Open Journal of Veterinary Medicine, 2012, 2, 129-136
http://dx.doi.org/10.4236/ojvm.2012.23022 Published Online September 2012 (http://www.SciRP.org/journal/ojvm)
Immunohistochemical Aspects of Ito and Kupffer Cells in
the Liver of Domesticated and Wild Ruminants
Valentina Carollo, Alessia Di Giancamillo, Francesca Vitari, Rainer Schneider,
Cinzia Domeneghini*
Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, Milan, Italy
Veterinary Practitioner, “Le Cornelle” Parco Faunistico, Bergamo, Italy
Received July 4, 2012; revised July 31, 2012; accepted August 7, 2012
ABSTRACT
The mammalian liver is a morphologically and functionally complex organ, made up of not only of the largely pre-
dominant parenchymal cells (hepatocytes) but also non-parenchymal cells. Although there are less non-parenchymal
cells than hepatocytes, they nevertheless play an important role in regulating many hepatocyte functions, as well as in
the immunology of the liver. We investigated the structural aspects of the liver and the morpho-functional characteris-
tics of Ito and Kupffer cells in two domesticated ruminant species (cattle and goat) in comparison with four wild rumi-
nant species living in captivity in a zoo in northern Italy. The liver specimens were studied using histological, histo-
chemical and immunohistochemical methods. The liver parenchyma was structurally normal. Immunohistochemistry
was performed for desmin, glial fibrillary acidic protein (GFAP), vimentin, α-smooth muscle actin (α-SMA), collagen I,
lysozyme, CD 68 and tumor necrosis factor α (TNF-α). In all the studied ruminants, Ito cells reacted with desmin and
vimentin antibodies, Kupffer cells were evidenced only with lysozyme-immunopositivity, and both displayed a charac-
teristic distribution in the hepatic lobular/acinar structure. The results obtained, not only contribute to the knowledge of
ruminant wild species, but also help to define a normal structure reference for the diagnosis and treatment of liver dis-
eases.
Keywords: Liver Non-Parenchymal Cells; Captive Wild Ruminants; Histology; Histochemistry
1. Introduction
The mammalian liver is a morphologically and func-
tionally complex organ, made up not only of the largely
predominant parenchymal cells (hepatocytes), but also
non-parenchymal cells (N-PCs), including Ito, Kupffer
and sinusoidal endothelial cells (SECs), as well as other
cell types that reside in the sinusoidal compartment [1-3].
Although present in a small percentage in the total liver
volume (around 6%), non-parenchymal cells play an im-
portant role in the regulation of many hepatocyte func-
tions [1,4] as well as in the immunobiology of the liver
[5], in both normal and pathological conditions [6]. Hepatic
stellate cells (HSCs) also called Ito cells, fat-storing cells,
lipocytes [7], are vitamin A-storing cells located in the
space of Disse between hepatocytes and sinusoidal
endothelial cells, which is why they are also called peri-
sinusoidal cells. These cells constitute approximately 5%
of the total number of liver cells [2], their cytoplasm is
especially rich in lipid droplets (long-chain fatty acid
esters of retinal, retinyl palmitate), and show long,
branched cytoplasmic processes that embrace the endo-
thelial cells [1,8]. In addition they are able to modulate
the turnover of parenchymal cells and regulate liver
regeneration. Owing to their smooth muscle α-actin when
contracting, these NPCs may reduce the lumen of si-
nusoid capillaries, in such a way modulating the liver
sinusoidal blood flow. When the liver is damaged, the
hepatic stellate cells change their shape and transform
(via a process named “activation”) into the myofibro-
blast-like cells, which are the major cell type responsible
for the onset of liver inflammatory fibrosis and even-
tually cirrhosis [9]. Myofibroblast-like cells are highly
proliferating and secrete a large quantity of extracellular
matrix proteins (collagens type I and III, proteoglycan,
adhesive glycoproteins), as well as extra-cellular matrix
degrading metalloproteinases, cytokines and chemokines,
but lose their function with regard to the vitamin A meta-
bolism [10]. They promote hepatic fibrogenesis, possibly
together with portal fibroblasts and parenchymal cells,
and parenchymal cells in parallel begin to be transformed
*Corresponding author.
