THE ANATOMICAL RECORD 211:192-197 (1985) Pit Cells in Extrahepatic Organs of the Rat KENJI KANEDA AND KENJIRO WAKE Department of Anatomy, Faculty of Medicine, Tokyo Medical and Dental University, Yushima 1-545, B u n k y e k y Tokyo, 113 Japan ABSTRACT Electron microscopic examination of the extrahepatic distribution of pit cells, a cell type found in the liver, revealed their existence in several other organs of the rat. They were 1)relatively frequent in lungs, spleen (red pulp), small intestine, epididymis, trachea, and peripheral blood; 2) much fewer in bone marrow and thymus (medulla); and 3) nonexistent in lymph nodes, spleen (white pulp), and thymus (cortex). The pit cells in these organs, as well as in the liver, contained characteristic dense granules and rod-cored vesicles in the cytoplasm. Our observations suggest that pit cells circulating in the peripheral blood adhere to the endothelium of capillaries in the various organs and migrate into the tissue, where they have some special immunological function. The pit cell is one cell type of the liver and is situated in the hepatic sinusoid (Wisse et al., 1976; Kaneda et al., 1982; Kaneda and Wake, 1983).These cells are characterized by electron-dense granules and rod-cored vesicles (Kaneda et al., 1982; Kaneda and Wake, 1983) in their cytoplasm. Although pit cells were initially thought to be endocrine in nature (Wisse et al., 1976), we have recently demonstrated that they belong morphologically to the “large granular lymphocytes” which are light microscopically designated by Saksela et al. (1979) and that a t least some of them were functionally natural killer cells in a conjugation test with target cells (YAC-1 tumor cells) (Kaneda et al., 1983). This relation of the pit cells to lymphocytes and the several reports in the literature on the existence of granule-containing lymphocytes in various organs (see Discussion) have led us to examine the extrahepatic distribution of pit cells. We have identified these cells on the basis of their ultrastructural characteristics, i.e., electron-dense granules, rod-cored vesicles, and so forth. We describe here their characteristic distribution pattern in various organs in comparison to that of conventional agranular lymphocytes. MATERIALS AND METHODS Untreated Wistar rats (male, 200-400 gm) were used. After cannulation of the left ventricle of the heart, saline and then fixative containing 1.5% glutaraldehyde in 0.067 M cacodylate buffer, pH 7.4, 1% sucrose (Wisse, 1970) were perfused for 4-5 min. Fragments of perfused tissue from the small intestine (duodenum, jejunum), epididymis (initial segment), trachea, thymus, submandibular lymph nodes, and bone marrow (femur) were then immersed in the same fixative for 1 h r at room temperature. The lungs and the liver were perfused for 1-2 min via the right ventricle of the heart and the portal vein, with flow rates of 10 ml/min and 15 ml/min, respectively. The spleen was similarly fixed via the blood vessels from the hilus. The tissue blocks were postfixed in 2% Os04 in phosphate buffer (pH 7.4) a t 4°C for 2 hr, dehydrated in 0 1985 ALAN R. LISS, INC ethanol, and embedded in Epon. Ultrathin sections were observed under a m O L 100 CX electron microscope at 100 kV. RESULTS Pit cells were observed in the following organs. 1) Liver (Fig. 1).They were usually located in the sinusoid and sometimes in the portal branch and central veins. They projected their well-developed pseudopodia in various directions and made contact with endothelial cells or Kupffer cells. Sometimes their cytoplasmic projections were inserted into the endothelial pores and contacted the microvilli of hepatocytes. 2) Lungs (Fig. 2). Pit cells were situated in the capillaries as in the liver, adhering to the endothelium. Their contours varied from round to elongated, and they possessed a variable number and size of pseudopodia. 3) Small intestine (Figs. 3,4), epididymis, and trachea. Pit cells were situated in the epithelium between the epithelial cells. They were mostly observed near the basement membrane, although they were sometimes found in the midportion or near the apex of the epithelium. They usually apposed epithelial cells with a very narrow intercellular space, but sometimes were separated by a wider space. They usually projected their pseudopodia into narrow spaces between epithelial cells, resulting in pleomorphic cell shapes. The granule-rich portions of the cytoplasm were randomly oriented. We could observe only a few pit cells in the lamina propria, where other white blood cells were often found, and almost no pit cells were observed adhering to the endothelium of the capillaries in the lamina propria. 