells of aspergillus granuloma associated with thymus hypoplasia and T-cell depression contained no lysozyme. The results suggest that cell-mediated immunology plays an important role for the lysozyme synthesis of macrophages in granuloma.     

IMMUNOHISTOCHEMICAL OBSERVATION OF LYSOZYME IN MACROPHAGES AND GIANT CELLS IN HUMAN GRANULOMAS

Lysozyme activity of macrophages and giant cells in various human granulomas were examined with immunoperoxidase bridge method in tissue sections. Various numbers of epithelioid cells and giant cells of epithelioid cell granulomas of tuberculosis, sarcoidosis and Crohn's disease exhibited intense granular cytoplasmic lysozyme activity. Foreign body granulomas induced with various substances showed negative or faintly positive lysozyme stain. Macrophages and giant cells of aspergillus granuloma associated with thymus hypoplasia and T-cell depression contained no lysozyme. The results suggest that cell-mediated immunology plays an important role for the lysozyme synthesis of macrophages in granuloma.     

Granulomatous inflammation in normal and athymic mice infected with schistosoma mansoni: an ultrastructural study.

Inflammatory cell types and their interrelationships were studied in hepatic schistosome egg granulomas by correlated light and electron microscopy in thymus-intact and athymic mice. Intact animals developed large granulomas composed of phagocytes, stimulated macrophages, epithelioid cells, eosinophils and mast cells. The lesions peaked in size between 10--14 weeks after infection and tended to heal after 16 weeks. In athymic mice only phagocytes, stimulated macrophages and possible epithelioid cells appeared in the granulomas which were much smaller and less well organised than in intact mice. Virtually no eosinophils or mast cells entered the granulomas. These findings support the idea that development of granulomatous inflammation per se is not determined by CMI, but T cell co-operation seems to be required for its full expression in schistosomiasis. Since CMI closely regulates ingress of eosinophils and mast cells into the granuloma, the nature of T cell co-operation probably occurs at several levels in this complex infectious granuloma.     

Immunohistologic comparison between armadillo-derived leprosin and standard lepromin skin tests in leprosy patients

A comparison was made on the in situ immunological characteristics of dermal infiltrates of early (24-hour) and late (3-4 weeks) skin reactions in leprosy patients. The skin reactions were induced by armadillo-derived leprosin coupled to liposomes and standard Dharmendra lepromin. Most lymphocytes in the early reaction induced by both antigens were positive for Leu 4, Leu 3a, OKT8 and Ia like antigens indicating thereby the presence of activated T cells. The ratio of Leu 3a/OKT8+ cells were similar. In the late reaction elicited by both antigens, the lymphocytes in the granulomas were predominantly activated T lymphocytes expressing Leu 4, Leu 3a, OKT8 and Ia like antigens. Leu 3a+ cells were scattered diffusely amidst the epithelioid cells. In contrast, the OKT8+ cells were present mainly as 'a ring' in the periphery of the granuloma. A similar ratio of Leu 3a+/OKT8+ cells was observed in these granulomas. Macrophages in the granulomas expressed Ia like antigens. These observations indicate that the immunological characteristics of dermal infiltrates in the skin reaction induced by armadillo-derived leprosin coupled to liposomes and standard Dharmendra lepromin appear to be identical.     

Macrophages in T cell line-mediated, demyelinating, and chronic relapsing experimental autoimmune encephalomyelitis in Lewis rats.

About 50% of the mononuclear cells in the perivascular lesions in the central nervous system (CNS) of rats suffering from experimental allergic encephalomyelitis (EAE) are blood-borne macrophages. In this study we investigated the role of these macrophages in different variants of EAE, using a liposome-mediated macrophage depletion technique. Intravenously injected liposomes containing dichloromethylene diphosphonate (Cl2MDP) are ingested by macrophages and cause temporary and selective elimination of these cells. Macrophage depletion during EAE induced by a T cell line specific for myelin basic protein (MBP; T cell-EAE) suppresses development of neurological signs of EAE. T cell-EAE with pronounced demyelination as induced by an additionally injected MoAb directed against myelin oligodendrocyte glycoprotein (MOG) was also significantly ameliorated after macrophage depletion. During chronic relapsing EAE (CR-EAE) the occurrence of relapses was prevented or suppressed, provided that the liposomes were injected before the initiation of a putative relapse. A chronic progressive course of CR-EAE was not modified by Cl2MDP containing liposome treatment. Histologic examination of the CNS of liposome-treated animals confirmed decreased infiltration of macrophages into the parenchyma in the rats with T cell and AD-EAE, whereas T cells were still present.     

