http://ajpgi.physiology.org/content/early/2007/12/06/ajpgi.00167.2007.full.pdf

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1 of 31 in PresS. Am J Physiol Gastrointest Liver Physiol (December 6, 2007). doi:10.1152/ajpgi.00167.2007
Articles
THE LIVER SINUSOIDAL ENDOTHELIAL CELL: A CELL TYPE OF CONTROVERSIAL
AND CONFUSING IDENTITY
Kjetil Elvevold1, Bård Smedsrød1, Inigo Martinez2
1
Department of Cell Biology and Histology; Institute of Medical Biology, Department of Orthopaedic
Surgery, Institute of Clinical Medicine, University of Tromsø, 9037 Tromsø, Norway
Running head: Confusions associated with LSEC
Contact information
Bård Smedsrød, Department of Cell Biology and Histology
Institute of Medical Biology
University of Tromsø
9037 Tromsø, Norway
Telephone: +47 77 64 46 87
Fax: +47 77 64 54 00
e-mail address: [email protected]
Copyright © 2007 by the American Physiological Society.
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Abstract
A look through the literature on liver endothelial cells (LSECs) reveals that there are several conflicts among
different authors of what this cell type is and does. Major controversies that will be highlighted in this review
include aspects of the physiological role, the characterization, and the protocols of isolation and cultivation of
these cells. Many of these conflicts may be ascribed to the fact that the cell was only recently established as a
distinct cell type, and that researchers from different disciplines tend to define their structure and function
differently. This field is in need of a common platform to obtain a sound communication and a unified
understanding of how to interpret novel research results. The aim of this review is to encourage scientists not
to ignore the fact that there are indeed different opinions in the literature on LSECs. We also hope that this
review will point out to the reader that some issues that may seem well established regarding our knowledge
about the LSECs, in reality are still unresolved and indeed controversial.
Keywords
Reticuloendothelial system, identification, endocytosis, serum
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Introduction
Review articles typically put forward unitary views and models as conclusions following a presentation of the
updated literature. The present review is different. Due to the many conflicting reports in the field of liver
sinusoidal endothelial cells (LSECs), we have in general chosen to identify the existing conflicts rather than
aiming at establishing unitary views. Some of these conflicts may not appear as obvious in the literature, and
are rarely discussed openly. It is particularly important that such occult conflicts are brought up in discussion
forums.
The study of LSECs is a rather young field, in fact, the first studies on LSECs can be traced back only about
35 years, when Eddie Wisse for the first time presented convincing evidence that the LSEC represents a
distinct cell type (85). Wisse showed that open fenestration without a diaphragm or basement membrane is a
typical feature of LSECs. He also was the first to report that LSECs contained unusually high amounts of
endocytic vesicles and suggested that they were engaged in uptake of protein from blood passing through the
sinusoids. The first physiological macromolecule shown to be cleared from the circulation by LSECs was
hyaluronan (18, 71). This marked the start of a series of experiments culminating in the notion that LSECs
are a specialized type of scavenger endothelium that uses clathrin-mediated endocytosis to clear an array of
physiological and foreign macromolecules and colloids from the blood (73). Until about 1990, most of these
studies were performed by scientists that had a special interest in the biology of these cells. Most of these
researchers were well informed about the young history of this field of research, and were open to new ways
of classifying LSECs. After about 1990 the general interest in LSECs increased, and the number of scientists
that were not raised in the tradition of cells of the liver sinusoid started to grow. With an increased number of
scientists coming in from other disciplines (cancer, immunology, virology, pharmacology, haematology and
more), showing an interest to include LSECs in their studies, there has been an increasing tendency to find in
the literature conflicting descriptions on LSECs. The understanding of what the LSEC is and does, and how
to identify and distinguish it, is not always obvious to new scientists in this field. The present review aims to
point out the major controversies associated with studies on LSECs. Knowledge about the existing conflicts
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is a prerequisite to understand the field, and a sound way to prepare for a future unitary view. Here we will
focus on the following four major issues of LSEC controversies:
I.
Ignored issues related to the role of LSECs in blood clearance
II.
Discrepancies in identification of LSECs
III.
Controversies associated with isolation and cultivation of LSECs
IV.
The confusing role of LSECs in immunity
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I. Ignored issues related to the role of LSECs in blood clearance.
