Significance of PAM Histochemical Reaction in Delineating Macrophages

Okajimas Folia Anat. Jpn., 84(1): 11–18, May, 2007

Signiﬁcance of PAM Histochemical Reaction in Delineating Macrophages

Yuji SHINYA1,KazuyaHAMADA1,P.D.GUPTA2 and Fumioki YASUZUMI1

1Department of Physiological Science Anatomy 2. Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa, 903-0215 Japan 2Atmiya Institute of Gerontology Research, Yogidham, Kalawad Road, Rajikot-360 005, Gujarat, India

– Received for Publication, December 29, 2006 –

Key Words: Macrophage, Fetal macrophage, Placental villi, Periodic acid methenamine silver, Glycol-methacrylate

Summary: Periodic acid methenamine silver staining has been used for proof of the existence of carbohydrates in both parafﬁn and resin embedded tissues. The staining was applied to delineate macrophages that have been well known as cells of mononuclear phagocyte system by stainable nature for lysosomes, and it is found to be useful method for de- tection of macrophage. Fetal macrophage, which is often observed in placental villi, is called the highly vacuolated cell. Vacuoles in the cytoplasm seem to be empty with light microscopic staining, but it is clariﬁed that they contain a full amount of carbohydrate. The present result leads us to the necessity of reconsidering the functional aspect of fetal macrophage.

Periodic acid Schiff (PAS) reaction is specific In the present study, without using enzyme his- histochemical staining for carbohydrates and was tochemistry in tissues embedded in GMA by PAM introduced by McManus (1948)1).Periodicacid staining we have delineated lysosomes in various methenamine silver (PAM) is a modified method of types of macrophages including fetal macrophages PAS reaction; the superiority of PAM over the which were not identified by this staining method so PAS was seen in glomerular basement membrane. far. Histochemically, positive objects in PAM reaction having very high contrast compare to the sur- rounding tissue components2). Rambourg and Leb- Materials and Method lond used this staining method directly on epoxy resin section, and examined specificities of the Fixation and embedding staining by taking proper controls i.e., staining the All tissues collected from various animals and tissue where periodic acid was ommited3).Intheir tissues, mentioned below were fixed for 12 hours in report one of the cell organelles, the lysosomes 2.5% glutaraldehyde adjusted to pH 7.4 with were stained by PAM staining method. The lyso- 0.05 M cacodylate buffer, and then washed with the somes also stained positive in PAM4) staining same buffer and dehydrated routinely with alcohol, method when glycol methacrylate (GMA) em- and embedded in GMA (Technovit 7100, Kulzer). bedded sections is used. Since proposed the mononuclear phagocytic sys- Kupffer cells (liver macrophages); Wister rat liver. tem (MPS) consisting of various types of macro- Intestinal macrophages from lamina propria; phages such as, histiocyte, Kupffer cell, osteoclast, monkey rectum. Langerhans cell, Hofbauer cell, etc5,6),arethemain Testicular macrophages from human (testis was defense cells in the body5). The macrophages have removed from prostate cancer patient). many lysosomes which show acid phosphatase Fetal macrophages (Hofbauer cells), from the activity as a marker enzyme5–7). human placenta of eight week gestation.

Yuji Shinya, Department of Physiological Science Anatomy 2. Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishi- hara, Okinawa, 903-0215 Japan.

11 12 Y. Shinya et al.

The specimen were sectioned at 4 mm thickness on the main cells for the defense system of the body, Sorvall JB-4 microtome fitted with Ralph’s glass called the mononuclear phagocyte system. One of knife8). Every section was re-sectioned from tissue the characteristic feature of these cells is that each blocks embedded in GMA for histological practices. cell contains a number of lysosomes5) in the cytoplasm. Acid phosphatase activity, the maker en- Staining zyme11) of the lysosomes is also very high in these HE staining cells. 1) Carazzi’s hematoxylin9) modified staining10): In 1967 Rambourg and Leblond used PAM Sections were stained with the modified solution for staining at ultrastructure level and were able to 24 hours. stain mucin, basement membrane, lysosomes, and 2) Eosin staining was followed as described ear- Golgi saccules however, when the section was not lier4). Toluidine blue10) was used for semi-thin resin subjected to periodic acid, lysosome was not stained sections. with methenamine silver, but nuclei and some pig- PAM staining and PAM with gold chloride ments were stained non-specifically even without method was followed according to Jones2). Control periodic acid treatment3). sections were not treated with periodic acid prior to treatment with methenamine silver3). Kupffer cells Photographs of the same section were taken Kupffer cells are located in the liver sinusoid, after different staining as described by Nakamura attached to the endothelial cells and contain many and Yasuzumi4). Olympus light microscope (BX50) black granules in the cytoplasm that are stained fitted with digital camera (PDMC Ie/OL) was used with PAM. These granules are homologous with for photography. granules of phagosomes6), that is, secondary lysosomes (acid phosphatase positive)11) (Figs. 1A, 1B).

