Section C
/ Sinusoidal Cells
30
ENDOTHELIAL AND PIT CELLS
FILIP BRAET
DIANZHONG LUO
ILAN SPECTOR
DAVID VERMIJLEN
EDDIE WISSE
LIVER SINUSOIDAL ENDOTHELIAL CELLS 437
Preparation of Endothelial Cells 438
Identification of Endothelial Cells 439
Biology of Endothelial Cells 439
Biology of Fenestrae 440
PIT CELLS, THE LIVER-SPECIFIC NATURAL KILLER
CELLS 444
LIVER SINUSOIDAL ENDOTHELIAL CELLS
Liver sinusoidal endothelial cells (LSECs) constitute the
sinusoidal wall, also called the endothelium, or endothelial
lining (see website chapter v W-26). The liver sinusoids
can be regarded as unique capillaries which differ from
other capillaries in the body, because of the presence of fenestrae lacking a diaphragm and a basal lamina underneath
the endothelium. The first electron microscopic observation of LSEC fenestrae was accomplished in 1970 by Wisse
(1), who applied perfusion fixation to the rat liver and
demonstrated groups of fenestrae arranged in sieve plates.
In subsequent reports, Widmann (2) and Ogawa (3) confirmed the existence of fenestrae in LSECs by using transmission electron microscopy (TEM). In general, endothelial fenestrae measure 150 to 175 nm in TEM, occur at a
frequency of 9 to 13 per μm2, and occupy 6% to 8% of the
F. Braet: Laboratory for Cell Biology and Histology, Free University of
Brussels, 1090 Brussels, Belgium.
D. Luo: Laboratory for Cell Biology and Histology, Free University of
Brussels, 1090 Brussels, Belgium; and Department of Pathology, First Affiliated
Hospital, Guangxi Medical University, Nanning 530021, Guangxi, China.
I. Spector: Department of Physiology and Biophysics, Health Science
Center, State University of New York at Stony Brook, New York.
D. Vermijlen: Laboratory for Cell Biology and Histology, Free University
of Brussels, 1090 Brussels, Belgium.
E. Wisse: Laboratory for Cell Biology and Histology, Free University of
Brussels, 1090 Brussels, Belgium.
Identification, Structure, and Tissue Distribution of Pit Cells
444
Surface Phenotype of Pit Cells 445
Isolation and Purification of Pit Cells 446
Heterogeneity and Origin of Pit Cells 446
Functions of Pit Cells 448
Mechanisms in Pit Cell-Mediated Cytotoxicity 448
endothelial surface in scanning electron microscopy (SEM)
(4). In addition, differences in fenestrae diameter and frequency in periportal and centrilobular zones were demonstrated; in SEM the diameter decreases slightly from 110.7
± 0.2 nm to 104.8 ± 0.2 nm, whereas the frequency
increases from 9 to 13 per μm2, resulting in an increase in
porosity from 6% to 8% from periportal to centrilobular
(5). Recent atomic force microscopic observations on glutaraldehyde-fixed and living LSECs in culture revealed that
the fenestrae diameter is about 240 to 280 nm (6,7).
Other ultrastructural characteristics of LSECs are the presence of numerous bristle-coated micropinocytotic vesicles
and many lysosome-like vacuoles in the perikaryon, indicating a well developed endocytotic activity. The nucleus sometimes contains a peculiar body, the sphaeridium (8).
On the basis of morphological and physiological evidence,
it was reported that the grouped fenestrae act as a dynamic
filter (4,9). Fenestrae filter fluids, solutes and particles that
are exchanged between the sinusoidal lumen and the space of
Disse, allowing only particles smaller than the fenestrae to
reach the parenchymal cells or to leave the space of Disse
(Fig. 30.1). At present, it has become clear that alteration of
the endothelial filter affects the bidirectional macromolecular
exchange, and therefore may determine the balance between
health and disease. Although the majority of the research has
been descriptive, the role of the liver sieve has been demonstrated in various diseases such as hyperlipoproteinemia, cirrhosis and cancer (Reviewed in ref. 10). Another functional
438
Chapter 30
A
B
D
C
FIGURE 30.1. Scanning electron micrographs of the sinusoidal endothelium from rat liver. A: Low
magnification showing the fenestrated wall, the space of Disse (SD) and the bordering parenchymal cells (Pc). Notice also the clustering of fenestrae in sieve plates (arrow). Scale bar, 1 μm.
B: Higher-power micrograph depicting fenestrae (arrow). Scale bar, 250 nm. C: Low magnification
showing the sinusoidal endothelium of a corn oil-fed rat. After administration of a dose of corn oil,
chylomicrons (arrow) with diameters in the range of 250 to 600 nm were present in the liver sinusoids. Scale bar, 1 μm. D: Higher magnification shows lipid particles passing the fenestrae (arrow),
illustrating the sieving effect of fenestrae. Scale bar, 250 nm. (Courtesy of Dr. R. De Zanger.)
characteristic of LSECs is their high endocytotic capacity.
This function is reflected by the presence of numerous endocytotic vesicles and by the effective uptake of a wide variety
of substances from the blood by receptor-mediated endocytosis [(11), reviewed in ref. 12]. This endocytotic capacity,
together with the presence of fenestrae and the absence of a
regular basal lamina, makes these cells different and unique
from any other type of endothelial cell in the body (13).
Preparation of Endothelial Cells
LSECs seem to be vulnerable cells, a fact that becomes obvious during the isolation, purification and cultivation proce-
dures for in vitro studies. LSEC suspensions from rats and
mice can be obtained through a variety of isolation and
purification techniques, all including perfusion of the liver
with one or more tissue-dissociating enzymes. Specific
methods can be chosen to satisfy particular requirements in
terms of cell yield, purity, intact morphology, responsiveness, and ability to survive in culture (Reviewed in ref. 14).