The following statements, commonly found in the literature, are in conflict with each other:
a) The hepatic reticuloendothelial system (RES) clears waste from the circulation.
b) The hepatic RES = Kupffer cells (KCs).
c) The LSEC represents the cell type that is mainly responsible for the clearance of most colloids and soluble
waste macromolecules from the circulation.
How can we comprehend these incompatible statements? The answer lies in knowledge about the history of
research on RES, and certain key concepts dealing with how cells internalize matter.
Phagocytosis or pinocytosis: A problem of semantics
Although regarded by many as an old-fashioned and ill-defined term, the reticuloendothelial system (RES) is
still a frequently used expression. The RES was for a long time commonly understood as just an alternative
way of naming the mononuclear phagocyte system (MPS), or macrophage system. Although recent studies
clearly show that a major arm of the RES consists of the LSECs, authors of most text books and scientific
publications still ignore this fact, and continue to bring forward the erroneous understanding that RES and
MPS are synonymous terms. As will be shown in the following, the way we today understand terms that were
launched more than a century ago, plays a central role to explain how this conflict came to. More than a
century ago Eli Metchnikoff introduced two terms that are still in use and form important parts of the
specialized vocabulary of cell biologists: “macrophage” (“cell type that eats a lot”) and “phagocytosis”
(“engulfment of material, often associated with macrophages”) (54). Thus, it is logical, and correct to
associate MPS with the specialized process of phagocytosis. However, Metchnikoff did not define
phagocytosis the way we do today. The idea of dividing cellular uptake into phagocytosis and pinocytosis,
was not introduced until 1932 when Lewis launched the concept of pinocytosis to describe the special
process internalizing solutes and soluble material (48). Thus, Metchnikoff and his contemporaries defined
phagocytosis without reference to the physical state of the material to be taken up. This circumstance,
combined with i) the fact that the term RES was also introduced before the idea of pinocytosis appeared, and
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ii) the universal view that cells of RES generally used phagocytosis to take up material from the circulation,
led to the conflict or confusion that was so clearly expressed by Ralph van Furth, who around 1970 advised
that there is no need to keep the old-fashioned term RES, since the MPS covers more precisely the structure
and function of RES (82). The establishment of the LSEC as a cell type distinct from the Kupffer cell (KC)
(85), combined with the discovery during the 80s that LSECs represent a major scavenger cell system in
liver, led Kawai and co-workers (36) to suspect that van Furth’s advice might be misleading, and decided to
repeat the experimental protocol used a century ago to identify the cell system known today as RES. Using
the original protocol of administering the colloidal vital stain lithium carmine (one of the most frequently
used test substances employed around a century ago to study the RES), and using modern methodology to
identify the cells that accumulated the dye, Kawai et al were able to show that the cells that most efficiently
accumulated vital stain in the liver were the LSECs, but not the liver macrophages, or KCs. And just as
important: Present-day research has revealed that the two cell types differ functionally based on their uptake
preferences: LSECs do not normally perform phagocytosis, but are extremely active in uptake of soluble or
colloidal materials, whereas the KCs are geared to uptake of larger particles (see the following section).
Size matters
A study on rat LSECs concluded that the cells are not able to engulf particles greater than 0.23 µm under
normal conditions, whereas the KCs take up larger particles (69). However, when the phagocytic function of
KCs was impaired, they found that LSECs took up particles larger than 1 µm. Although colloids have too
small size to be taken up by phagocytosis in macrophages, colloidal carbon, probably the most frequently
used RES test substance, accumulates primarily in KCs upon intravenously injection. This would appear to
contradict the rule that large particles home to KCs, whereas colloids and soluble macromolecules end up in
LSECs. However, Donald reported in 1975 (13) that colloidal carbon rapidly stick to platelets and activates
them to form aggregates. Such free or aggregated platelets with large amounts of colloidal carbon attached to
them, are rapidly taken up by phagocytosis in KCs. Present-day scientists should realize that colloidal carbon
interacts very differently than other vital stains with the blood and RES. If Donald’s report on the unusal
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interaction of colloidal carbon with platelets had been considered by authors on RES, we might have avoided
the misconception that colloidal carbon behaves like most other vital stains in its interaction with RES.