Results and Discussion Intestinal macrophages There are many kinds of mesenchymal cells, such Since the report of van Furth et al.5) had been as ﬁbroblast, macrophages, lymphocytes, plasma published, the macrophages has been recognized as cells, neutrophils, eosinophils, mast cells and adi-

Fig. 1. Kupffer cell of the rat liver. HC: liver parenchymal cell, K: Kupffer cell, S: sinusoid. A. Kupffer cell stained with PAM. In the cytoplasm of the cell, dense granules considered secondary lysosomes are stained with PAM specifically. Both nuclei of liver parenchymal cell and Kupffer cell are stained non-specifically. Lysosomes of parenchymal cell are stained palely (short arrow). Â1,350. B. Same field of A is stained with additional HE staining. Liver structures in the lobules is clarified increasingly by HE staining. Adjusted to liver cell cord the sinusoid, in which condensed erythrocytes and the Kupffer cell locates in the lumen, is clearly observed. Â1,350. PAM Staining in Delineating Macrophages 13

Fig. 2. Intestinal macrophages in the monkey rectum. The same section is stained with different ways of staining. e: eosinophil, f: ﬁbroblast, ly: lymphocyte, m: macrophage, ms: mast cell, p: plasma cell. A. A section which is stained with toluidine blue differentiates mast cell by metachromasia, plasma cell by basophilic cytoplasm, and macrophage by blueish lysosomes. Â830. B. The section of A is stained with HE after A. HE stains the granules of eosinophil positively. Â830. C. The section of B is stained with PAM after B. PAM stains lysosomes black. Â830.

pose cells in the lamina propria of the intenstine12). somes6) and in fact contain acid phosphatase, but Especially macrophages aggregated on the tips of PAM does not stain them (Figs. 2A, 2B, 2C). the intestinal villi and arranged at the base the epi- thelium were proved by means of acid phosphatase Testicular macrophages activity of lysosome13,14). When a GMA section of The seminiferous tubule which destines to be the rectal mucosa is stained with different ways of collapsed by ageing process undergoes atrophy, staining successively, toluidine blue, HE, and then thickening of the basement membrane, and dis- PAM, in the same visual field4), the mast cell is dif- appearing of all epithelial cells finally from the ferentiated with toluidine blue from other cells by tubule16). In the process of collapsing, the macro- metachromasia (Fig. 2A). Macrophages in the lam- phages impinge the basement membrane, enter the ina propria are in larger number in which lyso- tubule and phagocyte degenerated cells and cell somes are stained basophilic and slightly bluish debris17). In the cytoplasm of the macrophage, a with toluidine blue, faint eosinophilic with HE, and number of condensed lysosome are detected by black intensely with PAM. The eosinophil is de- PAM stain (Fig. 3A). With HE staining, normal tected by staining nature of specific granules which spermatogenic cell is well demarcated (Fig. 3B). A are stained negatively with toluidine blue but eo- completely collapsed residual tubule show thick- sinophilic with HE15). Nevertheless specific granules ened basement and luminal membranes, which is of eosinophil have been considered one of the ly- considered to be the surface of the lamina lucida of sosomes6) and in fact contain acid phosphatase, the basement membrane (Figs. 3C, 3D). PAM does not stain then (Figs. 2A, 2B, 2C). Therefore, lysosomes, specific granules of the eo- Fetal macrophages or Hofbauer cells sinophil, are thought to be primary lysosomes. The Hofbauer cells are one of the component of eosinophil is detected by staining nature of specific mononuclear phagocytes system17), which is based granules which are stained negatively with toluidine on the mesenchymal stromal cell of placenta and blue but eosinophilic with HE15). However, they fetus. These cells are highly vacuolated cells. The are not stained with PAM. Specific granules of eo- vacuoles show positive reaction to acid phosphatase sinophil have been considered one of the lyso- activity18). The cells are more prominent in the first 14 Y. Shinya et al.

