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Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 520-528
Expression of P-Selectin on Hepatic Endothelia and Platelets
Promoting Neutrophil Removal by Liver Macrophages
By
Jialan Shi,
Yoshihiro Kokubo, and
Kenjiro Wake
From the Department of Anatomy, School of Medicine, Tokyo Medical and
Dental University, Tokyo 113, Japan.
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ABSTRACT |
The role of P-selectin on polymorphonuclear leukocyte
(PMN) adhesion-induced PMN elimination in the liver is
unclear. Our objectives were to show the expression and distribution of
P-selectin in rat liver, as well as to evaluate the changes in the
modulation of the expression of P-selectin and its role in the
accumulation and sequestration of PMNs. The intravenous administration
of endotoxin markedly increased the expression of P-selectin on the
venous and sinusoidal endothelial cells, as well as on the platelets trapped in the liver. Its expression peaked at 6 hours postinjection and was associated with a rapid increase in the aggregation and elimination of PMNs in the hepatic sinusoids. Combined treatment with
an antibody to P-selectin or with low molecular weight heparin, a
P-selectin antagonist, blocked the P-selectin, significantly reduced
the arrest of PMNs, and delayed their removal in the liver. Pretreatment with gadolinium chloride inhibited phagocytosis of PMNs by
the Kupffer cells, decreased the expression of P-selectin, and limited
the hepatic accumulation of PMNs. Thus, P-selectin played a role in
accumulation and elimination of PMNs from the liver. Results also
suggest that activated Kupffer cells can modulate the expression of
P-selectin in the liver and influence the homeostasis of PMNs in the
circulation during acute inflammation.
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INTRODUCTION |
RELATIVELY LITTLE is known about the
modulation, kinetics, and role of P-selectin expression in the hepatic
microvasculature. Using tissue extracts to analyze the upregulation of
P-selectin synthesis1,2 cannot identify or localize the
expression of P-selectin at the cellular level, nor can the
intralobular heterogeneity of P-selectin expression on the endothelial
cells be studied. The absence of endothelial P-selectin has been found
to severely affect the homeostasis of polymorphonuclear leukocytes
(PMNs), with the resulting neutrophilia caused, at least in part, by a delay in the removal of PMNs.3-5 However, these studies did
not address the questions of how such homeostasis is maintained, where and how the increased PMNs are eliminated, or what the relationship between P-selectin expression and PMN sequestration might be. Unlike
the microvasculature of other organs, the sinusoids in the liver
contain Kupffer cells, which normally represent 70% to 80% of the
tissue macrophages.6 However, a definitive role for the
modulation of P-selectin expression and PMN adhesion and sequestration
by Kupffer cells in the process of acute hepatitis remains unknown,
although interactions between the leukocytes and endothelia have been
reviewed.7,8 In addition, the role of platelets in the
arrest and elimination of PMNs in vivo also remains to be investigated.
To investigate the death of PMNs and their elimination by the Kupffer
cells, we previously developed an animal model that shows an increase
in the number of circulating PMNs.9 This model shows that
after an injection of lyophilized streptococci, the circulating PMNs
undergo apoptosis and are phagocytosed by Kupffer cells in the liver.
This observation remains unexplained because a detailed analysis of the
causes of PMN aggregation in the liver has not been performed, and the
expression and effects of P-selectin on the removal of PMNs have not
been evaluated. Therefore, we decided to study further the expression
of P-selectin in the hepatic lobules and its role in maintaining PMN
homeostasis.
Methodically, we injected lipopolysaccharide (LPS) intravenously to
produce an expression of P-selectin and an increase in number of PMNs
in the circulation. We assumed that the resulting expression of
P-selectin and excess of the PMNs provided visible and morphological
evidence of P-selectin-induced PMN adhesion and sequestration in the
liver. This approach enabled us to characterize the time course and
magnitude of P-selectin expression, define the heterogeneous expression
of P-selectin, and quantify and examine the localization and
elimination of PMNs in the liver. Blocking the P-selectin and
phagocytic activity of Kupffer cells enabled us to delineate the
relative contribution of the P-selectin to PMN accumulation and Kupffer
cells to PMN clearance. The current results lead us to believe the
important role of P-selectin and the Kupffer cells in maintaining the
homeostasis of PMNs in the circulation.
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MATERIALS AND METHODS |
Rats.