4) Bone marrow (Fig. 5). Pit cells were observed in close apposition to the precursor cells of various other types of blood cells. We recognized no junctional specializations between pit cells and neighboring cells. The pit cells contained the characteristic features as seen in other locations, i.e., electron-dense granules and rodcored vesicles in the cytoplasm. Received December 19, 1983; accepted August 16, 1984 PIT CELLS IN EXTRAHEPATIC ORGANS Fig. 1. Pit cell (Pi) in the hepatic sinusoid with several cytoplasmic projections inserting into the endothelial pores and contacting the hepatic microvilli (arrows). It contains well-developed Golgi apparatus (Go),centrioles (Ce), and characteristic electron-dense granules in the cytoplasm. Most cell organelles are gathered at one side of the nucleus. Pa, parenchymal cell; E, endothelial cell. X8,lOO.Inset, the rod-cored vesicles (arrows) and electron-dense granules (GI in the cytoplasm. ~31,000. 193 Fig. 2. Pit cell (Pi) in the lung. It is located in a pulmonary capillary (PC) and adheres to the endothelial cells (E). It also contains several granules. GA, great alveolar cell; B, basement membrane; SA, squamous alveolar epithelial cell; PA, pulmonary alveolus. pl, platelet. x 11,000, 194 K. KANEDA AND K. WAKE Fig. 3. Pit cell (arrow) in the epithelium of the small intestine. It is situated at the bottom of the epithelium (Ep). A conventional agranular lymphocyte (Ly) is also seen in a similar position. Other types of white blood cells are often found in the lamina propria (LP). IL, intestinal lumen; Gb, goblet cell; A, absorptive cell; C, capillary, x 1,600. Fig. 4. Higher magnification of the pit cell (Pi) in Figure 3. It lies just on the basement membrane (B) and projects well-developed pseudopodia in several directions (arrows) between the epithelial cells (asterisks). It also contains several granules (GI in the cytoplasm. C, capillary. X8,300. Inset, aggregative forms of the rod-cored vesicles (arrows)found in the perinuclear region of this cell. ~ 4 5 , 0 0 0 . PIT CELLS IN EXTRAHEPATIC ORGANS Fig. 5. Pit cell (arrow) in the bone marrow. It is located among other differentiating blood cells and contains granules and rod-cored vesicles (arrow in inset) in the cytoplasm. ~4,300.Inset, ~57,000. 195 Fig. 6. Pit cells (large arrows) in the medulla of the thymus. They are located near the lymphatic vessel (LV) and project several pseudopodia (small arrows). E, endothelial cell of the lymphatic vessel. ~3,900. Inset, rod-cored vesicle (arrow) and electron-dense granule (GI to which a tubular attachment (arrowhead) is seen in the cytoplasm. ~56,000. 196 K. KANEDA AND K. WAKE 5) Thymus (Fig. 6). In the medulla, the pit cells often projected their pseudopodia into the narrow intercellular spaces between neighboring cells. In this study, we could not observe them in the cortex. 6) Spleen (Fig. 7). Pit cells were found in the splenic cords and sinuses. The frequency of the pit cells adhering to the endothelium of splenic sinuses was low. But they were more often recognized within the splenic cords, where they contacted reticular cells, macrophages, and other blood cells with no particular junctional specializations. They directed their pseudopodia and granulerich portions in various directions with regard to the sinus. We failed to identify pit cells in the white pulp. 7) Peripheral blood. Pit cells made up nearly 10% of the cells in the lymphocyte fraction prepared by Ficoll from peripheral blood (see Kaneda et al., 1983). There were common morphological characteristics for the pit cells observed in various organs such as rod-cored vesicles and electron-dense granules whose diameter ranged from 0.25 to 0.6 pm. Although quantitative examination was not carried out, the following trend in the frequency of the pit cells found adhering to the capillary endothelium or located in the parenchyma of various organs was seen. 1)We frequently observed them in liver, spleen (red pulp), and lungs. 2) Intraepithelially pit cells were most numerous in the small intestine. 3) We recognized only a few pit cells in bone marrow and thymus (medulla) and none in lymph nodes, spleen (white pulp), or thymus (cortex). DISCUSSION In this study we identified pit cells in various organs of the rat chiefly by their characteristic granules and rod-cored vesicles. Previously, several authors reported granule-containing lymphocytes in various organs-these studies are divided into two by their viewpoints. 