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Pit cells in extrahepatic organs of the rat.

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THE ANATOMICAL RECORD 211:192-197 (1985)
Pit Cells in Extrahepatic Organs of the Rat
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
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.
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.
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
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.
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,
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 .
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.
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.
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).
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. [1973] indicated
the existence of granule-containing multivesicular
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,
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
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).
To establish the identity of the pit cells with natural
killer cells in these organs, we need to examine whether
the pit cells possess the surface antigens of the natural
killer cells, for example asialo GM1 (Shimamura et al.,
1982). We believe that the pit cells in various organs
have some special immunological function at each
We express our sincere thanks to Dr. C. Dan in our
laboratory for her valuable discussion during the pres-
ent study. This work was supported by the Grant-In-Aid
for Scientific Research No. 5737001 from the Ministry
of Education, Science and Culture of Japan.
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