Seven- to 9-week-old male Wistar rats were obtained from Nippon
Biological Supplies (Tokyo, Japan) and were bred in the animal facilities at Tokyo Medical and Dental University.
Antibodies and drugs.
Polyclonal antibodies to P-selectin and CD61 monoclonal antibodies
(MoAbs) to platelet were obtained from PharMingen (San Diego, CA). To
label the macrophage-related antigens (Ags), we used ED1
and ED2 MoAbs (Serotec, Kidlington, Oxford, UK). To label major
histocompatibility complex (MHC) class II Ag, we used MRC OX6 MoAb
(Cedarlane, Ontario, Canada). LPS, low molecular weight (LMW) heparin,
an antagonist to P-selectin, and gadolinium chloride (GdCl3), an agent used to block the function of Kupffer
cells, were all obtained from Sigma (St Louis, MO). Proteinase K and biotin-16-2 -deoxyuridine-5-triphosphate (Biotin-16-dUTP)
were obtained from Boehringer Mannheim (Mannheim, Germany). Terminal deoxynucleotidyl transferase (TdT) was obtained from GIBCO BRL (Gaithersburg, MD). Other Abs, including biotinylated goat antimouse Ig, biotinylated goat antirabbit Ig, goat Ig to mouse IgG, goat Ig to
rabbit IgG, mouse peroxidase antiperoxidase (PAP) Ab complex, rabbit
PAP Ab complex, and peroxidase-conjugated streptavidin were obtained
from Dako (Glostrup, Denmark).
Experimental design.
In the first experiment, animals were injected with LPS (0.1 mg/kg
intravenously [IV]) and perfusion-fixed at 1, 3, 6, 12, 24, and 48 hours postinjection. Control rats were either untreated (day 0) or
injected with sterile saline at 1, 3, 6, 12, 24, and 48 hours. In the
second experiment, animals were treated with anti-P-selectin Ab (1.5 mg/kg, IV) or LMW heparin (70-125 IU/mg, 30 mg/kg, IV) 10 minutes after
the injection of LPS. Some animals were administered GdCl3
(7.5 mg/kg, IV) 12 hours before the LPS injection. The liver of each
animal was perfusion-fixed at 1, 3, 6, and 12 hours after the LPS
injection. Each value is expressed as a mean ± standard
deviation (SD) of three to five rats, and for some data, statistical
analyses were performed using Student's t-test. A level of
P < .05 was considered significant.
Immunohistochemistry.
The liver of the control and treated animals was perfused through the
portal vein with a peristaltic pump at a rate of 10 mL/min. Perfusion
consisted of saline for 20 seconds followed by phosphate-buffered 4%
paraformaldehyde for 2 minutes. After perfusion, slices of liver 3-mm
thick were immersed in fixative for 6 hours at 4°C, then embedded
in paraffin. Selected sections (4 µm) of the control and treated
liver were immunostained by the streptavidin-biotin-peroxidase complex
method. These sections were pretreated with 0.3%
H2O2 in methanol to block endogenous peroxidase
activity and were then incubated with normal goat serum (1:5 dilution).
The sections were then incubated overnight with primary Abs (diluted
1:1000), rinsed in phosphate-buffered saline (PBS) containing 0.03%
Triton X-100, incubated for 30 minutes with either biotinylated goat
antimouse Ig (1:600 dilution) or biotinylated goat antirabbit Ig (1:800
dilution, for anti-P-selectin), rinsed again in PBS, and finally,
incubated for 30 minutes with peroxidase-conjugated streptavidin (1:300
dilution). All Abs were diluted in 0.1 mol/L PBS containing 0.03%
Triton X-100 and 1% bovine serum albumin (BSA), and all steps were
performed at room temperature. Tissue peroxidase activity was
visualized after a 5-minute exposure to 0.025% 3,3-diamino-benzidine
tetrahydrochloride (DAB) in 0.05 mol/L Tris-HCl buffer (pH 7.4)
containing 0.01% H2O2, or a 10-minute exposure
to 3,3,5,5-tetramethylbenzidine in buffer. Some sections
were counterstained with hematoxylin or nuclear fast red.
DNA TUNEL.