We found that the use of a low-rate nonrecirculating perfusion of rat liver with collagenase plus fetal calf serum, followed by purification using isopycnic sedimentation in a
two-step Percoll gradient and removal of contaminating
cells by selective adherence, gave a vital and morphologic
intact LSEC population of high purity, enabling the study
Endothelial and Pit Cells
439
FIGURE 30.2. Scanning electron micrograph of a spread liver sinusoid endothelial cells after 8hour culture on a collagen-matrix (asterisk). Nucleus (N); Sieve plate (arrow). Scale bar, 2 μm.
of structure and function of these cells in vitro (Fig. 30.2)
(15). Comparable isolation and purification methods have
been described to obtain LSEC cultures from pig (16) and
humans as well (17).
Identification of Endothelial Cells
To discriminate LSECs from other liver cells (pit cells, stellate cells, Kupffer cells (KC), parenchymal cells, and bile
duct cells), immunocytochemical, receptor ligand, and
ultrastructural studies can be used. RECA-1 and SE-1 have
been reported as suitable antibodies to stain rat LSECs in
vitro and in situ (18,19). However, RECA-1 antibodies do
in fact label other microvascular endothelia as well (18). A
commercial LSEC-specific human monoclonal antibody
named HM 15/3 (BMA Biomedical AG, Switzerland) is
available as well. Staining with von Willebrand factor or
factor VIII-related antigen is commonly used as a cytochemical marker for vascular endothelium and has been
used in attempts to identify LSECs (16,20–21). However,
this marker should be used with caution, because the presence of von Willebrand factor in LSECs is controversial.
Variations in activity and presence among various species
make it inadvisable to use this marker. Until this controversy has been settled, markers other than von Willebrand
should be used to identify LSECs.
LSECs possess cell membrane receptors, which enable
them to clear rapidly from the blood specific substances
such as hyaluran and other extracellular matrix components, which can be used to specifically label LSECs (12).
Besides hyaluronan, Smedsro/d et al. (22) conjugated fluorescein isothiocyanate to chondroitin sulphate proteoglycan, collagen alpha chains, and N-terminal propeptide of
procollagen type I. All these substances are endocytosed
exclusively and with a remarkable efficiency by LSECs.
Therefore, in systems with viable cells, this way of distinguishing LSECs from other types of cells is probably the
most reliable and specific method available at present.
LSECs in culture display their normal morphologic and
functional characteristics, such as fenestrae and pinocytotic
vesicles (1), and can also be identified easily from other liver
cells by their ultrastructural morphology (Fig. 30.2)
(13,15).
Biology of Endothelial Cells
At present, studies are focused on the molecular biology
and the clinical aspects of this intriguing class of cells
(23–24). LSECs can be regarded as outlined earlier: (a) as
a “selective sieve” for substances passing from the blood to
parenchymal and stellate cells, and vice versa (10), and (b)
as a “scavenger system” which clears the blood from many
different macromolecular waste products, which originate
from turnover processes in different tissues (22). In addition, LSECs have (c) a variety of adhesion molecules
(25–26), (d) a capacity to secrete cytokines (26–27), and
(e) an important role in various diseases such as liver cancer (28), posttransplantation rejection (29), alcoholic liver
disease (30), lipoprotein metabolism (31), fibrosis (32),
and viral infection (23).
440
Chapter 30
Interactions between leukocytes or cancer cells and
LSECs have been known to be involved in the pathogenesis of liver injury (26). In these cell-to-cell interactions, a
wide spectrum of adhesion molecules play a key role, and
their expression is mainly regulated by inflammatory
cytokines such as interleukin-1, tumor necrosis factor-α
and interferon-γ (27). LSECs in normal liver express intercellular adhesion molecule-1, intercellular adhesion molecule-2, leukocyte function-associated-3, very late antigen-5,
and CD44. In patients with acute or chronic liver disease,
intercellular adhesion molecule-1 and vascular cell adhesion
molecule-1 expression are markedly enhanced in the
inflamed liver tissue (27,33–34). Selectins are not present in
normal conditions, but are induced after lipopolysaccharide
administration (35).
Colorectal tumors often metastasize in the liver. In general, it has been supposed that single tumor cells get stuck
in the sinusoids when entering the liver, because their size
largely exceeds the diameter of a sinusoid. After plugging,
their adhesion molecules might react with the surface molecules of the LSECs, enabling them to extravasate and enter
the liver parenchyma. Therefore, the adherence of tumor
cells to LSECs is a crucial step in early stages of hepatic
metastasis [(36), reviewed in ref. 37].
The success of liver transplantation depends at least partially on the functioning of LSECs in the transplanted liver.
Preparing the liver for transplantation includes perfusion of
preserving fluid, lowering the temperature, stagnant flow
during preservation, and reperfusion with warm oxygenated
blood when connecting the liver to the donor circulation
(29). Many studies point to the LSECs as being very sensitive
in this procedure, possibly leading to essential and lethal deficiency during the posttransplantation period (23,38).
Experimental data have accumulated suggesting that
alcohol-induced pathological changes of LSECs precede the
pathological changes of the hepatocytes. Moreover, due to
its strategic position in the liver sinusoid, LSEC dysfunction and structural alterations have far-reaching repercussions for the whole liver (39). There is evidence suggesting
that alcohol-induced LSEC alterations are mostly due to
KC activation induced by alcohol rather than to a direct
action of alcohol on LSEC. In alcoholemia, the activated
KC secrete a spectrum of mediators that affect both the
structure and function of LSECs. Alcohol-induced LSEC
dysfunction comprises a dramatic decrease in hyaluronan
uptake by the scavenger receptors of LSECs, a decreased
porosity of the liver sieve, and an increment in the secretion
of the vasoconstrictor endothelin-1 (40).
LSECs also have important functions in the handling of
lipoproteins and the regulation of lipoprotein metabolism
(31). They possess several cell membrane receptors for the
various types of apoproteins. Moreover, these scavenger
receptors have been implicated in adhesion, clearance of
dying cells, and host defense against foreign organisms [(41),
reviewed in ref. 42].
The role of LSECs in liver fibrosis seems to be quite passive; that is, the cells might become involved in the process
of capillarization, but active participation in the synthesis of
extracellular matrix products seems to be the task of activated
stellate cells (reviewed in ref. 32). However, in cirrhotic liver,
compared to normal liver, LSECs exhibit as much as five
times the collagen type I mRNA, whereas mRNA for type IV
collagen and laminin is decreased by up to 50% (43). Moreover, transforming growth factor-β1 stimulates the synthesis
of basement membrane proteins laminin, collagen type IV
and entactin in rat LSEC cultures (44).