C
opyright © 2012 SciRes. OJVM

V. CAROLLO ET AL.
130
into mesenchymal cells (epithelial to mesenchymal tran-
sition) [11]. Kupffer cells are the liver resident macro-
phages. They are intrasinusoidal and display huge endo-
cytic and non specific phagocytic activities, since they
are a part of the reticulo-endothelial system. The liver
contains one of the largest resident populations of
macrophages [12], which are key components of the
innate immune system [13] and derive from circulating
monocytes [2]. Kupffer cells represent approximately
30% of NPC fractions and approximately 15% of all liver
cells [2]. The distribution of Kupffer cells within the
hepatic lobules/acini is variable and perhaps species-
specific: in the rat, the periportal area contains 43% of
the cells, the midzonal region approximately 28% and the
remaining 29% of Kupffer cells are located in the centre
of the hepatic lobules/acini [14]. They remove senescent
and damaged erythrocytes from circulation, which may
lead to an excess of cellular iron deposits in some storage
diseases, as the effect of either seasonal variations or
metabolic dysregulation phenomena [15,16]. Kupffer
cells phagocyte the great majority of bacterial products
coming from the gut, and consequently are responsible
for the onset of the acute phase response and produce a
large variety of inflammatory mediators (IL-1, TNF-α,
TGF-β), which in turn may induce liver injury. As a
response to inflammatory inputs, Kupffer cells (and in
some species, also Ito cells) release prostaglandins from
arachidonic acid via cyclooxigenases (COX)-1, -2. Pros-
taglandins affect the hepatic glucose and lipid meta-
bolisms [17], and elicit oxidative stress molecules that
are read by hepatocytes as apoptogenic stimuli. They
have a limited local proliferating ability and, together
with SECs, express scavenger, mannose, and membrane
receptors for the Fc region of IgG and for the com-
plement [2,6]. In summary, both Ito and Kupffer cells
share a fundamental role in the occurrence of some
pathological liver conditions, however, paradoxically, the
histochemical and immunohistochemical aspects of both
these cell types are better known in terms of their
relationship to pathological rather than normal conditions.
Thus, also bearing in mind the complex heterogeneity
(sometimes species-specific) of these non parenchymal
cells, our aim was to investigate the structural aspects of
the liver, and to detail the morpho-functional charac-
teristics of Ito and Kupffer cells in two domesticated
ruminant species (cattle, goat: browsers) in comparison
with wild ruminant species (grazers and foliage selectors)
living in captivity at a zoo in northern Italy. In addition
the aim was to identify the fundamentals of the normal
liver structure in mammals that to date have not been
fully investigated, in order to improve the present
structural framework, to which one can refer for des-
cribing possible hepatic diseases. This might then lead to
a better quality of care and management of zoo animals,
where captivity is fundamental for safeguarding endan-
gered species, but which can also involve stressful envi-
ronmental conditions.