Paraffin sections 4-µm thick were collected on glass slides coated
with poly-L-lysine. The nuclear DNA fragmentation of apoptotic cells
was labeled in situ by the TUNEL method.10 Briefly, the sections were first deparaffinized and treated for 15 minutes with 20 µg/mL proteinase K in 0.1 mol/L Tris-HCl buffer (pH 7.4). After being
rinsed with distilled water, the sections were treated for 5 minutes
with 2% H2O2 to block endogenous peroxidases.
The sections were again washed with distilled water, then incubated with 0.3 U/µL TdT and 0.04 nmol/µL biotinylated dUTP in TdT buffer at 37°C for 60 minutes. After another rinse with distilled water, the sections were incubated for 10 minutes with 2% BSA in 0.1 mol/L
PBS (pH 7.4) and then for 30 minutes in peroxidase-conjugated streptavidin diluted 1:300 with PBS. Peroxidase activity in the sections was visualized after incubation in 0.025% DAB in 0.05 mol/L
Tris-HCl (pH 7.4) containing 0.01% H2O2 for 5 minutes. For double-staining (TUNEL and ED1, ED2, OX-6, or
anti-P-selectin Ab staining), the TUNEL-positive cells were stained
brown or dark blue by adding 2 mL of 0.5% CoCl2 to the DAB
solution. The sections were then processed by the PAP method in a
manner similar to that described previously. In brief, sections treated
with primary Ab were successively incubated with goat Ig to mouse IgG
(diluted 1:100) or goat Ig to rabbit IgG (diluted 1:50) and mouse PAP
complex (1:200 dilution) or rabbit PAP complex (1:100 dilution) before exposure to the DAB solution for visualization. All steps were performed at room temperature unless otherwise indicated.
Analysis of P-selectin expression.
The endothelial cells were separately analyzed in location of
interlobular portal vein, periportal area, pericentral area and central
vein. The staining intensity was rated negative ( ), occasionally
positive or weakly positive (+/), positive (+), and strong
positive (++) antigen staining by light microscopy.
Cell counting.
The number of PMNs was counted by a method described by Bouwens et
al.11 For each animal, a total tissue area of approximately 4 mm2 (65 fields, 0.0625 mm2 per field) was
counted from a random sample of three sections. The paraffin sections
(4 µm) stained with the TUNEL method as well as the semithin sections
(0.2 µm) stained with toluidine blue were analyzed by light
microscopy at ×250. The number of PMNs per mm2 was
counted.
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RESULTS |
Expression of P-selectin and PMN accumulation in the hepatic lobule
after LPS injection.
Controls, both on day 0 or treated with saline, showed no difference
among the two groups. Endothelial cells in the control liver showed no
staining or, at best, slight anti-P-selectin Ab staining on the
endothelial cells of periportal veins. PMNs were rare in sinusoids.
After LPS-treatment, the liver showed the expression of P-selectin on
endothelial cells in the portal and sublobular veins by 1 hour, with
extensive and strong expression observed in the lobule after 3 to 6 hours (Fig 1a and b). Its expression on
hepatic venous and sinusoidal endothelial cells peaked at 6 hours (Fig 1c). The expression of P-selectin varied within regions of the liver.
The density of expression on the endothelial cells was ranked as
follows: interlobular portal veins, sublobular veins, and central veins
greater than pericentral area greater than periportal area. Many of the
platelets that were trapped in the periportal and midzonal areas in
density expressed P-selectin (Fig 1d). They were labeled by platelet
marker, a MoAb to CD61 (F11) (Fig 1e). F11 MoAbs react with integrin
 3 chain (CD61), which associates with integrin
 IIb chain to form the gpIIb/IIIa (CD41/CD61) complex. Platelets were closely associated with the trapped PMNs
(Fig 2a). No staining or only very weak
staining could be found in arteries 6 hours after LPS injection (Fig
2b). Serial sections showed that the areas of P-selectin expression
correlated with the presence of PMNs in the intralobular area. Twelve
hours after the LPS injection, weak P-selectin reactivity was
restricted to the endothelia of portal, central, and large veins, and a
few platelets were in the sinusoids. Twenty-four hours after the
injection, little expression of P-selectin was found.
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| Fig 1.
Expression of P-selectin in rat liver 3 to 6 hours after
the administration of LPS. Endothelial cells of the large portal vein
(PV) and its branchings showed intense reaction to anti-P-selectin Ab
(arrows; a). The endothelia of sublobular veins (V) and central veins
(CV) also expressed P-selectin (b). P-selectin was also present along
the walls of sinusoids and veins. A strong Ab reaction in the
endothelia of central veins, as well as the pericentral sinusoids, was
observed (c). The Ab reaction was relatively weak in the endothelium of
periportal sinusoids but strong in adherent platelets (arrows, d),
which were labeled by MoAb CD61 to platelet (arrows; e).