LSECs are permissive for mouse hepatitis virus 3, at the
same time showing a decrease in the number of fenestrae in
vivo and in vitro (45). Therefore, it has been hypothesized
that viral infection of LSECs may cause hyperlipoproteinemia. However, feline immunodeficiency virus, as a model
for human immunodeficiency virus type 1, did not change
the number of fenestrae upon infection. Moreover, the infection of LSECs with the feline immunodeficiency virus contributed to the progression of the infection (46). When
human LSECs were infected with human immunodeficiency virus type 1 in vitro, they showed the budding of new
viral particles, indicating the production of new viruses. This
infection was probably facilitated by CD4 surface antigens
on LSECs, as were shown to be present by immunogold-EM
(47). Therefore, it is concluded that LSECs might be a target and a reservoir for human immunodeficiency viruses.
Biology of Fenestrae
To date, one of the widely accepted hypotheses maintains
that drugs which dilate fenestrae, such as pantethine, acetylcholine and ethanol improve the extraction of dietary cholesterol from the circulation while drugs such as nicotine,
long-term ethanol abuse, adrenalin, noradrenalin and serotonin, which decrease the endothelial porosity, play a role in
the development of drug- and stress-related atherogenesis
(48). As a consequence, alterations in the number or diameter of fenestrae by drugs, hormones, toxins, and diseases
can produce serious perturbations in liver function (10).
Current interest focuses on the role of the actin
cytoskeleton in regulating the diameter and number of fenestrae. Immunoelectron microscopic studies of LSECs in
the early 1980s revealed the first information regarding the
structural basis of the contraction and dilatation machinery
of fenestrae. Oda et al. (49) described in 1983 the presence
of actin filaments in the neighborhood of fenestrae, indicating that the cytoskeleton of LSECs plays an important
role in the modulation of fenestrae. At present, it is widely
accepted that contractile bundles of actin and myosin
around fenestrae regulate fenestrae diameter under the control of intracellular calcium levels (Fig. 30.3) (50–53).
In 1986, Steffan et al. (54–55) provided the first evidence that LSEC fenestrae are inducible structures. Treatment of LSECs in situ and in vitro with the microfilament-
Endothelial and Pit Cells
441
FIGURE 30.3. Scheme of the serotonin signal pathway showing the steps in fenestral
contraction and relaxation, as postulated by
Arias and co-workers (50–53): (I) serotonin
binds to a ketanserin-inhibitable receptor,
coupled to a pertussis-toxin sensitive G-protein; (II) a calcium channel opens, causing an
influx of calcium ions; (III) the intracellular
calcium level increases rapidly, and (IV) calcium binds to calmodulin, (V) the calciumcalmodulin complex activates myosin light
chain kinase, and (VI) as a result phosphorylation of the 20-kd light chain of myosin
occurs, resulting in (VII) an increased actinactivated myosin ATPase activity, which
finally initiates contraction of fenestrae. The
mechanism for the relaxation of liver
sinoidal endothelial cell fenestrae is
presently unclear and probably involves
dephosphorylation of myosin light chains
(MLC) as represented by dashed lines: a
decrease in the cytosolic free calcium concentration leads to dissociation of calcium
and calmodulin from the kinase, thereby
inactivating myosin light chain kinase
(MLCK); under these conditions myosin light
chain phosphatase, which is not dependent
on calcium for activity, dephosphorylates
myosin light chain and finally causes relaxation of fenestrae. ADP, adenosine diphosphate; ATP, adenosine triphosphate.
inhibiting drug cytochalasin B resulted in an increased number of fenestrae. SEM observations of detergent-extracted
LSECs revealed that the increase in the number of fenestrae
was related to an alteration of the cytoskeleton. Moreover,
the effect of cytochalasin B on the number of fenestrae and
cytoskeleton could be reversed after removal of the drug.
However, when LSECs were treated with various microtubule-altering drugs, there was no effect on the number of
fenestrae, thereby demonstrating that microtubules are not
involved in the formation of the endothelial pores (55–56).
These observations indicate that fenestrae are dynamic structures which may undergo changes in number in response to
local external stimuli and that the actin-cytoskeleton has a
major role in this process. Later, Bingen et al. (57) noted, in
freeze-fracture replicas of cytochalasin B-treated LSECs,
areas which were more or less devoid of intramembrane particles having the size of fenestrae. Those authors proposed
that fenestrae are formed by fusion between opposite sheets
of plasma membrane which are depleted of intramembrane
particles. In addition, Taira (58) elaborated the study of Bingen et al. (57) and found some new evidence about the formation of sieve plates or clustered fenestrae. The study of the
luminal cell membrane of freeze-fractured LSECs revealed
the presence of trabecular meshworks which were attached
to the E- and P-face of the cell membrane of both the cell
body and the attenuated cell processes. The author demon-
strated that these meshworks give rise to a stepwise formation of sieve plates by ballooning, fusion and flattening of
the cell membrane and cytosol among these plasmalemmal
invaginations.
The recent availability of a battery of new actin binding
drugs that affect the polymerization of actin by different
mechanisms greatly enhances the precision with which the
dynamics and functions of the actin cytoskeleton in various
cell types can be dissected (59). The study of the numerical
dynamics of LSEC fenestrae is an example of such a cellular process, in which the use of several microfilament-disrupting drugs was necessary to identify one that could selectively reveal the process of fenestrae formation. In the past
we demonstrated that treatment of LSECs with cytochalasin B, latrunculin A, swinholide A, misakinolide A or jasplakinolide induce an increased number of fenestrae
(59–61). However, only by treating LSECs with misakinolide were we able to visualize the process of fenestrae formation and to identify a new structure involved in the
process of fenestrae formation, as we describe below (61).
