Immunohistochemical Reactivity of Phagocytic and. Non-phagocytic Histiocytes in Lymph Nodes with Lysozyme, Alpha-1-antichymotrypsin, S-100 Protein,

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1 Okajimas Folia Anat. Jpn., 66 (2-3): 61-68, August, 1989 Immunohistochemical Reactivity of Phagocytic and Non-phagocytic Histiocytes in Lymph Nodes with Lysozyme, Alpha-1-antichymotrypsin, S-100 Protein, Alkaline Phosphatase, and Acid Phosphatase By Masachika SENBA and Kioko KAWAI Department of Pathology, Institute of Tropical Medicine, Nagasaki University, 12-4 Sakamoto-machi, Nagasaki 852, Japan Department of Pathology, Nagasaki University School of Medicine, 12-4 Sakamoto-machi, Nagasaki 852, Japan -Received for Publication, January 17, Key Words: Yellow-brown body, Phagocytic histiocyte, Non-phagocytic histiocyte, Lymph node, Immunohistochemistry, Lysozyme, Alpha-1-antichymotrypsin, protein, Alkaline phosphatase, Acid phosphatase Summary: Yellow-brown bodies were observed in the sinusoids of lymph node and histiocytes. The authors confirmed immunohistochemical reactivity of lysozyme, alpha-1-antichymotrypsin, S-100 protein, alkaline phosphatase, and acid phosphatase in non-phagocytic and phagocytic histiocytes which contained yellow-brown bodies. Phagocytic histiocytes (histiocytes with yellow-brown bodies) were not reacted with lysozyme, alpha-1-antichymotrypsin, S-100 protein, alkaline phosphatase, and acid phosphatase. On the other hand, non-phagocytic histiocytes were reacted with lysozyme, alpha-1-antichymotrypsin, S-100 protein, alkaline phosphatase, and acid phosphatase. Yellow-brown bodies were first described by Hamazaki in 1938 in the mesenteric lymph nodes of a patient with appendicitis. Wesenberg reported similar structures, such as round or spindle shaped bodies in the lymph nodes of patients with sarcoidosis in Yellow-brown bodies are significantly more common in sarcoidosis than in other conditions (Hamazaki, 1938; Wesenberg, 1966; Boyd and Valentine, 1970; Doyle et al., 1973; Sieracki and Fischer, 1973; Ro et al., 1987). However, their pathogenesis and significance are not well understood. The lysozyme, alpha-l-antichymotryp sin, and S-100 protein have been introduced by many authors as helpful markers in the diagnosis of malignant and benign histiocytic proliferative disorders (Meister et al., 1980; Carbone et al. 1981; Watanabe et al., 1983; Aoyama et al. 1984; Ducatman et al., 1984; Ide et al., 1984; Weiss et al., 1986; Flint et al., 1986; Wakuya, 1987; Wiettinen et al., 1987). The authors examined immunohistochemical properties of histiocytes with and without yellow-brown bodies, which were observed in two cases of lymph nodes with sarcoidosis and with ileus. In addition, immunohistochemical staining was used for studying intracytoplasmic lysozyme, alpha-l-antichymotrypsin, S-100 protein, alkaline phosphatase, and acid 61