Bars, a, b, and d = 50 µm; c = 50 µm; e = 10 µm.
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| Fig 2.
Localization of P-selectin in intralobular area
of the liver 3 to 6 hours after the administration of LPS.
Immunostaining was with anti-P-selectin Ab (blue) and counterstaining
was by nuclear fast red (red). Many platelets expressing P-selectin
were found in the periportal area, correlating closely with the
presence of PMNs (arrows; a). P-selectin was expressed on the
endothelial cells of portal veins (PV), but not on ones of the
periportal arteries (A; arrow) in the periportal tract (b). Bars = 40 µm.
Fig. 4.
Double immunostaining of Kupffer cells and cells
expressing P-selectin in the liver 6 hours after the administration of
LPS. Endothelial cells and platelets (blue), Kupffer cells (brown), and
PMNs (red) were labeled with anti-P-selectin Ab, ED2 Ab, and nuclear
fast red, respectively. PMNs were adhering to endothelial cells of
sinusoids (arrows; a). Platelets expressing P-selectin were in contact
with Kupffer cells (arrows), endothelial cells (arrowhead), and PMNs
(large arrowhead; b). Bars = 10 µm.
Fig. 5.
In situ detection
of DNA fragmentation in PMNs in the liver 6 hours after the
administration of LPS. The PMNs with DNA fragmentation were labeled
brown (arrows) by the TUNEL method, whereas PMNs without DNA
fragmentation were stained blue by hematoxylin (arrowheads; a).
TUNEL-positive PMNs (brown, arrows) showed a close relationship with
sinusoidal endothelial cells (blue) expressing P-selectin (b). Bar
= 10 µm.
Fig. 7.
PMN-laden cells in the liver 6 hours after
the administration of LPS. Double staining with TUNEL and ED1 Ab showed
that numerous TUNEL-positive PMNs (dark brown, arrows) were trapped by
ED1+ Kupffer cells (brown; a). Parallel with the
phenomenon of phagocytosis (a), numerous PMN-laden Kupffer cells were
stained with ED2 Ab (brown, arrows; b) and Ia Ag+
PMN-laden cells were labeled with OX6 Ab (brown, arrows; c). Bars =10
µm.
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The kinetics of PMN accumulation in the liver has been evaluated by
counting PMNs in 65 fields (0.0625 mm2 per field) from
three sections per rat. The number of PMNs in the liver after treatment
with sterile saline and LPS was counted on semithin section (0.2 µm).
PMNs were identified and localized in the lumen of the sinusoids. The
PMNs adhered to the endothelial cells, platelets, and Kupffer cells, or
were phagocytosed by the Kupffer cells. The control liver exhibited few
PMNs, but the number increased rapidly and peaked at 6 hours after the
injection of LPS. This increase, compared with the untreated animals or
animals treated with saline, was significant (P < .05 and
P < .01). The number gradually decreased over a 12-hour
period (Fig 3). The number of PMNs
paralleled the increase or the decrease in the expression of P-selectin
in the liver.
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| Fig 3.
Number of PMNs in the liver after treatment with sterile
saline ( ) or LPS ( ). A slight increase of PMNs at 1 to 3 hours and a significant increase at 6 hours in the LPS-treated group were
observed. The number of cells is expressed per mm2 of
semithin section (0.2 µm). Data are expressed as mean ± SD. Bars
represent SD. *P < .01, **P < .05 versus saline
treatment. Five rats per group were examined.
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Expression of P-selectin and sequestration of PMNs.
The PMNs in the sinusoidal lumen were seen to contact the endothelial
cells (Fig 4a) and the platelets (Fig 4b) that expressed P-selectin.