Fig. 3. Testicular macrophage. B: basement membrane, m: macrophage, SL: Sertoli cell, SP: spermatogenic cell. A. A section is stained with PAM. Testicular macrophage impinges the basement membrane and enter into the seminiferous tube which destines to be collapsed. Lysosomes of macrophage are stained intensely. Non-speciﬁc stainabilities of nuclei of both spermatogenic cells and Sertoli cell are not equal. Â540. B. The section of A is stained with HE after A. The seminiferous tubule seems to progress normal spermatogenesis. Â540. C. Collapsed seminiferous tubule in a section stained with PAM. All of the epithelial cells disappeared from the tubule, and a macrophage and thickened basement membrane are observed. Â540. D. The section of C is stained with HE after C. Â540.

trimester18). The most characteristic feature of these with toluidine blue (Figs. 5A, 5B, 6A). The stain- cells is the number of vacuoles, which have an inti- ability of eosin is useful for identification and mate relationship to larger vacuoles19) through quantification of protein analysis21). Accordingly, short narrow tubules formed by a well developed vacuoles do contain a little amount of protein. smooth endoplasmic reticulum. Enders and King Many of the vacuoles stained faintly with HE or reported the vacuoles containing flocculent precip- toluidine blue are also stained intensely with PAM itate, secondary lysosomes and lipid droplets are (Figs. 5C, 6B, 7). The PAM positive material in present in the cytoplasm20) of these cells. When these cells is considered to be derived from syncy- chorionic villi are stained with PAM (with and tiotrophoblasts, which also show similar PAM pos- without periodic acid treatment) a number of va- itive granules probably absorbed from maternal cuoles and a few lysosomes are stained specifically blood as nutrient (Fig. 7). and intensely black, except a few unstained vacu- A variety of functions have been assigned to oles (Figs. 4A, 4B). PAM stains carbohydrate-rich Hofbauer cells, including the macrophages func- material and/or glycoprotein3). The vacuoles are tions and immunological reactions17,19). However, stained eosinophilic faintly with HE, or faintly blue the contents of cytoplasmic vacuoles have not been PAM Staining in Delineating Macrophages 15

Fig. 4. Placental villus. CT: cytotrophoblast, HF: Hofbauer cell, ST: syncytiotrophoblast. A. Control PAM staining without treatment of periodic acid. Vacuoles of the Hofbauer cell appear to be empty, but a few non- speciﬁc granules are scattered in the cell. Nuclei of both Hofbauer cell and trophoblast are stained non-speciﬁcally. Â600. B. The section of A is stained with PAM after A. Morphological details of cellular components and vacuoles are clearly stained. Â600.

Fig. 5. Placental villus. HF: Hofbauer cell, TB: trophoblast. A. A section is stained with HE. Vacuoles of the Hofbauer cell are stained faintly eosinophilic. Â630. B. The section of A is stained with toluidine blue after A. The staining nature of metachromasia does not appear in the section. The vacuoles still show faintly blue. Â630. C. The section of B is stained with PAM. Staining nature of villus components remarkably decreases after HE and toluidine blue. Nuclei of cellular component still remain non-speciﬁc staining nature. Vacuoles of Hofbauer cell are stainable, but PAM positive granules decrease in volume and number. Â630. 16 Y. Shinya et al.

Fig. 6. Placental villus. BL: blood vessel, HF: Hofbauer cell. A. A section is stained with toluidine blue. Vacuoles of Hofbauer cell are stained faintly blue, and the blood plasma and erythrocytes in venules are stained more intensely than the vacuoles. Â630. B. The section of A is stained with PAM after A. The vacuoles are stained intensely with PAM. Â630.