It is common to find that present-day studies on LSECs do not take into consideration the special scavenger
features of these cells. A recent enzyme replacement therapy-report described the use of mannose receptor
deficient mice to study the distribution of acid lipase that was injected intravenously (14).
Immunohistochemistry performed on liver sections was, by the use of light microscope, interpreted to mean
that the enzyme was taken up foremost in KCs. This conclusion was drawn without any further attempt to
distinguish between KCs and LSECs, and is in contrast to a number of other studies where other injected
lysosomal enzymes were observed to be taken up foremost in LSECs (2, 30, 72). Knowing that acid lipase is
a soluble molecule, it is doubtful that it is mainly cleared from the circulation by phagocytosis. Further, the
use of plain light/fluorescence microscopy of liver sections following i.v. administration of stained material
does not allow a clear distinction among sinusoidal liver cells. It may be found in the literature that staining
along the sinusoids is taken to prove uptake in either LSECs or KCs, or both. To illustrate such typical
staining patterns we have included a figure showing fluorescence micrographs of liver sections following i.v.
injections of a TRITC-labelled soluble ligand and FITC-labelled particles (Fig. 1). In the example shown in
the figure we have injected soluble TRITC-labelled formaldehyde treated serum albumin (TRITC-FSA) and
FITC-labelled beads (2 µm). Some authors would claim that continuous staining along the sinusoids (Fig.
1B) represents uptake in KCs. Other authors would argue that the same staining pattern is typical of LSECs.
Knowing that FSA and 2µm particles distinguish LSECs and KCs, (32, 60), it is advisable to employ
coinjection with fluorescently labelled FSA and particles when the goal is to localize and identify the type(s)
of hepatic cells that is responsible for uptake of certain materials.
LSECs and uptake of virus
The majority of studies on interaction of virus with LSECs deal with transfection and infectivity (4, 65, 76).
Very few studies – if any - focus on the general clearance of blood borne virus particles in these cells. Studies
on uptake of phage particles and distribution of virus used in gene therapy all show that the liver has a very
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high capacity to remove these particles from the circulation (6, 65, 86, 90). In fact, it has been noted as a
problem in gene therapy that virus carrying the therapeutic gene material must be administered in huge doses
to overcome hepatic clearance, allowing virus to reach their target organs. This problem is not often
mentioned in the literature, but nevertheless represents an important obstacle in virus-based gene therapy. On
this basis it is surprising that very few studies have been carried out to study the mechanism of elimination of
virus in liver. Interestingly, a study in 1969 concluded that injected phage particles were eliminated very
efficiently by “phagocytic uptake” in the KC (28). It should be noted here that virus represents colloidal
particles, most of them <100 nm, that are too small to be taken up by phagocytosis. Yet, it is frequently seen
in the literature that ”virus is phagocytosed by macrophages”. Clearly, studies should be carried out to
specifically address the different roles of LSECs and KCs in the elimination of virus in general. By regarding
uptake of virus in LSEC as part of the innate immune system, it is conceivable that LSECs may function as a
sink for elimination and degradation of virus rather than representing the gate of infection. In fact, it is highly
likely that “silent” elimination of virus is a major task of LSECs, whereas the more dramatic scenarios
leading to infection may represent exceptions. Yet, since viral infection is easily detected and much studied,
whereas silent elimination is not, the literature gives a false picture: Silent elimination of virus in these cells
may be a far more important issue than interaction with those few types of virus that may result in infection.
For instance, many viruses are highly mannosylated (91), which makes them a likely ligand for the mannose
receptors (a typical pattern recognition receptor) present on these cells.