1)The first is the electron microscopical observations of the lymphocytes situated in the liver (Scheuermann and De Groodt-Lasseel, 1977), lungs (Scheuermann, 1982), small intestine (Toner and Ferguson, 1971; Marsh, 19751, epididymis (Hoffer et al., 19731, and mammary gland (Seelig and Billingham, 1980).The granule-containing lymphocytes described in the literature were, however, characterized only by their manifest lysosomal granules and no attention was paid to the other characteristic organelles, like rod-cored vesicles in pit cells; also, there was usually no difference demonstrated between pleomorphic lysosomes and the characteristic multivesicular body-related granules (although Hoffer et al.  indicated the existence of granule-containing multivesicular bodies). 2) The second is the studies done from the immunological aspects; the “large granular lymphocytes” that show azurophile granules light microscopically were considered to be the morphological correspondence of natural killer activity (Saksela et al., 1979).Although the tissue distribution of large granular lymphocytes has been examined under the light microscope (Reynolds et al., 1982; Si and Whiteside, 1983; Ward et al., 1983), the ultrastructural characteristics of these lymphocytes situated in each organ have not been demonstrated. As described above, the granule-containing lymphocytes so far reported have been collectively recognized as the lymphocytes that are characterized only by their lysosomal granules shown under the light or electron microscope. Among these granule-containing lympho- Fig. 7. Pit cell (arrow) in the splenic cord of the spleen. It is situated outside the venous sinus (VS) and possesses cytoplasmic granules (G, inset) and rod-cored vesicles (arrow, inset). R, reticular cell; M, macrophage; E, endothelial cell of the venous sinus; r, red blood cell; pl, platelet. Material in square is shown in inset. ~ 5 , 0 0 0Inset, . ~48,000. PIT CELLS IN EXTRAHEPATIC ORGANS cytes, we have first identified in various organs the pit cells, one independent subpopulation of the lymphocytes more strictly defined by their ultrastructural criteria (Kaneda and Wake, 1983; Kaneda et al., 1983): 1)electron-dense granules, 0.3-0.6 pm in diameter, which often show a n intermediate form between a wholly dense granule and a multivesicular body; 2) rod-cored vesicles, which are 0.17-0.2 pm in diameter and contain moderately dense rod structures bridging the internal space completely (this type of vesicle is exclusively found in pit cells); 3) high cell-polarity; and 4) well-developed pseudopodia. Pit cells were observed in many organs, and no particular morphological difference was recognized among them. They were divided into several groups based on their location in the particular organ. 1) In the liver, lungs, and spleen: these organs are richly- vascularized and contain numerous macrophages which participate in the clearance of foreign bodies or antigens and in immunological responses. Here, pit cells were found in the capillaries adhering to the endothelium. 2) In the small intestine, trachea, and epididymis: the epithelium of these organs surrounds the lumen and receives antigenic stimulations directly from the luminal contents. Here, pit cells were found in the epithelium (between epithelial cells). 3) In the bone marrow, where many types of blood cells are produced: if pit cells are also formed and matured in this organ, only a few of them would be discerned here as the granule-containing mature forms. 4)In the typical lymphoid organs or tissues: we could recognize a very small number of pit cells in the medulla of the thymus, possibly migrating out of the lymphatic vessels, but there were no pit cells in the cortex of the thymus, white pulp of the spleen, or the lymph nodes. 5) One group was found in the peripheral blood, circulating in the whole body (Kaneda and Wake, 1983; Kaneda et al., 1983). Pit cells were frequently found in the liver, lungs, small intestine, and so forth, which are exposed to foreign bodies and antigens directly through the epithelium or the blood stream. In these organs, they were usually observed together with conventional agranular lymphocytes. We consider that the former, like the latter, circulate in the bloodstream through the whole body, some of them adhering to the capillary endothelium or migrating into the parenchyma of the organs. Pit cells were, however, few in lymphoid organs like lymph nodes and thymus, where conventional agranular lymphocytes are numerous. Such a tissue distribution of pit cells was mostly in accordance with that of large granular lymphocytes or natural killer activity reported so far (Kiessling et al., 1975; Riccardi et al., 1979; Puccetti et al., 1980; Reynolds et al., 1981, 1982; Cohen et al., 1982; Tagliabue et al., 1982; Petit et al., 1983)-although some immunohistochemical studies showed large granular lymphocytes also in lymphoid tissues (Si and Whiteside, 1983; Ward et al., 1983). 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