Actin filament staining of untreated LSECs displays
intense circular bundles lining the cell periphery and few
straight bundles oriented parallel to the long axis of the cell
(Fig. 30.4A). The maximal effect of misakinolide on F-actin
was obtained as soon as 10 to 15 minutes after treatment
and caused loss of F-actin bundles. Further incubation did
Chapter 30
442
A
B
C
D
E
F
Endothelial and Pit Cells
not result in additional alterations in actin organization or in
adverse effects on cell shape and viability (Fig. 30.4B).
Remarkably, within one hour of misakinolide treatment, we
could observe with the aid of SEM small cytoplasmic unfenestrated areas, surrounded by rows of very small fenestrae
within the area of fenestrated cytoplasm (Fig. 30.4C). To
study these areas in more detail we applied whole-mount
TEM to LSECs cultured on collagen-coated grids, after
slight prefixation and extraction with detergent, as this technique allows the visualization of the cytoskeleton with minimal disruption of the cells (62) (Fig. 30.4D-F). Examination of control LSECs at low magnification showed the
existence of an extensive network of cytoskeletal elements
that fills and structurally organizes the cytoplasm (Fig.
30.4D). Treatment with misakinolide for 10 to 30 minutes
resulted in the disappearance of microfilaments, and in the
appearance of small cytoplasmic unfenestrated areas of intermediate electron density (gray centers) within the cytoplasm
of all treated cells. In several of these unfenestrated areas, a
singular structure could be observed, consisting of rows of
fenestrae with increasing diameter, emanating from the gray
centers and fanning out into the surrounding cytoplasm
(Fig. 30.4E). These structures are suggestive of de novo fenestrae formation, and we therefore named them “fenestraeforming center” (FFC). By two hours of treatment, the burst
of fenestrae formation has subsided and the small unfenestrated areas (gray centers) did not show the presence of rows
of fenestrae (Fig. 30.4F). Thorough investigation of swinholide A-, jasplakinolide-, cytochalasin B-, and latrunculin
A-treated LSECs revealed only small unfenestrated areas, but
no sign of connected fenestrae rows. The disassembly of
actin filaments in LSECs by these compounds, together with
the increase in the number of fenestrae and the presence of
inactive FFCs, suggests nevertheless a common mechanism
of fenestrae formation for all actin binding agents. Appar-
443
ently, specific alterations in actin organization at particular
locations and at particular times are required to bring to
light nascent fenestrae emerging from the FFCs.
When the short period of time necessary to form new
fenestrae is taken into account, it is most likely that this
process does not involve de novo protein synthesis. We have
performed experiments with cycloheximide, an inhibitor of
protein synthesis, to check this hypothesis. In this set-up,
the use of cycloheximide in combination with misakinolide
resulted also in the appearance of active FFCs and in an
increment in the number of fenestrae. Therefore, it is obvious that the process of fenestrae formation is not driven via
protein synthesis. Instead, we consider a reorganization of
preexisting FFCs. By using dry-cleaving of LSECs, we
recently observed a sponge-like framework of three-dimensional organized fenestrae grouped along the nucleus, as
early as 5 minutes after microfilament-disruption (59). We
suppose therefore that FFCs are normally anchored to the
actin cytoskeleton in the perinuclear area of LSECs, where
they cannot be resolved in whole-mount TEM or TEM sections due to the mass thickness and/or the complex threedimensional organization of the cytoskeletal proteins in this
area (59). Disruption of the actin cytoskeleton can, therefore, free the preexisting FFCs from their anchor in the perinuclear region, resulting in their translocation into the
300- to 400-nm–thick peripheral cytoplasm, thereby giving
rise to flattened FFCs as depicted in Figure 30.4E.
The fusion of two opposing cell membranes to form fenestrae in LSECs requires the presence of unique compositional membrane microdomains (57,63) and a cell membrane-associated cytoskeletal structure (61). Several theories
have been used to model the possible mechanisms of membrane fusion and pore formation. In general, the process leading to membrane fusion is subdivided into the following:
adhesion-dehydration; disappearance of the hydration bar-
FIGURE 30.4. A: Actin distribution in control liver sinusoidal endothelial cells (LSECs) showing
the presence of stress fibers (arrow), mainly oriented parallel to the long axis of the cells, and
peripheral bands (arrowhead) of actin bundles that line the cell margin. Scale bar, 5 μm. B: LSECs
treated with 25 nM misakinolide A for 10 minutes show a loss of actin bundles and the appearance of curly actin aggregates (arrow). Peripheral actin bands (arrowhead) are less dense and
interrupted. Scale bar, 5 μm. C: High-power scanning electron micrograph of the fenestrated
cytoplasm obtained after 1 hour of exposure to 25 nM misakinolide. Note a typical cytoplasmic
unfenestrated area (asterisk), surrounded by rows of very small fenestrae (arrow). Scale bar, 250
nm. D–F: Transmission electron micrographs of whole-mount, formaldehyde-prefixed, cytoskeleton buffer-extracted LSEC of control (D) and misakinolide-treated LSECs. (E–F) D: Low magnification showing the cell nucleus (N) and extracted cytoplasm. Note that the sieve plates are well
defined by a dark border (arrowheads). Inside the sieve plates, fenestrae can be observed
(arrow). Scale bar, 2 μm. E: Treatment with 25 nM misakinolide A for 30 to 60 minutes resulted
in the appearance of small cytoplasmatic unfenestrated areas of intermediate density (asterisk)
within the fenestrated cytoplasm and showing the initial step of fenestrae formation (arrow).
Scale bar, 200 nm. F: Low magnification showing the cell nucleus (N) and the highly fenestrated
cytoplasm (small arrow) after 120 minutes of 25 nM misakinolide treatment. Compare with Fig.
4D for the difference from control. Note the thin cytoplasmic arms (arrowheads) which run from
the nucleus into the cytoplasm. In the cytoplasm, inactive FFCs (large arrow) can be observed.
Scale bar, 5 μm.