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II. Discrepancies in identification of LSECs
Membrane markers
During the 70s and the 80s some laboratories reported the existence of membrane molecules that specifically
stained endothelia of numerous organs, including both large vessels and capillaries (26, 33, 59, 81, 84). Since
then these markers have been frequently used by many laboratories to characterize endothelial cells in
different tissues and species. The expression of many of these marker molecules by LSECs remains
controversial (Tables 1 and 2). Experiments in vitro (Table 2) have revealed discrepancies in the expression
of CD31, von Willebrand Factor (vWF), CD106 and immunomarkers such as MHC class II, CD4, CD40,
CD80, and CD86 in different species. Expression of vWF has been generally reported on human liver
sections, whereas in cultured human LSECs the expression of this marker has been variably detected. This
shows that there are differences between in vivo and in vitro observations in addition to differences between
species. Another controversial LSEC marker is the CD31. EM studies in rat showed that CD31 is located
intracellularly shortly after establishing cultures, but later, after defenestration, CD31 is expressed on the
surface as in conventional endothelial cells (10). Thus, immunohistochemistry gives positive staining for
CD31 in LSECs in liver sections, whereas freshly isolated LSECs do not stain for CD31 unless the cells are
permeabilized prior to staining. The absence or presence of surface markers in LSECs is of major importance
in immunomagnetic isolation of the cells. This issue will be discussed in greater detail in the section dealing
with isolation and cultivation. Of note, certain treatments of animals may induce the expression of markers
that are not normally present on LSECs. For instance, injection of colloidal carbon have been observed to
induce the expression of vWF in LSECs in vivo (37). Variation in the expression of most of these molecules
by LSECs has been observed during the development of certain liver diseases including chronic
inflammatory disorders, fibrosis, viral infection, or tumour development (19, 23-25, 79, 80, 83, 87, 88).
Normal aging is associated with sinusoidal capillarization (5, 9, 31, 47), leading to changes in the expression
of marker molecules along the sinusoidal endothelium. This fact is not often taken into consideration in
studies of human liver that frequently involve samples from individuals of different age.
The presence of markers normally associated with cells of the immune system will be discussed later.
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Fenestration
A characteristic feature of LSECs is the presence of numerous transcytoplasmic canals arranged in sieve
plates called “fenestrae” (Figs. 2A and 2C). Fenestration is generally considered to be a reliable marker of
LSECs, making them clearly distinguishable from all other types of liver cells including endothelial cells
from larger vessels (3). However, preparation of cells for scanning electron microscopy might easily result in
the formation of holes in the cell surface (Fig. 2B), that sometimes are wrongly interpreted as fenestration.
Such non-fenestral transcytoplasmic holes are often observed in long time cultured LSECs. This observation,
along with the well documented (yet by many authors ignored) fact that that fenestration in LSECs of most
species studied is rapidly lost upon in vitro culturing (see also Fig. 2) may lead some authors to misjudge
mere transcytoplasmic holes for true fenestration. Additionally, a considerable variation in the number, size
and localization of fenestration is observed among different species, and also during the development of
some liver disorders or during aging (27, 47, 51, 56). Moreover, fenestration can be induced in vascular
endothelial cells upon stimulation with VEGF or hepatocyte conditioned medium (17, 20, 43, 89). In
conclusion, the LSEC-specific feature of fenestration must be used with some caution because it is a highly
dynamic marker that is sensitive to the protocol of preparation for electron microscopy.
Uptake of latex particles
Uptake of microbeads has been used as a functional marker for identification of LSECs (21, 75). During
some abnormal situations in which the hepatic scavenger system is either saturated, or KCs are selectively
depleted, LSECs may develop a limited phagocytic capacity, a phenomenon that is also observed during
special in vitro conditions. The observation that LSECs under certain abnormal conditions may be induced to
carry out limited phagocytosis is not surprising, since most cells – even the non-phagocytic hepatocyte – may
become phagocytic (38).
Under normal conditions LSECs employ only receptor-mediated endocytosis to eliminate soluble or colloidal
substances smaller than 0.23 µm (69). Under any circumstance, it would seem useless to employ
phagocytosis as a functional marker of LSECs given the fact that they are located next to the KCs, the body’s
largest population of macrophages.
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Uptake of soluble macromolecules
A unique feature of LSECs is their high capacity to eliminate colloids and soluble waste macromolecules
from the circulation. Although this function can be used as a reliable marker to identify LSECs, it is
important to be aware of the fact that every cell in the body is in principle able to perform endocytosis.
However, the unsurpassed speed and capacity of endocytosis in LSECs set these cells apart from all other cell
types. By challenging LSECs for a limited period (<20 min) (16) and with low amounts of ligand (<10
µg/ml), only this cell type will endocytose detectable amounts of the ligand. Of note, it is not uncommon to
see in the literature that LSECs claim to be functionally identified by endocytosis using ligand concentrations
of mg/ml, and incubating periods of several h. Clearly, under such conditions many cell types will
accumulate detectable levels of ligand, which will make it difficult to distinguish LSECs. It should be noted
that the characteristic high activity receptor-mediated endocytosis is rapidly lost in cultured LSECs. This
phenomenon is particularly prominent in rodents (Fig.4).