2. Materials and Methods
2.1. Animals and Tissues Processing
Approximately 1 cm3 of liver samples (similar lobes)
from different ruminant species (two adult individuals for
each species) were collected, promptly after death: Hol-
stein Freisian cattle (Bos taurus) and Saanen goat (Capra
hircus) livers were obtained at slaughter; giraffe (Giraffa
camelopardalis), reindeer (Rangifer tarandus), scimitar
oryx (Oryx dammah) and Mrs Gray’s lechwe (Kobus
megaceros) livers were obtained during necropsies, which
were performed on the animals in 2010-2011 years at
“Le Cornelle” park in the north of Italy. The captive wild
ruminants live in mono-specific large enclosures planned
to recreate conditions similar to wildlife. The diet of
these captive wild ruminants is the same, and is com-
posed by barley, bran, pellet and some seasonal fruit and
vegetables. Only giraffe meal is enriched with maize and
carob and with acacia apical branches sup- plied with
high mangers. These captive wild ruminants had died for
reasons unrelated to gastrointestinal diseases: these var-
ied from traumatic lesions from conspecific to post-par-
tum complications as well as to heart dysfunction. The
gross anatomy of the livers was in all cases judged to be
normal. The hepatic liver samples were fixed by im-
mersion in 10% neutral-buffered formalin, routinely em-
bedded in paraffin, and then sectioned 4 μm thick. The
paraffin sections, after dewaxing and rehydration, were
treated with histological, histochemical and immunohis-
tochemical stains, as described below.
2.2. Histological and Histochemical Analyses
Dewaxed sections were stained with Haematoxylin and
Eosin (HE) sequential stain, Masson’s trichromic stain,
and Gordon and Sweet’s modified procedure for reticu-
lum [18], the latter for revealing the liver architecture.
2.3. Immunohistochemical Analyses
After dewaxing and endogenous peroxidase blocking
with H2O2, 10% for 10 minutes, slides were pre-treated
with either a microwave treatment (twice for 5 minutes at
450 W in a citrate buffer pH 6, with a 20 minute interval
between the two treatments; Table 1) or proteinase K
(0.2% proteinase K in PBS pH 7.4 at room temperature
for 5 minutes; Table 1) to induce antigen retrieval. Sec-
tions were then incubated overnight at room temperature
in a humid chamber with the primary antibodies (see
Table 1). Sections were subsequently incubated with
EnVision™ Detection Systems, Rabbit or Mouse (Dako-

V. CAROLLO ET AL. 131
cytomation, Italy) and the reaction products were visua-
lized with a freshly prepared solution of 3.3-diamino-
benzidine tetrahydrochloride (DAB, Sigma, Italy), 10 mg
in 15 ml of a 0.5 M Tris buffer at pH 7.6, containing 1.5
ml of 0.03% H2O2. To ascertain structural details, sec-
tions were slightly counterstained with Mayer’s haema-
toxylin. Porcine liver sections were used as a positive
control. For the negative controls, other sections were
processed simultaneously with the procedure described
above, except that the primary antibodies were substi-
tuted with 1 (PBS, 2) preimmune sera. Both these proce-
dures gave negative results.
3. Results
3.1. Histological and Histochemical Analyses
The histological and histochemical observations con-
firmed the observations following gross anatomy exami-
nation: The liver parenchyma was structurally normal, the
central vein and the portal spaces were always evident, and
the connective component that accompanies the lobular
structure was rather scarce (Figures 1(a) and (b)).
Masson’s trichromic stain showed that in the cattle,
goat and reindeer livers, the connective tissue was pre-
sent small quantities in the capsule, portal areas, and de-
lineating the lobular septae (Figure 2(a)). In the giraffe,
scimitar oryx and Mrs Gray’s lechwe livers, the portal
areas were more clearly characterized by the presence of
connective tissue (Figure 2(b)).
The histochemical stain aimed at highlighting the re-
ticular fibres that support the hepatic parenchyma also
showed the normal architecture of the lobular structure
(Figures 3 (a) and (b)).
3.2. Immunohistochemical Analyses
Immunohistochemistry was used to demonstrate the pre-
sence of Ito and Kupffer cells (see Table 2).
Table 1. Primary antisera used, aimed at identifying Ito and
Kupffer cells (overnight at room temperature for all of
them; PK = Proteinase K).