444
Chapter 30
rier; contact between phospholipid bilayers, and molecular
rearrangement, resulting in pore formation (64–66). As for
LSECs, the first step corresponds to the formation of
intramembrane protein-free zones (57), while the appearance
of peristomal rings of sterols around fenestrae probably corresponds to the final step (63). It seems reasonable to consider
that these events take place in the rim of FFCs. However,
although nothing realistic can be said about the molecular
composition of FFCs, we speculate that the presence of fusion
proteins (64), which pull the bilayers of the cell membranes
together on the rim of FFCs, may contribute as well to the
fusion-fission process. In addition, based on our gallery of
electron micrographs taken from misakinolide-treated
LSECs, it became clear that the nascent fenestrae emanating
from an FFC are already decorated with the earlier described
fenestrae-associated cytoskeleton ring (FACR) (Fig. 30.4E)
(62). This probably indicates that FFCs already contain the
necessary cytoskeletal proteins for assembling the FACR.
In conclusion, despite the multidisciplinary approach
taken to study the structure, origin, dynamics and formation of fenestrae, there are still important gaps in information. Our knowledge needs considerable consolidation and
expansion at the structural and biochemical level to reveal
how the different proteins form a contractile unit, and
which signal transduction pathways are involved in fenestral dilatation and relaxation. Moreover, further investigation is needed to determine the molecular composition of
the FACR, the FFC, and the exact mechanism of fenestrae
formation in the FFCs.
For the near future, we expect that forthcoming research
will focus on therapeutic strategies by altering the sieve’s
porosity. For example, the discovery of new drugs that
increase the porosity of the liver sieve may be of great benefit. Such agents may not only improve the extraction of
atherogenic lipoproteins from the circulation; they could
also be used for enhancing the efficiency of liposome-mediated gene or drug delivery to parenchymal cells.
such as the spleen, lung, intestine, lymph nodes, bone marrow and liver (70). NK cells in the liver, also called pit cells
(71), constitute a unique resident population in the liver
sinusoids. Their immunophenotypical, morphological and
functional characteristics differ from those of blood NK
cells (72). Morphologic and cytotoxic differences between
pit cells of different species have been observed (73).
Identification, Structure, and Tissue
Distribution of Pit Cells
Pit cells were first described in 1976 by Wisse (71). The
name pit cell was introduced because of the characteristic
cytoplasmic granules, which in Dutch language are called
pit, resembling the pits in a grape (71). The hypothesis that
pit cells might possess NK activity was formulated by
Kaneda et al. (74), and was based on their morphologic
resemblance to LGL. The isolation and purification of pit
cells from rat liver and the evidence of spontaneous cytotoxicity against YAC-1 cells confirmed these cells to be
hepatic NK cells (75–76).
Pit cells inhabit the liver sinusoids and often adhere to
LSECs, although they incidentally contact KCs (Fig. 30.5).
PIT CELLS, THE LIVER-SPECIFIC NATURAL
KILLER CELLS
Natural killer (NK) cells are functionally defined by their
ability to kill certain tumor cells and virus-infected cells
without prior sensitization (67). NK cells comprise about
10% to 15% of lymphocytes in the peripheral blood, and
most of these cells in human and rat have the morphology
of large granular lymphocytes (LGL) (68). However, it has
been demonstrated that small agranular lymphocytes, lacking CD3 expression, have cytolytic activity comparable to
that of NK cells (69). These variations may be related to the
stage of NK cell differentiation or heterogeneity (70).
Moreover, some cytotoxic T lymphocytes (CTL) also display LGL characteristics (70). Besides NK cells in peripheral blood, NK cells are also found in tissue compartments
FIGURE 30.5. Transmission electron micrograph of a pit cell in a
rat hepatic sinusoidal lumen. The pit cell (P) shows a polarity
with an eccentric nucleus. The cytoplasm is abundant and contains characteristic electron-dense granules and other organelles
lying mainly on one side of the nucleus. The cell contacts an
endothelial cell (E) and a portion of a Kupffer cell (K) with a positive peroxidase reaction product in the rough endoplasmic reticulum. Note the relation with parenchymal cells (Pc). Scale bar,
1μm. (From Bouwens L, Wisse E. Tissue localization and kinetics
of pit cells or large granular lymphocytes in the liver of rats
treated with biological response modifiers. Hepatology 1988;8:
46–52, with permission.)
Endothelial and Pit Cells
They face the blood directly. Pseudopodia of pit cells can
penetrate the fenestrae of the LSECs and enter the space of
Disse, and can directly contact the microvilli of hepatocytes
(4,71). Their appearance in the space of Disse is not a common feature (77). By morphological investigation, the frequency of pit cells in rat liver tissue was found to be about
an average of 1 pit cell per 10 KCs. The number of pit cells,
in untreated rats, is therefore estimated to be 1.4 to 2 × 106
cells per gram liver weight (75). By immunohistochemistry,
using mAb 3.2.3 against NKR-P1A (a specific marker of
NK cells), the number of pit cells in frozen sections of rat
liver was counted as 13.7 per mm2 (78). After intravenous
injection of biological response modifiers (BRM), the number of pit cells increases 4- to 6-fold in rat liver treated with
zymosan (79), and 43-fold with interleukin-2 (IL-2) (80).
The surplus of pit cells is considered to originate from local
proliferation and from the bone marrow (79–80). Pit cells
were found to be more numerous in the periportal than in
the pericentral region of the liver lobule (74,78).
Pit cells have essentially the same morphology as NK
cells from blood and other organs, that is, LGL morphology
(Fig. 30.5). The cells are characterized by a relatively large
size compared to other lymphocytes, the presence of granules in the cytoplasm, a pronounced asymmetry of the cell
and an indented or kidney-shaped nucleus of high density
(81). Pit cells in the rat are about 7 μm in diameter and vary
in shape, possessing well-developed pseudopodia. They
show a pronounced polarity with an eccentric nucleus and
most organelles lying at one side of the nucleus.
The most conspicuous organelles are the electron-dense
granules. These granules have several characteristics. They are
azurophilic and can be seen after Giemsa staining of a cell
smear or cytospin preparation with light microscopic examination. As measured by electron microscopy, the granules dif-
445
fer in size between different pit cell subpopulations [low-density (LD) and high-density (HD) pit cells], but within one cell
type the granules are rather homogeneous with respect to size,
shape and electron density (82). The granules are membranebound and range in size between 0.2 μm in LD pit cells and
0.5 μm in lymphokine-activated killer (LAK) cells. The granules contain lysosomal enzymes, such as acid phosphatase
(75,83). Although perforin and granzymes, which have been
isolated from NK cell granules (84–85), have not yet been
identified in pit cell granules, it is believed by analogy that
these molecules are present in the granules of pit cells.