Endocytosis of acetylated low-density lipoprotein (AcLDL) has been used by several authors as a specific
functional marker for LSECs (29). AcLDL is cleared by the scavenger receptors on LSECs, but scavenger
receptors are also expressed on KCs and a number of other cell types, and endocytosis of AcLDL has
frequently been used as a functional marker of several types of extrahepatic endothelial cells. Therefore,
AcLDL is a useful, but not specific endocytic marker for LSECs. However, other ligands have been reported
to be taken up exclusively by receptor-mediated endocytosis in LSECs, such as denatured -collagen chains
(16, 70). If LSECs are to be identified by virtue of their specific endocytic ability, the ligands chosen should
be of the type that is taken up preferentially or exclusively by LSECs.
New markers related to specific LSEC functions
A new member of the scavenger receptor family that is expressed exclusively on the surface of LSECs is
stabilin-2 (22), also called the liver hyaluronan receptor. LYVE-1 is another member of the scavenger
receptor family that is constitutively expressed on LSECs and absent on other hepatic cells and conventional
endothelium. (58).
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Recent literature has identified two new lectins expressed on LSECs. L-SIGN (liver/lymph node-specific
ICAM-3-grabbing non-integrin) (1, 44, 74) is strongly and constitutively expressed on LSECs and lymph
node endothelium, but not on other hepatic cells or other endothelia. LSECtin (liver and lymph node
sinusoidal endothelial cell C-type lectin), has been shown to have the same cellular distribution and similar
binding capacities as L-SIGN(12, 49). These new marker molecules claimed to be exclusively expressed on
LSECs were described only very recently, and since the literature on these markers is still rather limited, the
use of these new marker molecules to identify LSECs should be carried out with some caution.
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III. Controversies associated with isolation and cultivation of LSECs
Isolation
Detailed studies on LSECs require reliable methods for cell isolation and cultivation. The first paper
describing immunoisolation of LSECs was published in 2002 (77). By using the LSEC-specific antibody SE1, 98% pure rat LSECs was reported. Unfortunately this monoclonal antibody has so far not been
commercially available. In a series of experiments where LSECs were isolated by counterflow elutriation
Knolle and coworkers reported that LSECs function as antigen presenting cells (APC) (41, 42). But by using
immunomagnetic beads to remove CD45+ KCs and CD11b+ dendritic cells (DC) in order to obtain highly
purified LSECs, Katz et al (35) found no evidence of antigen presenting function of the highly purified
LSECs, and speculated that the contradictory results might be explained by the possibility that Knolle and coworkers used LSEC-preparations that were contaminated by other cell types. Recently, the isolation methods
used by both Katz and coworkers and Knolle and coworkers were questioned by Onoe et al (62), who
employed the membrane marker CD105+ for positive selection of LSECs by using MACS. I.e. Onoe et al
used positive selection to obtain their cells whereas Katz and coworkers obtained their LSECs by negative
selection. However, CD105 is also expressed on liver stellate cells and myofibroblasts (55) and it has been
advised to exercise caution when using this marker to identify or purify LSECs (46). Another endothelial cell
marker, CD31, has been used for immunomagnetic isolation of human LSECs (45). But in rat, the selection
of CD31 positive cells yielded a population of LSECs with fewer fenestrae than LSECs isolated by
elutriation (11). The latter study concluded that “cells isolated by anti-CD31 and immunomagnetic sorting
lacked the hallmark features of LSEC”. Although all isolations methods mentioned above may yield high
relative proportions of LSECs, it is likely that the degree of purity of LSECs, and the main contaminating cell
types vary from one method to the other.
Cultivation
Cultivation procedures may represent the chief source of conflict in the studies on LSECs today. Some
laboratories culture LSECs under conditions that allow cell proliferation and subsequent splitting and
passaging, before the cells are used in experiments several days or even weeks after the isolation (34, 64).