Code Source Dilution
Antigen
retrieval
CD 68 H7122 Dakocytomation 1:50 Heat
Collagen I 7066 Chondrex 1:400 Heat
Desmin H7094 Dakocytomation 1:50 Heat
GFAP 20334 Dakocytomation 1:500 PK
Lysozyme A0099 Dakocytomation 1:400 PK
α-SMA H7114 Dakocytomation 1:50 Heat
TNF-α Ab6671 Abcam 1:100 Heat
Vimentin 674M Biogenex 1:10 Heat
(a) (b)
Figure 1. Ruminant livers. HE sequential stain. (a) Giraffe:
the liver structure is normal. Two central veins (asterisks)
are visible. Scale bar 200 µm; (b) Reindeer: the hepatic
parenchyma is normal. A portal area is present (arrow).
Scale bar 200 µm.
(a) (b)
Figure 2. Ruminant livers. Masson’s trichromic stain. (a)
Reindeer: the connective tissue component is very scarce
(thin arrows). Scale bar 200 µm; (b) Scimitar oryx: the
connective tissue is relatively abundant in a portal area
(arrow). Scale bar 200 µm.
(a) (b)
Figure 3. Ruminant livers. Gordon and Sweets’ modified
method for reticulum. (a) Cattle; (b) Scimitar oryx: the
normal architecture of the liver is demonstrated by the re-
ticular fibers running. Scale bar 200 µm.
None cell type was revealed by applying anti-α-SMA
and anti-Collagen I antibodies (data not shown), whereas
anti-GFAP immunohistochemistry showed small, roun-
dish to irregular perisinusoidal cells in the cattle and re-
indeer livers (Figure 4).
In addition, a number of similar, variously sized, peri-
sinusoidal cells were found to be immunopositive to both
anti-desmin (Figure 5) and anti-vimentin (Figure 6) an-
tibodies in all the animals. In the bovine, goat and oryx
livers these desmin (Figure 5(a)) and vimentin (Figure
6(a)) immunopositive cells were prevalently roundish,
and contained cytoplasmic vacuoles that appeared to be
devoid of contents after the applied routine procedure for

V. CAROLLO ET AL.
132
paraffin embedding, and were thus interpreted as rich in
lipid content. Giraffe, reindeer and Mrs Gray’s lechwe
presented another immunohistochemical feature: Desmin
(Figure 5(b)) and vimentin (Figure 6(b)) immunoposi-
tive cells predominantly exhibited a stellate shape with
extensive long cytoplasmic processes running along or
encircling the sinusoids. In the latter animals, roundish
desmin and vimentin-immunopositive cells were also pre-
sent, however in fewer numbers than the immunopositive
stellate cells.
The distribution of this type of perisinusoidal cells was
different in the animals studied: in the cattle, giraffe
(Figure 7(a)) and scimitar oryx, desmin and vimentin-
immunopositive cells were more numerous in the peri-
central than the periportal areas of the hepatic lobules. In
contrast in the goat (Figure 7(b)), reindeer and Mrs
Gray’s lechwe, these cells were more numerous in the
periportal areas.
One other non-parenchymal cell type was immunohis-
tochemically found in all the studied ruminants, with a
characteristic localization (in the sinusoid wall), and
Table 2. Ito and Kupffer cells immunoreactivities in the six
ruminant species.
Ca Go Gi Re SO MGL
α-SMA - - - - - -
Collagen I - - - - - -
GFAP + - - + - -
Desmin + + + + + +
Ito cells
Vimentin + + + + + +
Lysozyme + + + + + +/-
TNF-α - - - + - -
Kupffer
cells
CD 68 - - - - - -
Ca = cattle; Go = goat; Gi = giraffe; Re = reindeer; SO = scimitar oryx;
MGL = MRs Gray’s lechwe
Figure 4. Ruminant liver. GFAP-immunohistochemistry.
Reindeer: small, roundish (arrow) to irregular (arrowhead)
immunopositive cells are visible in perisinusoidal localiza-
tions. Scale bar 100 µm.
Figure 5. Ruminant livers. Desmin-immunohistochemistry.