Rod-cored vesicles are small inclusions, ranging in diameter from 0.17 to 0.2 μm, and are exclusively found in LGL
(74,83). They contain a straight rod structure that is 30 to
50 nm in length, which bridges the entire diameter of the
vesicle (74,83). Rod-cored vesicles derive from and distribute preferentially around the Golgi apparatus. Possibly rodcored vesicles may also contain cytotoxic factors functioning in natural cytotoxicity (77).
Pit cells also exist in human and mouse liver, but their
identification is difficult because they contain a low number of small granules and only a very few rod-cored vesicles
(86–88). On the other hand, 5% to 25% of human pit cells
contain “parallel tubular arrays” (PTA), which were also
reported in human blood NK cells and are considered as a
characteristic of these cells (72,87).
Surface Phenotype of Pit Cells
Most surface antigens found on rat pit cells are similar to
those found on spleen or blood NK cells (Table 30.1)
(76,78,89,90). Pit cells were found to express NKR-P1 (78)
(Fig. 30.6). NKR-P1 was first identified in the rat (91) and
has now been shown to be expressed by mouse and human
TABLE 30.1. CHARACTERISTICS OF RESTING NK CELLS, LD, HD PIT CELLS, AND IL-2-ACTIVATED-NK CELLS
Morphology
Size of the cell (øin μm)
Number of rod-cord vesicles per cell
Size of specific granules (øin μm)
Number of granules per cell
Surface antigensb
CD2
CD8
CD11a
CD18
CD54
Asialo-GM1
NKR-PI
Cytotoxicity
NK activity
P815 cell killing
Resting NK Cells
HD Pit Cells
LD Pit Cells
IL-2-Activated-NK Cells
6–7
0.5
Large (0.3–0.5)
Low (n = 10)
6–7
0.8
Intermediate (±0.2)
Intermediate (n = 20)
6–7
1.0
Small (±0.17)
High (n = 50)
8–13
n.d.a
Very Large (0.5–0.9)
n.d.
80
40
54
90
35
100
94
80
100
90
90
35
70
95
80
100
90
90
35
36
95
100
n.d.
90–100
90–100
97
n.d.
95
+
−
++
−
+++
+
++++
+
Data summarized from references 68, 76, 78, 82, 83, 89, and 91; an.d., no data; bApproximate percentage of cells that
express antigen.
446
Chapter 30
cells using mAbs against surface antigens found on T and B
cells (72,95). Since pit cells are apparently not heavily
anchored in the liver sinusoids, the cells can be washed out
by a nonenzymatic, high-pressure (50 cm water) perfusion
of the liver via the portal vein with phosphate-buffered saline
supplemented with 0.1% EDTA (75). The washout is collected from the vena cava. The erythrocytes, granulocytes
and cell debris in the washout are removed by Ficoll-Paque
gradient centrifugation. The mononuclear cells recovered
from the interface of Ficoll-Paque gradient are composed of
T cells, pit cells, B cells, monocytes, and a few LSECs.
Adherent monocytes and B cells in this population can be
selectively removed in a nylon wool column (75). Pit cells
are further purified by magnetic cell sorting (95). With this
system, a highly purified population of pit cells can be
obtained by negative selection, that is, by elimination of
remaining monocytes, T and B cells using specific antibodies and immunomagnetic beads. By this method, pit cells
with a purity of more than 90% and a viability of more than
95% can be obtained (Fig. 30.7). Moreover, this nonenzymatic method does not destroy cell surface molecules.
FIGURE 30.6. Immunotransmission electron micrograph showing a 3.2.3-positive LGL (pit cell) (arrowhead) and a 3.2.3-negative agranular cell. The 3.2.3+ pit cell shows characteristic electron-dense granules in the cytoplasm and immunoperoxidase
reaction product on the surface. Scale bar, 1μm. (From Luo D,
Vanderkerken K, Bouwens L, et al. The number and distribution
of hepatic natural killer cells (pit cells) in normal rat liver: an
immunohistochemical study. Hepatology 1995;21:1690–1694,
with permission.)
Heterogeneity and Origin of Pit Cells
A considerable set of data indicates that rat pit cells constitute a heterogeneous population. Based on the cell density,
pit cells can be separated into LD and HD cells by apply-
NK cells (92–93). NKR-P1 is present on 94% of rat LGL
and serves as a molecule triggering cytotoxic action (91).
The anti-NKR-P1 monoclonal antibody (mAb) 3.2.3 is
considered to be useful for NK cell identification (91).
However, a subset of T lymphocytes and polymorphonuclear leukocytes (PMN) also expresses NKR-P1 (91,93).
CD11a is present on 90% of rat pit cells, which is different
from rat peripheral blood NK cells (54%) (89). Approximately 90% of rat pit cells express CD18, 35% express
CD54 and 80% express CD2 (89). Asialo-GM1, which is
expressed by all rat blood NK cells (82), is present on 36%
of LD pit cells and 70% of HD pit cells (82). CD8, a
marker of NK cells and cytotoxic T lymphocytes (68), is
present on all rat pit cells (76). However, the composition
of CD8 in NK cells and T lymphocytes is different. Most
CD8+ NK cells express CD8α/CD8α homodimers rather
than the CD8α/CD8β heterodimers prevalent on cytotoxic
T cells (94). In addition, rat pit cells do not express T cell
receptor and CD5 antigen (a pan T cell marker) (76–77).
Isolation and Purification of Pit Cells
The isolation method for rat pit cells is based on a nonenzymatic, high-pressure washout technique (75) followed by
purification, based on the magnetic negative selection of
FIGURE 30.7. Light micrograph of an isolated and purified pit
cell population in a May-Grünwald-stained cytospin. The cells
contain cytoplasmic granules, which can be used to recognize
and count the number of pit cells in freshly isolated liver-associated lymphocyte population. Scale bar, 5 μm.