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This frequently used practice is associated with conflicts that can be sorted in two ”schools”: i) the one
claiming that LSEC features can only be studied in short term (true primary) culture, and ii) the one that
frequently makes use of long term cultures (including use of cell lines). Although these schools rarely – if
ever – discuss openly their disagreements, it appears important to point out this conflict. To illustrate this, we
show some of the contradictory statements that are often seen in the literature related to time of cultivation of
LSECs:
a) LSEC cultures can be propagated by trypsin splitting and subcultivation (21, 34, 64).
b) Studies on LSECs can be performed using cell lines originating from native LSECs (53, 64).
c) Important features of LSECs, such as scavenger function and fenestration, are severely decreased or
disappear completely in LSECs that are cultivated for more than 1-2 days (15, 43, 61).
d) Serum (5-20%), which is normally supplemented in long term cultures of cells (and often also in short
term primary cultures), has been noted to act toxic to dividing LSECs (43, 61).
e) LSECs proliferate only very slowly or not at all in culture (15, 43).
A fact that is often underscored is that many phenotypic features of LSECs change gradually when the cells
are placed in a culture dish, and most importantly, many of the signature functions of LSECs are also rapidly
lost during in vitro culture (15, 43) (see also Figs. 2 and 4). The drastic change in environment associated
with the transfer of the LSECs from the intact liver to culture conditions is clearly responsible for the many
of these alterations. The use of light microscopy to determine the quality of a LSEC culture is at best semioptimal. In Fig. 3B, the 3 days old cells appear very similar to the fresh cells in Fig. 3A. However,
ultrastructural studies by SEM reveal a clear loss of fenestrae already after 1 day (Fig. 2B). In addition, the
very high endocytic activity is lost after some days in culture (Fig. 4). Given that serum is lethal to dividing
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LSECs (43) the cultures propagated in the presence of serum will gradually change from being composed of
mainly LSECs to a stromal like culture (Fig. 3C). This puts forward the following questions: Should serum
be used to cultivate LSECs? How soon after seeding of freshly isolated LSECs should experiments be carried
out on these cells? Do the results obtained with long term cultivated LSECs reflect the function of these cells
in the intact liver? How is the term ”primary culture of LSECs” defined? Is there a possibility that cells taken
to represent proliferating LSECs are instead contaminating non-sinusoidal or non-endothelial cells? If some
authors claim that LSECs do not proliferate in vitro, then how come other authors use terms as
”subcultivation of LSECs” and ”LSECs grown to confluence”? Do LSECs from different species differ in
their proliferative potential? And how come some authors use LSEC cell lines when other authors claim that
cell lines that originate from primary LSEC cultures have lost their original scavenger activity and
fenestration? And finally, how many specialized differentiated LSEC characteristics can be lost before the
cells loose their status as LSECs?
These conflicts exist due the lack of unified criteria to characterize LSEC. It is therefore advisable that
workers in this field openly discuss these issues.
Page 16 of 31
IV. The confusing role of LSECs in immunity.
The liver is enriched in macrophages (KCs), NK cells, and NKT cells which have traditionally been regarded
as key cellular components of the innate immune system. However, the LSEC also promotes active antigen
uptake via its Fc gamma receptor, and pattern recognition receptors such as the mannose receptor and
scavenger receptor. For this reason alone these cells must be considered as an important part of the innate
immune system. In recent years the LSECs have also been implicated as inducers of tolerance in the liver. In
a series of experiments, Knolle and co-workers reported that LSECs express molecules that promote antigen
presentation, including MHC class I and II and the co-stimulatory molecules CD40, CD80 and CD86 in
addition to their endocytic scavenger receptors (reviewed in (39)). The LSECs were reported to perform
receptor-mediated endocytosis and/or phagocytosis, antigen processing and presentation with similar efficacy
as those of DC. But in contrast to conventional APC populations, LSECs failed to induce differentiation of
naive CD4+ T cells toward a Th1 phenotype, but promoted development of Th0 cells. The failure of LSECs to
induce differentiation toward Th1 together with the production of negative immunomodulatory cytokines by
LSEC-primed T cells upon restimulation, was thought to contribute to the unique hepatic immune tolerance
(7). In this model, the LSEC represented the sessile, organ-resident APC inducing local tolerance in the liver
towards soluble antigens such as food antigens or self-proteins. The liver DCs were thought to take up
antigen in the liver as well, and then migrate to local lymph nodes mediating immune tolerance in the
lymphoid compartment. This view of LSECs as functional APC was seriously challenged by Katz et.al. that
employed new efficient separation techniques to obtain highly purified LSEC cultures (35). They found that
unlike DCs, LSECs had low or absent expression of MHC class II, CD86, and CD11c. However, they found
that LSECs demonstrated a high capacity for antigen uptake in vitro and in vivo, and although uptake of
AcLDL has been reported to be a specific function of the LSEC, they found that DCs captured AcLDL to a
similar extent in vivo. Consistent with their phenotype, LSECs were poor stimulators of allogeneic T cells.