2 62 M. Senba and K. Kawai phosphatase in assessing the cellular nature of phagocytic and non-phagocytic histiocytes in lymph nodes associated with yellow-brown bodies. Materials and Methods Two cases of yellow-brown body specimens were obtained from Nagasaki University Hospital with sarcoidosis and with ileus. The materials were fixed in 10% formalin and embedded in paraffin. Sections were cut at four micron and stained with hematoxylinand-eosin, periodic acid Schiff (PAS), and Fontana-Masson 's silver. Histiocytes with and without yellow-brown bodies were stained with lysozyme (Dako PAP Kit, K504, Lot ), alpha-1 -antichymotrypsin (ACT) (Dako PAP Kit, K534, Lot ), protein (Dako PAP Kit, K524, Lot ), alkaline phosphatase (Alkaline phosphatase conjugated Strept-Avidin: Bio Genex Laboratories, Lot. 126A; Naphtol AS- MX phosphate: Sigma Chemical Company, N-4875, Lot. 85F-5046; Fast Red TR Salt: Sigma Chemical Company, F-1500, Lot. 114F-0262), and acid phosphatase (Bio Genex Laboratories PAP Kit, Lot CMR-2057). Results Yellow-brown bodies were diffusely present in the sinusoids of the lymph nodes, and located within the cytoplasm of histiocytes. The larger bodies were within the cytoplasm of histiocytes. These bodies were stained intensely black with Fontana-Masson's silver method (Fig. 1) which could be suitable for rapid screening and identification of yellowbrown bodies. The structures were stained with periodic acid Schiff (PAS). Histiocytes without yellow-brown bodies were strongly stained with lysozyme (Fig. 2), alpha-l-antichymotrypsin (Fig. 3), S-100 protein (Fig. 4), and alkaline phosphatase (Fig. 5). These bodies were weakly stained with acid phosphatase. On the other hand, histiocytes with yellow-brown bodies were not stained with lysozyme (Fig. 2), alpha-l-antichymotrypsin (Fig. 3), S-100 protein (Fig. 4), alkaline phosphatase Discussion (Fig. 5), and acid phosphatase. The lysozyme and alpha-1 -antichymotrypin for histiocytes of mononuclear phagocytic system origin and protein for those belonging to Langerhans' cells and T-zone histiocytes have been reported (Meister et al., 1980; Carbone et al., 1981; Watanabe et al., 1983; Aoyama et al., 1984; Ducatman et al., 1984; Ide et al., 1984: Weiss et al., 1966; Flint et al., 1986; Wakuya, 1987; Miettinen et al., 1987). The immunocytochemical detection of lysozyme and alpha-l-antichymotrypsin activity has been considered to be helpful confirmatory test in malignant histiocytosis and benign histiocytic proliferative disorders. Watanabe and coworkers (1983) suggested the hypothesis that lysozyme negative, but protein positive histiocytes such as Langerhans' cells and interdigitation reticulum cells, may constitute a subgroup independent of the ordinary mononuclear phagocytic system. They used S-100 protein as a valuable marker and proposed the concept of neoplasms of T- zone histiocytes with S-100 protein. Ide and colleagues (1984) stated that irnmunohistochemical study for S-100 protein in histiocytosis X cells and Langerhans' cells, they observed the presence of this protein in both cells. The presence of protein in histiocytosis X cells seems to be supported the possible origin of Langerhans' cells. Therefore, the immunohistochemical demonstration of S-100 protein in routine histologic sections is of great value in the identification of Langerhans' cells and histiocytosis X cells.