Fig. 7. Placental villus of fetus in eight week gestation shows two types of trophoblast, syncytiotrophoblast and cytotrophoblast. The former contains a number of PAM positive granules which are considered nutrients absorbed from maternal blood, and the latter, which locate under the former, shows clear cytoplasm and a large clear nucleus. Hofbauer cell is seen in close apposition to the trophoblast. Â1,080. CT: cytotrophoblast, HF: Hofbauer cell, ST: syncytiotrophoblast. PAM Staining in Delineating Macrophages 17 analyzed except the facts that the vacuoles contain tem: a new classification of macrophages, monocytes, and flocculent precipitate20) and shows positive acid their precursor cells. Bull WHO, 1972; 46:845–852. th phosphatase activity. The results of present study 6) Fawcett DW: A textbook of histology. 11 ed. W. B. Sa- unders, Philadelphia, 1986; 156–158. suggest that the role of Hofbauer cell is preferably 7) Naito M: Macrophage heterogeneity in development and a carrier of nutrients, saccharide rich material and/ differentiation. Arch Histol Cytol 1993; 56:331–351. or glycoprotein. We consider that the acid phos- 8) Bennett HS, Wyrick AD, Lee SW and McNeil JH: Science phatase activity of vacuoles is due to lysosomes and art in preparing tissues embedded in plastic for light binding to vacuoles for processing of nutrients by microscopy, with special reference to glycol methacrylate, glass knives and simple stains. Stain Technol 1976; 51:71– the enzyme activity. 97. The methods of successive staining on the same 9) Carazzi D: A new hematoxylin solution. Zschr Wiss Mik- section is preferred to the following sequence, first rosk 1911; 28:273 (in German). toluidine blue, second HE, and final PAM, and is 10) Richardson KC, Jarett L and Finke EH: Embedding in able to differentiate into many of mesenchymal epoxy resins for ultra-thin sectioning in electron microscopy. Stain Technol 1960; 35:313–323. cells and cell organelles (Figs. 2A, 2B, 2C). When a 11) De Duve C: Lysosomes, a new group of cytoplasmic par- section is stained with the successive sequence of ticles. T. Hayashi ed. Subcellular particles. Ronald Press, staining, HE, toluidine blue and PAM, the nature New York, 1959; 128–159. of metachromasia with toluidine blue disappears 12) Deane HW: Some electron microscopic observations on the and the stainability with PAM decreases remarkably lamina propria of the gut, with comments on the close as- sociation of macrophages, plasma cells, and eosinophils. for both specific and non-specific staining (Figs. 5A, Anat Rec 1964; 149:453–474. 5B, 5C). 13) Han H, Iwanaga T, Uchiyama Y and Fujita T: Aggregation of macrophages in the tips of intestinal villi in guinea pigs: their possible role in the phagocytosis of effete epithelial cells. Cell Tissue Res 1993; 271:407–416. Acknowledgments 14) Iwanaga T, Han H and Fujita T: Macrophages possibly in- volved in the disposal of apoptotic epithelial cells in the The authors thank Mrs. Makiko Sekiguchi and monkey small and large intestine. Acta Medica et Biologica Yukiji Yabiku for their technical assistance. 1992; 40:105–113. 15) Yasuzumi F and Gupta PD: Indispensable practical atlas of histology and embryology, Capricon, Jaipur, 2007 (in press). 16) Stieve H: Urinary and genital organs. Handbuch der mik- References roskopischen anatomie des menschen. Springer Verlag, Berlin, 1930; 126–134 (in German). 1) McManus JFA: Histological and histochemical uses of pe- 17) Wood GW: Mononuclear phagocytes in the human pla- riodic acid. Stain Technol 1948; 23:99–108. centa. Placenta 1980; 1:113–123. 2) Jones DB: Nephrotic glmerulonephritis. Am J Path 1957; 18) McKay DG, Hertig AT, Adams EC and Richardson MV: 33:313–329. Histochemical observations on the human placenta. Obstet 3) Rambourg A and Leblond CP: Electron microscope ob- Gynecol 1958; 12:1–36. servations on the carbohydrate-rich cell coat present at the 19) Jones CJP and Fox H: Ultrastructure of the normal human surface of cells in the rat. J Cell Biol 1967; 32:27–53. placenta. Electron Microsc Rev 1991; 4:129–178. 4) Nakamura N and Yasuzumi F: Staining of the Reinke 20) Enders AC and King BF: The cytology of Hofbauer cells. crystalloids in the human testis – Re-evaluation study. Anat Rec 1970; 167:231–251. Okajimas Folia Anat Jpn 2005; 82:103–110. 21) Waheed AA and Gupta PD: Application of an eosin B dye 5) Van Furth R, Cohn ZA, Hirsch JG, Humphrey JH, Spector method for estimating a wide range of protein. J Biochem WG and Langevoort HL: The mononuclear phagocyte sys- Biophys Methods 1996; 33:187–196.