Furthermore, in the absence of exogenous co-stimulation, Katz et al found that LSECs induced negligible
proliferation of CD4+ or CD8+ TCR-transgenic T cells. They concluded that LSECs alone are insufficient to
activate naive T cells. A year later, a study by Onoe et al. contradictory to the results of Katz et al, but in
agreement with Knolle and co-workers, concluded that primary LSECs from mice do indeed express MHC
Page 17 of 31
class II and CD86, but not CD11c (62). The contradictory findings were believed to occur due to the
separation technique used by Katz et al that according to Onoe was unsatisfactory (as mentioned in section
III above). However, selection of CD105+ as used by Onoe, may not result in pure LSECs as indicated by
others (46) because this marker is also expressed on liver stellate cells (55). It is also very important to note
that the length of cultivation and presence of serum were different in the studies reported by Knolle, Katz and
Onoe groups. These parameters have been shown previously to seriously affect the outcome of LSEC studies.
Recently it was reported that LSECs endocytose splenocytes injected via the portal vein. The LSECs that
endocytosed the splenocytes showed enhanced expression of MHC class II molecules and CD80 (78). Of
note, the notion that LSECs are capable of endocytosing/phagocytosing splenocytes is in contrast to the
general concept that the cells under normal conditions are not able to take up material exceeding approx 0.23
µm (69). The finding that neutrophils are removed by KCs and not by LSECs is in agreement with this notion
(68). On this basis it is difficult to explain LSEC phagocytosis of splenocytes.
The conflicts associated with the expression of MHC class II in LSECs is further underlined by a number of
earlier studies that failed to demonstrate this molecule on rat or human LSECs (36, 50, 66) (see also Table 1
and 2). Obviously, further studies are needed to elucidate the role of LSECs in the immune system.
Page 18 of 31
Conclusions
The study of LSECs is still a young discipline. Different laboratories have different opinions about the
identity of LSECs, and therefore design methods and interpret results differently. Some of the conflicting
views can be readily identified and discussed in a scientific way. But other conflicting issues are based on the
fact that some authors do not even appreciate those issues of conflict that other authors identify as highly
conflicting.
Role in blood clearance. A large body of evidence strongly suggests that the two major scavenger cell
types of liver are KCs, which take up particulate matter, and LSECs, that clear soluble macromolecules and
colloids of size <0.23 µm. However, most authors of textbooks and scientific publications still ignore this,
and maintain the old notion that ”hepatic RES = KCs”. A meaningful further development of this field is
difficult unless the correct notion (the hepatic RES = LSECs + KCs) is generally accepted.
Identification. Some markers used to identify LSECs are also expressed on KCs, stellate cells or
endothelial cells from different vascular beds, and are therefore inadequate as LSEC-specific markers. In
addition, phenotypic and functional variations are observed in LSECs from diseased livers, as well as in
healthy livers of different age groups. Characterization of LSECs based on their unique scavenger function
seems to be a reliable approach; however, caution should be taken for choosing the adequate experimental
settings.
Isolation and cultivation. Some authors use long term cultures and cell lines, and subcultivate LSECs,
whereas other authors claim that LSECs loose their unique native characteristics upon cultivation for more
than 1-2 days and differ too much from native LSECs. Some authors cultivate LSECs in medium with serum,
whereas others claim that serum is toxic to the cells. Clearly, the results of in vitro experiments depend to a
large extent on the isolation and cultivation procedures used by the different authors.
Role in immunity. Some authors claim that LSECs carry MHC class II molecules and present antigens to
generate hepatic immune tolerance. Other authors have not found evidence that LSECs express MHC class II
Page 19 of 31
nor present antigens. These conflicts are largely based on different views on isolation and cultivation
procedures.
The present review was written to remind scientists dealing with LSECs about the important controversies
that still exist regarding these cells. Both old scholars and new scientists entering the field should consider
that some issues that may seem well established regarding our knowledge about the LSECs, are in reality still
unresolved and controversial.