3 Immunohistochemical Reactivity of Histiocytes 63 Immunoreactivity of phagocytic histiocytes is clearly different from non-phagocytic histiocytes. A possible explanation may be that some enzyme are released from phagocytic histiocytes, or immunologically active sites are marked as a result of phagocytosis. Moreover, immunological nature of histiocytes varies in different organs, different disease, and subpopulation of histiocytes. Further study from this point of view might lead to a significant contribution. Yellow-brown bodies must be distinguished from hemosiderin pigments which show yellowish color in hematoxylin-and-eosin stained sections. There is difference between yellow-brown bodies hemosiderin pigments. Hemosiderin pigments react with iron stain and not autofluorescent under ultraviolet illumination. Yellow-brown bodies are mainly located in sinusoids of lymph nodes, but hemosiderin pigments are not. References 1) Aoyama, K., Terashima, K., Imai, Y., Katsushima, N., Okuyama, Y., Niikawa, K., Mukada, T. and Takahashi, K.: Sinus histiocytosis with massive lymphadenopathy: A histogenic analysis of histiocytes found in the fourth Japanese cases. Acta Pathol. Jpn., 34: , ) Boyd, J.F. and Valentine, J.C.: Unidentified yellow bodies in human lymph nodes. J. Pathol., 102: 58-60, ) Carbone, A., Micheau, C., Caillaud, J. and Carlut, C.: A cytochemical and immunohistochemical approach to malignant histiocytosis. Cancer, 47: , ) Doyle, W.F., Brahman, H.D. and Burgess, J.H.: The nature of yellow-brown bodies in peritoneal lymph nodes. Arch. Pathol. Lab. Med., 96: , ) Ducatman, B.S., Wick, M.R., Morgan, T.W., Banks, P.M. and Pierre, R.V.: Malignant histiocytosis: A clinical, histologic, and immunohistochemical study of 20 cases. Hum. Pathol., 15: , ) Flint, A., Lloyd, R.V., Colby, T.V. and Wilson, B.W.: Pulmonary histiocytosis X: Immuno-- peroxidase staining for HLD-DR antigen and S-100 protein. Arch. Pathol. Lab. Med., 110: , ) Hamazaki, Y.: Ober ein Neues saurefeste Substanz fiihrendes SpindelkOrperchen der Menschlichen Lymphdrusen. Virchows Arch. Pathol. Anat., 301: , ) Ide, F., Iwase, T., Saito, I., Umemura, S. and Nakajima, T.: Immunohistochemical and ultrastructural analysis of the proliferating cells in histiocytosis X. Cancer, 53: , ) Meister, P., Huhn, D. and Nathrath, W.: Malignant histiocytosis: Immunohistochemical characterization on paraffin embedded tissue. Virchows Arch. A Pathol. Anat. Histol., 385: , ) Miettinen, M., Paljakka, P., Haven, P. and Saxer', E.: Sinus histiocytosis with massive lymphadenopathy: A nodal and extranoda1 proliferation of S-100 protein positive histiocytes? Am. J. Clin. Pathol., 88: , ) Ro, J.Y., Luna, M.A., Mackay, B. and Ramos, 0.: Yellow-brown (Hamazaki-Wesenberg) bodies mimicking yeasts. Arch. Pathol. Lab. Med., 111: , ) Sieracki, J.C. and Fischer, E.R.: The ceroid nature of the so-called "Hamazaki-Wesenberg bodies". Am. J. Clin. Pathol., 59: , ) Wakuya, J.: Immunohistochemical and ultrastructural studies on histiocytosis in children. Acta Pathol. Jpn., 37: , ) Watanabe, S., Nakajima, T., Shimosato, Y., Sato, Y. and Shimizu, K.: Malignant histiocytosis and Letterer-Siwe disease: Neoplasms of T-zone histiocyte with S-100 protein. Cancer, 51: , ) Weiss, L.M., Azzi, R., Dorfman, R.F. and Warnke, R.A.: Sinusoidal hematolymphoid malignancy ("Malignant histiocytosis") presenting as atypical sinusoidal proliferation. Cancer, 58: , ) Wesenberg, W.: Ober saurefeste "SpindelkOrper Hamazaki" bei Sarkoidose der Lymphknoten und fiber doppellichthrechende Zelleinschltisse bei Sarkoidose der Lungen. Arch. Kiln. Exp. Dermatol., 227: , 1966.

4 64 M. Senba and K. Kawai Explanation of Figures Plate I Fig. 1. Varying sizes and shapes of yellow-brown bodies are seen in the sinusoids of the lymph node and in the histiocytes. Fontana-Masson's silver stain x 400.

5 Immunohistochemical Reactivity of Histiocytes 65 Plate I

6 66 M. Senba and K. Kawai Plate II Fig. 2. Histiocytes without yellow-brown bodies are stained red with lysozyme, but histiocytes with yellowbrown bodies are not stained. Yellow-brown bodies are seen in the sinusoids of the lymph node. Immunoperoxidase stain for lysozyme x 400. Fig. 3. Histiocytes without yellow-brown bodies are stained red with alpha-1-antichymotrypsin, but histiocytes with yellow-brown bodies are not stained. Yellow-brown bodies are seen in the sinusoids of the lymph node. Immunoperoxidase stain for alpha- 1-antichymotrypsin x 400. Fig. 4. Histiocytes without yellow-brown bodies are stained red with S-100 protein, but histiocytes with yellow-brown bodies are not stained. Yellow-brown bodies are seen in the sinusoids of the lymph node. Immunoperoxidase stain for S-100 protein x 400. Fig. 5. Histiocytes without yellow-brown bodies are stained red with alkaline phosphatase, but histiocytes with yellow-brown bodies are seen in the sinusoids of the lymph node. Stain for alkaline phosphatase x 400.

7 Immunohistochemical Reactivity of Histiocytes 67 Plate II