Platelets adhered to the endothelial cells, the Kupffer cells, and the
PMNs (Fig 4b). PMNs with DNA fragmentation were labeled brown by TUNEL,
whereas those without DNA fragmentation were counterstained blue by
hematoxylin (Fig 5a). TUNEL-positive PMNs were in contact with the
endothelial cells (blue) that expressed P-selectin (Fig 5b). The number
of TUNEL-positive and TUNEL-negative PMNs were low in the control
liver; however, the number of both increased rapidly and peaked 6 hours
after the injection of LPS. The TUNEL-positive PMNs comprised more than
50% of the PMNs that had accumulated at 6 hours in the LPS-treated
liver, and decreased also rapidly after 6 hours
(Fig 6). This decrease, compared with the
number of TUNEL-positive PMNs at 6 hours, was significant (P < .05). Numerous TUNEL-positive PMNs were phagocytosed by the macrophages/Kupffer cells that were labeled by ED1 (Fig 7a) or ED2 Ab 6 hours after the injection of LPS (Fig 7b). PMNs were also seen in the
phagocytes labeled by OX6 Ab to MHC class II Ag (Fig 7c). The number of
free and phagocytosed PMNs in the liver was evaluated on semithin
sections (0.2 µm) and increased in parallel and reached a maximum 6 hours after the injection of LPS (Fig 8).
This increase, compared with the untreated animals, was significant (P < .05 and P < .01).
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| Fig 6.
Number of TUNEL-positive and TUNEL-negative PMNs in the
liver after the administration of sterile saline () and LPS (). Few TUNEL-positive and TUNEL-negative PMNs were observed in the liver
after sterile saline injection. However, the number of both TUNEL-positive and TUNEL-negative PMNs increased rapidly and peaked at
6 hours after LPS injection. The percentage of TUNEL positive PMNs
exceeded 50% of the total (TUNEL-positive and TUNEL-negative PMNs) at
6 hours after the LPS injection. The number of cells is expressed per
mm2 of TUNEL-stained paraffin sections (4 µm). Data are
expressed as mean ± SD. Bars represent SD. *P < .01 versus
saline treatment. Three rats per group were examined.
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| Fig 8.
Number of free and phagocytosed PMNs in the liver after
the administration of LPS. The number of free () and phagocytosed () PMNs increased rapidly and reached maximal levels at 6 hours. The
number of cells is expressed per mm2 of semithin section
(0.2 µm). Data are expressed as mean ± SD. Bars represent SD.
*P < .01, **P < .05 versus time 0 group. Five rats
per group were examined.
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Blocking of P-selectin and depletion of Kupffer cells affected
LPS-induced sequestration and elimination of PMNs in the liver.
Heparin chains containing four or more monosaccharide residues bind to
P-selectin and inhibit the function of P-selectin. Pretreatment with an
Ab to P-selectin or LMW heparin blocked the P-selectin, as observed by
slight immunostaining in the hepatic lobule, positive staining was
occasionally found on the endothelial cells of the portal veins and
blocked the arrest of PMNs in the liver. Pretreatment with
GdCl3 depleted the Kupffer cells, as observed by no
immunostaining of ED2 to Kupffer cells, few phagocytosis of PMNs, and
also weak staining of P-selectin (Table 1).
The number of free PMNs in the liver 3 or 6 hours after administration
of LPS was significantly reduced by the concomitant administration of
anti-P-selectin Ab or of LMW heparin or by pretreatment with
GdCl3 compared with LPS treatment alone (P < .01). A significantly greater decrease was observed after concomitant
treatment with anti-P-selectin Ab or GdCl3 versus that
with LMW heparin (Fig 9). The number of
phagocytosed PMNs observed in the liver 3 or 6 hours after the
injection of LPS was also significantly decreased by the combined
administration of anti-P-selectin Ab or of LMW heparin or by
pretreatment with GdCl3. However, the decrease in the
number of phagocytosed PMNs was greater at 6 hours than at 3 hours;
GdCl3 pretreatment showed the greatest decrease (Fig 9).
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| Fig 9.
Number of free and phagocytosed PMNs in the liver at 3 and 6 hours after treatment with LPS, LPS anti-P-selectin Ab, LPS LMW heparin, and GdCl3 LPS. The number of free and phagocytosed
PMNs was reduced at 3 hours, and significant reduction was observed 6 hours after combined treatment with anti-P-selectin Ab or LMW heparin
or GdCl3, compared with LPS treatment alone. The number of
phagocytosed PMNs was near to 0 level 3 or 6 hours after combined treatment with GdCl3. The number of cells is expressed per
mm2 of semithin section (0.2 µm). Data are expressed as
mean ± SD. Bars represent SD. *P < .01, **P < .05 versus LPS treatment only. Three rats per group were examined.
