Epithelioid Cell Granulomas Experimentally Induced by Prototheca in the Skin of Mice: a Light Microscopic Study
Total Page:16
File Type:pdf, Size:1020Kb
21 Hiroshima J. Med. Sci. Vol.44, No.2, 21-27, June, 1995 HIJM 44-5 Epithelioid Cell Granulomas Experimentally Induced by Prototheca in the Skin of Mice: A Light Microscopic Study Yasuhiro HORIUCHI and Mikio MASUZAWA Department of Dermatology, Kitasato University School of Medicine, Sagamihara, Japan ABSTRACT Prototheca wickerhamii, an achlorophyllous algae, was previously found to induce massive epithelioid cell granulomas in the skin of mice. By means of light microscopy, examination was made of the histological reactions involved in epithelioid cell granulomas induced by intrader mal and/or subcutaneous inoculation of Prototheca wickerhamii in BALB/c and ICR mice. Six BALB/c mice showed granuloma nodules while only three of six ICR mice did so. Based on the results of the present and previous studies, BALB/c mice may be considered a strain particularly vulnerable to contracting epithelioid cell granuloma and ICR mice, a resistant strain. In very early lesions at one week following initial prototheca inoculation, cellular infiltration with varying numbers of polymorphonuclear leukocytes, lymphocytes and some macrophages was observed throughout the dermis and subcutaneous fat tissue. In early lesions at one to two months after inoculation, focal granulomas composed of histiocytic cells and/or macro phages were observed. Mast cells were occasionally present among the histiocytic cell infil trates. In the granulomatous lesions at two to three months, scattered eosinophils and some lymphocytes were seen. Central necrosis, with numerous neutrophils and many endospores surrounded by the granuloma, was often observed. In late stage lesions at six months, massive lymphocyte and plasma cell infiltration surrounding and/or intervening between vacuolated epithelioid cell clusters was evident. Histological reactions in epithelioid cell granuloma and the ultimate course of this disease can be staged from the histological point of view as follows: 1) diffuse inflammation, 2) cell proliferation leading to epithelioid cell formation, 3) hypertro phy of epithelioid cells with consequent formation of cell aggregates and/or organized granulo ma and 4) degeneration of granuloma. Histological reactions were found in this study to be essentially the same in ICR and BALB/c mice, though a time lag was noted in the course of epithelioid cell granuloma formation. Key words: Epithelioid cell granuloma, Prototheca, Mice Epithelioid cell granuloma has been studied mastitis, in marine water and sewage. Human widely in sarcoidosis and in tuberculosis 17). In protothecosis is extremely rare10). The histolog sarcoidosis, the bacillus or other factors causing ical reactions in most reported cases with cuta this disease have not been determined, although neous protothecosis are chronic granulomatous certain substances 12) and metals such as zirco inflammation with a mixed inflammatory cell nium15 ) cause a sarcoid type granuloma. Epithe infiltration10). A number of human cases with a lioid cells derive from tissue macrophages histology of epithelioid cell granuloma, however, (histiocytes)17), which come from bone marrow have been reported14 ). Experimental prototheco precursors via circulating monocytes16). Little sis in animals appears in some reports, one of attention has been directed, however, to the which maintains that this condition occurs only histological reactions involved in the onset of in the liver of prednisolone-treated mice as a early epithelioid cell granuloma, particularly at result of Prototheca zopfii strain inoculation8). P. the start of characteristic lesion formation, and in wickerhamii, isolated from skin specimens of a the late degenerative phase of granuloma. patient with cutaneous protothecosis, was found Prototheca wickerhamii, an achlorophyllous in a previous study to induce massive epithelioid algae10), is often found in the milk of cows with cell granulomas in the skin of mice6). This may Reprint requests to: Dr.Y.Horiuchi, Division of Dermatology, Higashi-Matsuyama Medical Association Hospital, 1-15-10 Shinmei-cho, Higashi-Matsuyama City, Saitama 355, Japan. 