Page 20 of 31
Acknowledgements
The excellent assistance by Cristina Øie is greatly appreciated. This work was supported in part by the
Norwegian Research Council, The Norwegian Cancer Association, and the Medical Faculty, University of
Tromsø
Page 21 of 31
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Figure legends
Figure 1:
Fluorescence micrographs of a rat liver section following double i.v. administration of 0.5 mg TRITClabelled formaldehyde-treated serum albumin (TRITC-FSA), a substance known to be taken up by LSECs
(32) (A), and 5x108 FITC-labelled beads (2 µm diameter), known to be taken up exclusively in KCs (60) (B).
Both probes can be observed to have accumulated along the sinusoids. By superimposing A and B it can be
seen that the beads have accumulated in sites that do not coincide with the presence of TRITC-FSA (C).
Figure 2:
Scanning electron micrographs of fenestration on LSECs. A and B are cultured on fibronectin-coated glass
slides, whereas C show the liver sinusoids with numerous fenestrae. Freshly isolated cells (A) show a welldefined and organized appearance of fenestrae in sieve plates (circle), whereas after 1 day in culture (B) the
cells are largely defenestrated and only sporadic fenestrae in sieve plates are observed (circle).
Transcytoplasmic holes are observed (arrows), but these structures are most likely artefacts resulting from the
preparation of the specimens, and should be cautiously interpreted. N=nucleus, bars are 2 µm.
Figure 3:
Effect of serum on primary rat LSECs cultured on fibronectin-coated dishes. LSECs were cultivated for 12 h
(A), and then 3 days in a special serum-free medium (B) (16), or 3 days in RPMI 1640 medium containing
10% fetal calf serum (C). The morphology of the cells in the serum-free medium (B) is very similar to the
cells at early phases of culture (A). However, incubation in the presence of serum for longer periods (C) had
dramatic negative effects on the viability of LSECs, also favouring the growth of contaminating fibroblast
like cells (arrows), and/or other types of endothelial cells (arrowheads).
Figure 4:
Progressive loss of endocytic capacity by LSECs in culture. Freshly isolated LSECs show high capacity for
internalization and degradation of radioactively labelled formaldehyde-treated serum albumin, a ligand for
the scavenger receptor. This specific LSEC function is gradually lost with time in culture and disappears
faster in LSEC cultures from small vertebrates like rodents (A), than larger vertebrates like pig (B).
Page 27 of 31
Table 1. Phenotypic markers used to identify liver sinusoidal endothelial cells on liver slices and on cultured
cells
Liver slices
Cultured cells
+
-/+
+
-
+++
-
+++
+++
+++
+
+
+
-
++
+++
++
++
+++
+++
++
Intensity of immunochemical staining: 0, absent; +, low; ++, medium; +++, high.
†Intensity of immunochemical staining only + or/and –
§ Flow cytometry
§§ RT-PCR
* performed both on liver slices and on cultured cells
-/+
-/+
+
-
-
+
+
† §§ Immortalized human
serum not reported (52)
-
† Rat, at day 1, no serum
(50)
+
† §Mouse, 5 days, serum
not reported (62)
†§Mouse, 3 days, 10%
serum (35)
-
Human, passage 3-7, 20%
human serum (8)
Human, passage 4-5, 10
% serum (34)
†Rat, 0-15 days, 0%, 1%
and 10% serum (29)
+
†§Mouse, 3 days, 10%
serum (40)
+++
†Rat (57)
+++
†Human (81)
+
+
+
+
+
Human (63)
Human (67)
CD4
CD14
CD16
CD31
CD32
CD34
CD54
CD62E
CD62P
CD106
AcLDL
vWF
Fact VIII
MHC II
CD11b
CD11c
CD40
CD80
CD86
CD105
HLA-I
L-SIGN
Human (66)
*
+
+
+
+
+
+
+
-
+
+
-
+
+
+
+
+
-
Page 28 of 31
Figure 1
80x182mm (300 x 300 DPI)
Page 29 of 31
Fig 2
143x42mm (300 x 300 DPI)
Page 30 of 31
Fig 3
55x127mm (300 x 300 DPI)
Page 31 of 31
Fig 4
53x28mm (600 x 600 DPI)