(a) Goat: several small roundish (arrows) immunopositive
cells are visible in perisinusoidal areas. The cytoplasm
clearly shows a lipid content. Scale bar 100 µm; (b) Giraffe:
numerous irregularly shaped immunopositive cells are pre-
sent in perisinusoidal localizations (arrowheads). Scale bar
100 µm.
Figure 6. Ruminant livers. Vimentin immunohistochemis-
try. (a) Goat: small roundish (thin arrow) immunopositive
cells are visible in perisinusoidal areas. The cytoplasm
clearly shows a lipid content. Scale bar 100 µm; (b) Giraffe:
numerous irregularly shaped immunopositive cells are pre-
sent in perisinusoidal localizations (arrowhead). A smaller
number of roundish immunopositive cells are also present
(asterisk). Scale bar 100 µm.
Figure 7. Ruminant livers. Desmin immunohistochemistry.
(a) Giraffe: Immunopositivity is mainly visible in a pericen-
tral area (asterisk). Scale bar 200 µm; (b) Goat: the im-
munopositivity is mainly visible in a periportal area (aster-
isk). Scale bar 200 µm.
shape (irregular or spindled): this cell type was ly-
sozyme-immunopositive (Figure 8), although the inten-
sity of the immunoreactions was not the same for all the
animals studied, and was particularly scarce in Mrs Gray’s
lechwe. These non-parenchymal cells were more nume-
rous in the periportal (Figure 8) than the pericentral areas,
and this distribution was uniformly observed in the liver
of all the animals studied.
This cell type was also TNF-α-immunopositive, but
limited to reindeer liver, and was not immunopositive to
the CD 68 anti-body (data not shown).

V. CAROLLO ET AL. 133
Figure 8. Ruminant liver. Lysozyme-immunohistochemis-
try. Cattle: numerous immunopositive, irregularly shaped
cells are visibl e in the sinusoid walls (arrow s). Scale bar 100
µm.
4. Discussion
In this study we examined the liver of six different rumi-
nants belonging to the Artiodactyla order. Judging from
the histology and histochemistry, the architecture of the
liver of these ruminants was normal. Its structure corre-
sponded to what is known for ruminant species, espe-
cially concerning the generally limited quantity of con-
nective tissue in the normal hepatic lobular/acinar struc-
ture. Considering the mutual relationship of the non-
parenchymal cells in developing hepatic diseases, special
attention was paid to the immunohistochemical features
of the Ito and Kupffer cells that were revealed by panels
of immunohistochemical markers. In all the ruminant
species analyzed in this study, the variously sized and
shaped Ito cells, with their characteristic content of cyto-
plasmic fat droplets, showed a positive reactivity to
anti-desmin and anti-vimentin antibodies, as also ob-
served by Neubauer et al. [19] and Uetsuka et al. [20]. In
the case of the cattle and reindeer, Ito cells were also
immunopositive for GFAP, in accordance with Neubauer
et al. [19], who identified this marker in the rat liver and
in vitro experiments. In humans hepatic stellate cells are
also detectable using GFAP-immunopositivity, however
the intensity of the reaction increases when fibrosis de-
velops [21]. Cattle, goat and scimitar oryx livers showed
rounded lipid vacuoles in the cytoplasm of Ito cells,
which were more abundant in the pericentral area. Gi-
raffe, reindeer and Mrs Gray’s lechwe liver Ito cells ex-
hibited a stellate shape with extensive long cytoplasmic
processes running along or encircling sinusoids, and
were more evident in the periportal area. No positive
immunostaining for α-SMA was observed in the Ito cells,
which is in line with Uetsuka’s study on bovine liver [20].