22 Y. Horiuchi and M. Masuzawa thus serve as a suitable animal model for study tion, 2) for biopsy at one week following a period ing epithelioid cell granuloma formation. The of inoculation of three months subsequent to ini same lesion was also observed in this study, in tial inoculation and 3) for biopsy at one or two which examinations were focused on ascertaining months following a period of inoculation of six specific histological reactions in the course of months subsequent to initial inoculation. The development of granulomas experimentally suspension was inoculated into the dermis and/or induced by prototheca in the skin of mice. subcutaneous tissue of the back (three separate sites) of each mouse once every week. On a speci MATERIALS AND METHODS fied day after inoculation, biopsy specimens were obtained and fixed in 10% formalin solution and Mice stained with hematoxylin-eosin, periodic acid Six female BALB/c mice and six female ICR Schiff (PAS), Giemsa solution and toluidine blue. (Institute of Cancer Research) mice, from SLC Japan CO.Ltd (Shizuoka, Japan) were used, in RESULTS order to determine differences in histological All six BALB/c mice developed a nodular granu reactions in the course of epithelioid cell granu loma by one to three months following the initial loma formation between BALB/c mice with spe prototheca inoculation. Only three of the six ICR cific immunity sensitive to X-ray radiation and mice showed the nodular granuloma at two to six ICR mice with normal immunity. Pelleted food months. As a result, BALB/c mice were consid was made available throughout the study. ered a strain particularly vulnerable to contract ing epithelioid cell granuloma and ICR mice, a Preparation and Inoculation of Prototheca resistant strain. Prototheca wickerhamii organisms6), obtained from skin biopsy specimens of a patient10) with Very early lesions (One week after initial inocula cutaneous protothecosis, were cultured in Sabou tion) raud's medium (peptone, lOg and glucose, 20g/li In both the BALB/c and ICR mice, polymorpho ter ). A 0.5ml aliquot of the organisms of 4.0 to nuclear leukocytes, some mast cells and eosino 8.0x106/ml was washed once with sterile saline phils were seen sparsely but diffusely scattered and resuspended in 0.5ml saline. The prototheca throughout the deep dermis or subcutaneous fat suspension was divided into three portions: 1) for tissue (Fig. la,b). No granulomatous reactions biopsy at one week following initial inoculation or could be detected. A few large macrophage-like at one week following a period of inoculation of cells were occasionally present (Fig. la). PAS one or two months subsequent to initial inocula: Fig. 1. Very early lesions (one week after initial inoculation). a: In the BALB/c mice, mononuclear cell infiltration, primarily polymorphonuclear neutrophils, eosinophils, histiocytes, macrophages and a few mast cells can be seen in some places throughout the deep dermis. Note three large macrophages mixed with mononuclear cell infiltrates. (Hematoxylin & eosin, x 200) b: In the ICR mice, mononuclear cell infiltration, histiocytes, neutrophils and eosinophils are evident throughout the subcutaneous fat tissue. (Hematoxylin & eosin, x 400) Epithelioid Cell Granuloma Formation 23 Fig. 2. Early lesions (one month after initial inoculation). a: In the BALB/c mice, extensive histiocytic infiltration with scattered multinucleated giant cells and macrophages are apparent.(Hematoxylin & eosin, x 100) b: Scattered mast cells stained with Giemsa can be seen among mononuclear/histiocytic infiltrates. (Giemsa stain, x 100) staining failed to show algal organisms. trophils mixed with numerous prototheca organ isms (Fig. 4b, c, PAS stain) surrounded by Early lesions (One to two months following initial histiocyte and epithelioid cell clusters could fre inoculation) quently be seen (Fig. 4b). Epithelioid cell nests In the BALB/c mice, the extensive histiocytic (Fig. 4a, b) were particularly evident in the infiltration contained epithelioid-like cells. Aggre BALB/c mice, and appeared to be tumorous (Fig. gates of histiocytic cells with multinucleated 4b). The mature epithelioid cells had large, pale giant cells were occasionally noted in the dense oval nuclei with prominent nucleoli and abun histiocytic infiltrate (Fig. 2a). Thereafter, the dant eosinophilic cytoplasm (Fig. 4d). In the ICR multinucleated giant cells ceased to be evident in mice (photographs not shown), dense histiocytic the late phase of granuloma. Scattered mast cells cell infiltrates mixed with scattered neutrophils staining with Giemsa solution and toluidine blue and eosinophils were noted, along with a number were seen among histiocytic/macrophage-like cells of small epithelioid cell nests. Hardly any proto in the deep dermis (Fig. 2b). Some prototheca theca organisms among or within the cellular organisms still intact were present among and infiltrates could be found, even following staining within some cells. In the ICR mice, extensive his with PAS stain. At this stage, some scattered tiocytic cell infiltration was apparent, and some mast cells were seen along the edge of epithelioid macrophage-like cells could be seen along the granuloma islands in some cases. Extensive retic edge of dense histiocytic cell infiltrates (Fig. 3a). ular fibrosis was evident among the epithelioid Focal aggregated histiocytes, macrophages and cell clusters