Experimental mycobacterial granulomas in guinea pig lymph nodes: Ultrastructural observations

A systematic ultrastructural study has been performed of the mononuclear phagocytes in granulomas induced by different types of mycobacteria, e.g. live BCG (Pasteur), irradiated M. leprae and irradiated BCG (Pasteur) in guinea pig lymph nodes. Live BCG (Pasteur) induces a granuloma which peaks at 2-3 weeks and by light microscopy, a large number of the infiltrating cells have the appearance of epithelioid cells. Ultrastructurally a large proportion of these cells have a distinct appearance. They are characterised by the presence of very large nuclei, and prominent nucleoli, paucity of cytoplasmic organelles and swollen rough endoplasmic reticulum. Cells from 2 week granulomas induced by live M. kansasii and BCG (Glaxo), had a similar ultrastructural appearance. BCG (Pasteur) granulomas slowly resolved and by light and electron microscopy fibrosis and collagen deposition was seen by 7 weeks and was very extensive in some nodes at 10 weeks. The nodes from M. leprae injected animals showed peak infiltration at 5 weeks and by light microscopy the infiltrating cell population was more mixed, with most of the cells having a macrophage appearance. Electron-microscopy showed these infiltrating cells to be mainly activated macrophages, containing phagocytosed organisms. Nodes from irradiated BCG (Pasteur) injected animals had peak infiltration at 1 week. The light and electron microscopic appearance showed areas consisting mainly of fibroblasts and mononuclear phagocytes. Thus, the granulomas induced by M. leprae are very different from those induced by BCG in the guinea pig. It is suggested that lymph nodes draining areas of local injection of BCG and irradiated M. leprae could respectively form a good model in the guinea pig for cellular and biochemical studies of the granulomas of tuberculoid and lepromatous leprosy.     

The Structure of Mononuclear Phagocytes Differentiating In Vivo: I. Sequential Fine and Histologic Studies of the Effect of Bacillus Calmette-Guerin (BCG)

Although the differentiation of mononuclear phagocytes is fundamental to their multifarious activities, their differentiation is incompletely understood—particularly in vivo. The development of an epithelioid granuloma may be hypothesized to represent such differentiation in vivo. To test this, the sequential ultrastructure of developing epithelioid granulomas was examined. Viable bacilli Calmette-Guerin (BCG) injected into the subcutaneum of guinea pigs produced epithelioid granulomatous inflammation, which was sampled for light and electron microscopy on alternate days until the 33rd day after injection. Initially, monocytes invaded the tissues and then coalesced, enlarged and formed small granulomas which ultimately evolved into epithelioid granulomas. The monocytes, ultrastructurally very simple cells, developed increased nuclear euchromatin, prominent nucleoli, extensive cytoplasm, free ribosomes, abundant Golgi profiles, many mitochondria and numerous large lysosomes and became macrophages. The macrophages in turn underwent further enlargement and became closely intertwined with one another to form epithelioid cells—large polygonal macrophages, containing euchromatic nuclei, numerous lysosomes, plentiful mitochondria and prominent synthetic apparatus. These changes undergone by monocytes during their development into epithelioid cells, which may be divided into five stages, are interpreted as differentiation in vivo of the mononuclear phagocytes. The observations demonstrate directly the differentiation of these cells in vivo and suggest some, if not all, characteristic features of granulomatous inflammation result from such differentiation.     

Plasmacytoid monocytes (so-called plasmacytoid T cells) in granulomatous lymphadenitis

Immunohistochemical evidence that the plasmacytoid T cell is closely related to the blood monocyte has been reported, and the term plasmacytoid monocyte has been proposed to describe this cell. The present study was undertaken to analyze the presence and distribution of plasmacytoid monocytes in human reactive lymph nodes showing epithelioid cell reactions. Numerous plasmacytoid monocytes (detected by a panel of monoclonal antibodies) were found in the majority of the lymph nodes studies, usually in close topographical association with epithelioid cells and multinucleated giant cells. The present findings suggest that plasmacytoid monocytes may give rise to epithelioid cells. This is further supported by the ultrastructural similarities between plasmacytoid monocytes and plasmacytoid epithelioid cells, a cell type that has been identified previously in granulomas and considered a direct precursor of the classical epithelioid cell.     