Endothelial and Pit Cells
ing 45% iso-osmotic Percoll gradient centrifugation (82).
These two cell populations have been shown to differ
immunophenotypically, morphologically and functionally
from each other and from blood LGL (Table 30.1)
A
B
FIGURE 30.8. Transmission electron micrograph of a typical
low-density (LD) pit cell (A) and a blood NK cell (B). A: The
main morphological characteristics of LD pit cells, compared to
high-density (HD) cells and blood NK cells, is the presence of
numerous small cytoplasmic granules. B: Note the few, but
large granules in blood NK cell. Scale bar, 1 μm. (From Vanderkerken K, Bouwens L, Wisse E. Characterization of a phenotypically and functionally distinct subset of large granular lymphocytes (pit cells) in rat liver sinusoids. Hepatology,
1990,12:70–75, with permission.)
447
(82,89,90,96). LD pit cells (Fig. 30.8A) contain more rodcored vesicles and more but smaller granules than blood
NK cells (Fig. 30.8B) (82). The number of rod-cored vesicles and granule composition (number and size) of HD pit
cells are intermediate between LD pit cells and blood NK
cells (82). Immunophenotypically, almost all blood NK
cells are asialo-GM1 positive, and 70% of HD pit cells are
strongly positive, whereas only 36% of LD pit cells are
weakly positive (82). Furthermore, functional differences
have been observed among these three populations. The LD
pit cells are more cytotoxic against YAC-1 cells and colon
carcinoma (CC531s) cells than blood NK cells (96). The
HD pit cells have intermediate cytotoxic activity between
LD pit cells and blood NK cells (96). In addition, LD pit
cells are able to lyse LAK-sensitive P815 mastocytoma targets, which are resistant to normal blood NK cells and
hepatic HD pit cells (96).
Pit cells are considered to originate from blood NK cells
(97). Several types of evidence support the concept that
blood NK cells immigrate into the hepatic sinusoids to
become HD pit cells, which further differentiate into LD pit
cells. Importantly, the characteristics and functions of HD
pit cells are intermediate between blood NK cells and LD pit
cells (97). Kinetic experiments with sublethal total body
irradiation (700 cGy) showed that blood NK cells and HD
pit cells were depleted about one week after irradiation,
whereas LD pit cells had totally disappeared at two weeks
after irradiation. Shielding of the liver gave similar results,
and splenectomy did not affect pit cell number (97). With
the use of intravenous anti-asialo-GM1 antiserum injection,
blood NK and HD pit cells totally disappeared within one
week of treatment, whereas LD pit cells disappeared from
the liver one week later (97). The direct evidence for LD pit
cells originating from asialo-GM1-positive precursors
(blood NK and HD pit cells) was given by the adoptive
transfer of fluorescent-labeled HD pit cells into syngeneic
rats. After three days, 5% of labeled cells were recovered in
the LD fraction, and these cells displayed typical LD pit cell
morphology (97). These observations also indicate that the
lifespan of pit cells in the liver is about two weeks (72,97).
The mechanism behind the migration of blood NK cells
to the liver sinusoids is not fully understood. Several adhesion
molecules were found to be involved in the process (89). Rat
blood NK and pit cells express LFA-1 (CD11a/CD18) and
CD2 (LFA-2) adhesion molecules (89). Their ligands, CD54
(ICAM-1) and CD58 (LFA-3) were found to be present on
liver LSECs (98). After intravenous injection of antibodies
against CD2, CD11a and CD18 into rats, the number of pit
cells in the liver decreased significantly, indicating that the
interactions of LFA-1/CD54 and CD2/CD58 are involved
in the recruitment of pit cells in the liver (89).
Once marginated in the liver sinusoids, blood NK precursors first differentiate into HD pit cells, then into LD pit
cells. The microenvironment of the liver sinusoid is believed
to be responsible for this differentiation process (99). Van-
448
Chapter 30
derkerken et al. (99) found that KCs were selectively eliminated three days after intravenous injection of liposomes
containing the cytotoxic drug dichloromethylene diphosphonate. This treatment kills the KCs, whereas other cells
remain unharmed. The number of HD pit cells declined
three days after the injection. At that time, the LD pit cell
population showed no change, but a decline of about 80%
was seen seven days after the injection (99). These data
indicate that pit cells are KC-dependent, and therefore it is
supposed that KCs play a role in the differentiation of pit
cells in the liver. However, it remains unclear what factor(s)
derived from KC are responsible for this differentiation. In
addition, LSECs may work synergically with KCs and may
contribute to pit cell differentiation, since co-culture of HD
pit cells with KCs failed to induce the full differentiation of
HD into LD pit cells (99).
Functions of Pit Cells
As a member of the NK cell family, pit cells have demonstrated cytotoxic activity against various tumor cells. The
production of cytokines and participation in the resistance
to microbial pathogens is less well known (67). Rat pit cells
have a high spontaneous cytotoxic activity against various
tumor cell lines, such as YAC-1, P815, CC531s, DHDK12, L929, 3LL, and 3LL-R (81). Compared with blood
NK cells, pit cells are four to eight times more cytotoxic
against YAC-1 and CC531s cells, and are able to kill the
NK-resistant but LAK-sensitive P815 cells (82,96,100).
This evidence supports the fact that pit cells become activated by the hepatic microenvironment. Furthermore, NK
activity in the liver could be augmented by BRM (79,88).
Interestingly, an increase in function seems to coincide with
a large increase in the number of LGL (79,88).
Mechanisms in Pit Cell-Mediated
Cytotoxicity
It is believed that, like NK cells (101), pit cell-mediated target killing is a multistep process, including recognition of
target cells, binding of effector to target cells (conjugation),
activation of effector cells, delivery of the lethal signal to
target cells, and effector cell detachment and recycling
(68,70,101).