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DISCUSSION |
We made three significant observations. First, we observed the
heterogeneity expression of P-selectin on endothelia in the intralobular area of the liver after the administration of LPS. Second,
a substantial number of PMNs and platelets were found in the liver, but
PMNs eventually became apoptotic. Third, we showed that the adherent
PMNs did not emigrate, but were phagocytosed by the Kupffer cells.
In most tissues, the maximal expression of P-selectin occurs 4 hours
after the administration of LPS in vivo.1,12 However, in
this study, P-selectin expression on venous and sinusoidal endothelial
cells and platelets trapped in the liver peaked 6 hours after the
injection of LPS. Because depletion of Kupffer cells by pretreatment
with GdCl3 decreased intensity and shortened the time
course of P-selectin expression, this prolonged expression may arise in
part from the LPS-induced generation of tumor necrosis factor-
(TNF-) and interleukin-1 (IL-1) by the Kupffer
cells,6,13,14 which acts to sustain the level of P-selectin
expression on the endothelial cells in the liver. In addition, rather
profound heterogeneous distribution of P-selectin expression was noted
between the periportal and pericentral areas (pericentral area > periportal area), which suggests that the role of P-selectin in the
adherence and migration of PMNs in the lobule may differ between the
two areas. Significant regional differences in the intensity of
P-selectin expression, with no expression on endothelia of lymph
vessels or weak/part expression on that of arteries reflect that there
were structural and functional heterogeneity in hepatic endothelia.
An interesting finding was the arrest of CD61+ platelets,
which expressed P-selectin in the lobule mainly in the periportal and
midzonal areas. The trapping of platelets with intense expression of
P-selectin increased the concentration of P-selectin in the areas,
although the expression of P-selectin was relatively weak on endothelia
in the periportal area. Intravital microscopic observations have
suggested that unstimulated as well as activated platelets roll on, but
do not adhere to, the venous endothelium unless the integrity of the
vessel wall is disrupted.15,16 However, activated platelets
expressing P-selectin could preferentially bind the PMNs (Fig 2a) and
roll together on the endothelial cells (Fig 4b, large arrowhead). In
this way, each may recruit the other to the surface of stimulated
endothelium. In addition, as shown by Wake et al,6 the
Kupffer cell population is distributionally and functionally
heterogeneous. When the liver lobule is divided in three regions from
periportal to pericentral, the Kupffer cells are distributed in an
approximate ratio of 4:3:2 as determined by morphometric and
phagocytotic characterization. The distribution of arrested platelets
is consistent with the areas of higher concentrations of Kupffer cells.
Pretreatment with GdCl3 or the concomitant treatment with
anti-P-selectin Ab or LMW heparin reduced the aggregation of platelets
in the liver. Thus, it is reasonable that both the Kupffer cells and
the expression of P-selectin on endothelial cells of sinusoids
contribute to the sequestration of platelets in the liver. The
concomitant aggregation of platelets to PMNs at sites of hemostasis and
inflammation may contribute to the induction of vessel wall
injury.17 On the other hand, P-selectin is important in the
recruitment of PMNs to the sites of platelet deposition both in vivo
and in vitro.18-20 The trapped platelets could promote the
attachment of PMNs to the sinusoidal wall by slowing the PMNs so that
platelet-opsonized PMN phagocytosis by Kupffer cells could occur.
Furthermore, an activated monocyte-macrophage system can increase the
rate of platelet removal,21 and as seen in this study,
platelet P-selectin expression and platelet-PMN binding in the
periportal and midzonal areas coincided with phagocytosis of PMN by the
Kupffer cells there.
The results from perfused liver showed that a number of PMNs were
TUNEL-positive (Fig 5 and 6), and comprised more than 50% of the PMNs
that had accumulated at 6 hours in the LPS-treated liver; the number of
TUNEL-positive PMNs fell off more rapidly than the that of
TUNEL-negative cells. One explanation is that chromatin condensation
and DNA fragmentation appear to occur within a few minutes, and the
phagocytosis of apoptotic PMNs leads to their marked degradation, at
which point, occurring also within minutes, they are no longer
recognized as PMNs.22 As regards TUNEL-negative cells,
there was a significant decrease, compared with the number at 6 hours
after treatment, although there were some in the liver at a time when
P-selectin staining of sinusoidal endothelial cells was negative.