Formation of Multinucleate Giant Cells in Organized Epithelioid Cell Granulomas

Organized epithelioid cell granulomas were produced experimentally by injecting intradermally dilute suspensions of zirconium and beryllium salts into individuals who had been previously sensitized to these metals. Biopsies were obtained at intervals of between 5 days and 13 months later, and the specimens were fixed and prepared for light and electron microscopy. Tritiated thymidine was injected into a number of the granulomas; the biopsy specimens were secured from 40 minutes to 2 or more weeks later, and the tissues were processed for light microscopic autoradiography. Giant cells occurred commonly, both within organized tubercles and in relation to areas of necrosis, and had markedly different cytoplasmic features from typical secretory epithelioid cells which enabled them to be readily recognized at scanning magnifications. The characteristic hallmark of these giant cells was the presence of myriads of small mitochondria adjacent to nuclei with numerous membrane-lined vesicles in the center of the cell. Giant cells occurred mainly in edematous disrupted tubercles. In these tubercles, epithelioid cells contained cytoplasmic components more like giant cells. Direct evidence of cell fusion was not seen, although fusion of membranes seemed to occur between cells having similar cytoplasmic features. The failure to find labeled nuclei in any giant cells 40 minutes after injection of tritiated thymidine indicates that normal nuclear division does not occur within giant cells. We postulate that epithelioid cells containing vesicles develop in damaged and necrotic areas, and that mainly this type of epithelioid cell fuses to form giant cells.     

Immunohistochemical characterization of tuberculous and non-tuberculous lesionsin naturally infected European badgers (Meles meles)

A panel of species cross-reactive antibodies was established for the immunohistochemical labelling of phagocytic and lymphoid cells in formalin-fixed normal badger tissues. These reagents were used to investigate the immunopathogenesis of both tuberculous and non-tuberculous granulomas in badgers. In normal badger tissues, antisera specific for the CD79a and CD79b epitopes strongly labelled follicular B lymphocytes and plasma cells in lymph nodes, bronchus-associated lymphoid tissue and Peyer's patches. Rabbit anti-dog IgG, IgM and IgA, and goat anti-human lambda light chain strongly labelled plasma cells, but goat anti-ferret IgA produced weak labelling. Interfollicular and occasional follicular lymphocytes and gut intraepithelial lymphocytes expressed the CD3 epitope. Mouse anti-human HLA-DR (MHC Class II) antigen strongly labelled macrophages, some follicular lymphocytes and some intestinal and respiratory epithelial cells. Mouse anti-human calprotectin (MAC387) labelled a limited number of macrophages. In infected badgers, all fusiform to angular macrophages (epithelioid cells) of all tuberculous granulomas strongly expressed HLA-DR antigen, but only a small, variable proportion of these were labelled by MAC387 antiserum. Lymphocytes in the peripheral rims of granulomas and those scattered sparsely amongst the epithelioid cells were labelled primarily with CD3 antiserum. Peripheral plasma cells were more common in larger than in smaller tubercles and usually expressed IgA or IgG. Small unencapsulated siliceous granulomas, which were present in both tuberculous and non-tuberculous badgers, consisted of aggregates of round to polyhedral epithelioid cells expressing the MHC Class II but not the MAC387 epitope. Granulomas caused by infection with presumed fungal adiaspores of Chrysosporium sp. consisted of aggregates of variably shaped macrophages that expressed MHC Class II antigen, but only a proportion expressed MAC387 antigen. The majority of lymphocytes within the peripheral rims of these granulomas were T cells, accompanied by sparse to moderate numbers of plasma cells that primarily expressed IgG or IgA. In conclusion, species cross-reactive antibodies can be used to identify the cellular components of tuberculous and non-tuberculous granulomas. Immunohistochemical examination failed to distinguish small tuberculous granulomas from adiaspiromycotic granulomas.