The prerequisite of pit cell killing is the binding of one
or more effector cells to a target cell (72). Adhesion molecules are considered to be responsible for this process. It has
been found that the interaction between β2 integrins
(CD11a-c/CD18) and ICAMs (intercellular adhesion molecules) is the most important mechanism of binding of NK
cells to their targets (102–103). In addition, LFA-1
(CD11a/CD18) also participates in the signal transduction
in NK cells required for NK cell activation (104). Crosslinking of LFA-1 on NK cells with its antibody is known to
induce a calcium influx, phosphoinositide turnover, and
tumor necrosis factor-α (TNF-α) production (104), and to
inhibit the target cell killing by NK cells (105). LFA-1 was
also found to be involved in pit cell-mediated cytotoxicity.
The antibody against LFA-1 inhibits not only the binding
of pit cells to target cells, but also the killing of target cells
by pit cells (106). Taken together, this information suggests
that LFA-1 on effector cells may have a dual function of
binding to target cells and triggering cytolysis.
CD2 is an adhesion molecule involved in T cell (107)
and NK cell cytotoxicity (108–109). Approximately 80%
of rat pit cells express CD2 (89). Anti-CD2 mAb had no
effect on the binding of pit cells to CC531s, or on the cytotoxicity against CC531s cells (106). However, the antiCD2 mAb enhanced the cytolytic activity of rat pit cells
against FcγR+ P815 target cells (106).
NKR-P1 is a well-known triggering receptor on NK cells
(102,110). mAbs against mouse and rat NKR-P1 were
found to trigger NK cell-mediated lysis of FcγR+ target
cells, termed re-directed antibody-dependent cellular cytotoxicity (ADCC) (91). This action also involves a rise in
intracellular Ca++ levels (111) and cytokine production
(112). Furthermore, mAbs to NKR-P1 stimulate phosphoinositide turnover (111), arachidonic acid generation (113)
and granule exocytosis (91). NKR-P1 has been found to be
involved in pit cell-mediated cytotoxicity against FcγR+
P815 target, but not in FcγR− CC531s target killing (114).
These data indicate that NKR-P1 and CD2 depend on subclass specificity of target cell IgG-FcR (109), and may serve
as activation structures on pit cells.
NK cytotoxicity was originally thought to be spontaneous and major histocompatibility complex (MHC) class
I-unrestricted. However, increasing evidence indicates that
NK cells preferentially kill cells lacking MHC class I
(115–119). Masking of MHC class I by an mAb enhances
pit cell-mediated cytotoxicity against CC531s cells, indicating that MHC class I on CC531s cells protects these cells
from being killed by rat pit cells (114). An explanation of
this observation is that the cytotoxic activity of NK cells is
regulated by positive and negative signals from triggering
and inhibitory membrane receptors. The final outcome,
that is, triggering of cytotoxic activity or inhibition of cytotoxicity, appears to depend on the balance between the positive and negative signals (102,120). Inhibitory receptors on
effector cells recognize MHC class I, and this recognition
generally inhibits the lysis of MHC class I+ cells
(110,120–123). However, inhibitory receptors like Ly49
have not been directly identified on pit cells yet.
Studies have demonstrated that NK cell-mediated cytotoxicity can mainly be implemented by two pathways, the
perforin/granzyme (granule exocytosis) pathway and the
Fas/FasL pathway (124,125). The perforin/granzyme pathway is a Ca++-dependent pathway and is mediated by the
pore-forming protein perforin and granzymes, especially
granzyme B, both of which are stored in NK cell granules
(125), of which pit cells have plenty (82). After contact
Endothelial and Pit Cells
between effector and target cells, perforin and granzymes are
released in a directed manner into the intercellular space
between these cells. Perforin alone induces lysis without
inducing apoptosis, that is, fragmentation of target cell
DNA. Granzymes play a critical role in the rapid induction
of DNA fragmentation by CTLs, NK cells and pit cells
(126,127). It has been shown that pit cells induce apoptosis
in CC531s tumor cells (Fig. 30.9) by the perforin/granzyme
pathway (126).
The Fas pathway of apoptosis is mediated by the interaction of Fas ligand (FasL, CD95L) with the apoptosisinducer Fas (CD95/APO-1) molecule expressed on target
cells (124,128,129). Fas is widely expressed on lymphoid
and nonlymphoid cells, and some tumor cells (126,129).
FasL is expressed by activated T cells, NK cells and pit cells
(126,129). It has been demonstrated that Fas/FasL plays an
important role in the killing of virus-infected cells and
tumor cells by CTLs and NK cells (130). Although Fas is
expressed on CC531s cells and FasL is expressed on rat pit
cells, pit cell-mediated CC531s apoptosis was found to be
exclusively implemented by the perforin/granzyme exocytosis pathway (126).
In conclusion, there is growing evidence that pit cells
are highly active, liver-specific NK cells. Pit cells are
located in the liver sinusoids and can be separated into LD
449
and HD fractions by 45% iso-osmotic Percoll gradient
centrifugation. These two subpopulations differ morphologically, phenotypically and functionally from each other
and from blood NK cells. LD pit cells contain a higher
number of small granules, have a higher expression of LFA1, are more cytotoxic against several tumor cell lines as
compared to blood NK cells, and are able to kill NK-resistant but LAK-sensitive P815 cells. The characteristics of
HD cells are intermediate between those of LD pit cells
and blood NK cells. Pit cells most probably originate from
blood NK cells, and the recruitment of pit cells in the liver
is mediated by adhesion molecules. A major challenge is to
achieve a better understanding of the mechanisms of pit
cell cytotoxicity and the cooperation between pit cells and
other cells in the liver (i.e., KCs, LSECs, T-, and NK-T
cells). Moreover, since pit cells are located in a strategic
position in the hepatic sinusoids, they represent a first line
of cellular defense against metastasizing colon cancer cells.
The role of pit cells in a number of liver pathologies, such
as viral hepatitis, deserves more attention.
ACKNOWLEDGMENTS
The authors thank Chris Derom, Marijke Baekeland, Carine
Seynaeve, and Ann De Dobbeleer for their photographic,
technical, and administrative support. F.B. is a postdoctoral
fellow of the Fund for Scientific Research — Flanders.
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FIGURE 30.9. Transmission electron micrograph of an apoptotic
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arrow). Scale bar, 2 μm. (From Vermijlen D, Luo D, Robaye B, et
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