Coincidence of distribution of Kupffer cells and PMNs suggest that the
Kupffer cells may also play a role in trapping PMNs in the liver. PMNs
have been shown to die by apoptosis in vivo.9,14,23 The
apoptotic PMNs show a reduced expression of L-selectin.24
L-selectin has been reported to be expressed on the microvilli of
leukocytes,25 and the loss of microvilli, a significant and
early change in apoptotic PMNs,9 suggests that L-selectin
is not important in accumulation of PMNs in the liver. Furthermore,
P-selectin can tether the PMNs to the endothelial cells without PMN
activation or the 2-integrin system.26,27 LMW heparin reduces the influx of PMNs into the peritoneal cavity by
binding to P- and L-selectin,28-31 which coincide with a
report32 that blocking of the L- and P-selectin inhibits
PMN migration into mouse peritoneum. In our experiments, combined
treatment with Ab to P-selectin or LMW heparin resulted in a
negative/weak staining of the P-selectin and blocking of PMN arrest in
the liver. Thus, in this study, it appeared that the P-selectin on the
endothelia and platelets mediated sequestration of PMNs in the liver.
The excessive accumulation of PMNs can damage tissues via the release
of proteases and oxygen radicals.33 Therefore, the elimination of PMNs from the vasculature is important for stopping the
development of lesions. In our study, the aggregation of PMNs by
P-selectin-induced adhesion in the hepatic sinusoids was followed by
phagocytosis of PMNs. These results suggest that phagocytosis of PMNs
by Kupffer cells occurs when the PMNs transit through the hepatic
sinusoids from portal to central veins. This elimination of PMNs could
be caused by several factors. One explanation may be that the adhesion
modulated by P-selectin was a prerequisite for phagocytosis by the
Kupffer cells in this study. Furthermore, the PMNs were aging and
undergoing apoptosis, a process that is closely followed in vivo by
phagocytosis. These results are supported by our previous
studies.9,14
The PMN-laden cells identified in this study were Kupffer cells and MHC
class II Ag+ cells. The Kupffer cells were labeled by MoAbs
to the macrophage-related Ag ED1 and ED2.9 The elimination
of the Kupffer cells with GdCl3 caused a delayed removal of
PMNs. MHC class II Ag+ cells were stained by MoAb OX6 to
Ia-nonpolymorphic Ag. It was recently reported that MHC class II
Ag+ phagocytes consist of activated monocytes and dendritic
cells, and that dendritic cells exhibit phagocytic activity for
particulates in vivo when they undergo a blood-lymph translocation via
the hepatic sinusoids.34
The roles that adhesion molecules play in sinusoidal PMN accumulation
remain unclear. Sequestration of PMNs in the hepatic vasculature during
endotoxemia is independent of 2-integrins and
intercellular adhesion molecule-1 (ICAM-1).33,35 A minimal role for selectins in the recruitment of leukocytes into the inflamed liver observed by Wong et al36 seems improbable, because
expression of P- and E-selectin on normal and FMLP-treated animals was
not determined. The results of this model showed that P-selectin was indeed required for the adhesion-induced elimination of PMNs. Results
suggest that the adherent platelets on the walls of the hepatic
sinusoids could act as receptors for the adhesion of flowing PMNs, or
P-selectin might act as the first step in delaying the PMNs so that
they become locally adherent to the endothelium via P-selectin-mediated binding. Margination, adhesion, and apoptotic changes of the flowing PMNs in hepatic sinusoids could, therefore, be a
prerequisite for phagocytosis by the Kupffer cells. Our results also
suggest that the Kupffer cells, together with the ability of
phagocytosis and the modulation of P-selectin expression, appeared to
play a role in the accumulation and homeostasis of PMNs in the
circulatory system.
| |
FOOTNOTES |
Submitted October 16, 1997;
accepted March 18, 1998.
Supported by Grant No. C08457002 from the Japanese Ministry of
Education, Science, Sports and Culture.
Address reprint requests to Jialan Shi, MD, PhD, Division 1, Department
of Anatomy, School of Medicine, Tokyo Medical and Dental University,
1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful to Prof Vinci Mizuhira and Katsuiku Hirokawa for their
valuable suggestions and encouragement. We acknowledge the technical
assistance of Akira Masuda and Yingli Yang.
| |
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Abstract
Introduction
Abstract