The absence of immunopositivity for both α-SMA and
Collagen I is likely due to the absence of Ito cell active-
tion, that is, Ito cells were not transforming into myofi-
broblast-like cells. Accordingly, the liver in all the rumi-
nants of this study appeared to be fully normal and did
not display fibrosis. Liver fibrosis has been described in
ruminant species, above all in cattle [22-24], in which, as
in other mammals, this disease represents the liver’s re-
sponse, via the activation of stellate cells, to inflame-
matory, toxic, infectious or metabolic stimuli [25,26].
Kupffer cells are fundamental in sustaining the immuno-
biology of the liver both in normal and pathological con-
ditions. They are also able to modulate systemic immune
tolerance, via their capacity to suppress T cell activation
[6,27]. Together with Ito cells, the liver resident macro-
phages contribute to an assessment of liver fibrosis [28].
Ruminant Kupffer cells showed a clear lysozyme-im-
munoreactivity; only in Mrs Gray’s lechwe’s did they
present a scarce immunopositivity to lysozyme. In the
reindeer, Kupffer cells were also immunopositive for
TNF-α. Kushibishi [29] demonstrated in cattle that TNF-
α from activated Kupffer cells regulates inflammatory
responses in mastitis, and affects metabolic disorders,
such as acidosis. None of the ruminants livers showed an
immunoreactivity to CD 68, in contrast with other studies
on different mammals [28,30]. This apparent immuno-
histochemical heterogeneity was expected, because it is
well known that macrophages are differently detected in
their various tissue localizations and species [31,32].
Ruminant Kupffer cells were located in the sinusoid
walls, showed an irregular shape, and were more nume-
rous in the periportal than the pericentral areas, in accor-
dance with studies in other mammals [33]. When the
liver is injured, Kupffer cells activate and contribute to
the immune cell responses [34-36]. Kupffer cells have
been shown to contain PrP (Sc) (a marker of prion dis-
ease) in experimentally infected sheep [35], and, when
activated, may release a lot of inflammatory mediators in
cattle [37]. Ruminant Kupffer cells are evidently da-
maged in intoxication phenomena, which sometimes give
rise to lysosomal storage diseases [38,39], and poisoning
[40,41]. To the best of our knowledge, this is the first
description of the morphofunctional aspects and dis-
tribution of non-parenchymal cell types in wild rumi-
nants. Since Sleyster and Knook’s study on the rat [42],
it has been well known that functional gradients exist in
the lobular distributions of liver nonparenchymal cells, in
particular of Kupffer cells, and these functional distrib-
uting differences may potentially help in elucidating
pathogenic mechanisms [2]. In conclusion, we have shown
the immunohistochemical features and morphological
distribution of Kupffer and Ito cells in the liver of six
different ruminant species. Four of these species were
originally wild ruminants kept in a zoo in northern Italy.
The examined ruminants belonged to three different
feeding habits (sometimes overlapping), according to the

V. CAROLLO ET AL.
134
classification by Hofmann and Stewart [43], updated by
Hackmann and Spain [44]: reindeer, scimitar oryx and
Mrs Gray’s lechwe are grass and roughage eaters (graz-
ers), the giraffe is a concentrate herbage and foliage se-
lector, and cattle and goat are intermediate feeders
(browsers). This classification, which above all concerns
the morphology and morphometry of the gastrointestinal
tract, does not seem to affect the liver parenchyma,
whose normal structure is fundamentally similar in the
six examined species. The non-parenchymal cells were
well evidenced by immunohistochemistry, although with
some differences possibly linked to either genetics or
feeding habits/plans, which need further research. Ito
cells were revealed with both desmin and vimentin im-
munohistochemistry, Kupffer cells were immunoposi-
tive to lysozyme, and both displayed a characteristic dis-
tribution in the hepatic lobular/acinar structure. We be-
lieve that our data contribute to the knowledge of wild
species, and also help to define a normal liver structure
reference for the diagnosis and treatment of liver dis-
eases.
5. Acknowledgements
The Authors are greatly indebted with Mister Paolo
Stortini for is valuable technical support, and with “Le
Cornelle” park direction for kindly providing facilities.
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