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-Induced Expression of Adhesion Molecules in the Liver Is Under the Control of TNFR1—Relevance for Concanavalin A-Induced Hepatitis1
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Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nürnberg, Erlangen, Germany;
Department of Experimental Medicine, University of Erlangen-Nürnberg, Erlangen, Germany; and
Department of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Hannover, Germany.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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has been clearly identified as central mediator of T
cell activation-induced acute hepatic injury in mice, e.g., Con A
hepatitis. In this model, liver injury depends on both TNFRs, i.e., the
55-kDa TNFR1 as well as the 75-kDa TNFR2. We show in this report that
the hepatic TNFRs are not transcriptionally regulated, but are
regulated by receptor shedding. TNF directly mediates hepatocellular
death by activation of TNFR1 but also induces the expression of
inflammatory proteins, such as cytokines and adhesion molecules. Here
we provide evidence that resistance of TNFR1-/- and
TNFR2-/- mice against Con A hepatitis is not due to an
impaired production of the central mediators TNF and IFN-
. Con A
injection results in a massive induction of ICAM-1, VCAM-1, and
E-selectin in the liver. Lack of either one of both TNFRs did not
change adhesion molecule expression in the livers of Con A-treated
mice, presumably reflecting the fact that other endothelial
cell-activating cytokines up-regulated adhesion molecule expression.
However, treatment of TNFR1-/- and TNFR2-/-
mice with murine rTNF revealed a predominant role for TNFR1 for the
induction of hepatic adhesion molecule expression. Pretreatment with
blocking Abs against E- and P-selectin or of ICAM-/- mice
with anti-VCAM-1 Abs failed to prevent Con A hepatitis, although
accumulation of the critical cell population, i.e., CD4+ T
cells was significantly inhibited. Hence, up-regulation of adhesion
molecules during acute hepatitis unlikely contributes to organ injury
but rather represents a defense mechanism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a pleiotropic cytokine that mediates host
defense as well as tumor necrosis. TNF is able to induce profound
changes of the vascular endothelium. These mechanisms are likely to
contribute to tissue injury in pathology such as disseminated
intravascular coagulation seen in septic shock, adult respiratory
distress syndrome, or cerebral malaria. The central role for TNF in
up-regulating cell surface adhesion molecules such as ICAM-1, VCAM-1,
or E-selectin, thereby promoting adhesion of leukocytes to and
subsequent transmigration through the endothelial barrier, is well
documented (1, 2). TNF exerts its actions via two distinct surface receptors, the 55-kDa receptor (also termed TNFR1 or TNFR p55) and the 75-kDa receptor (TNFR2 or TNFR p75) (3). The biological actions of TNFR1 have been extensively studied (4, 5), whereas the role for TNFR2, which preferentially binds the 26-kDa membrane-bound (m)3 precursor form of soluble (s) TNF (6), is not completely understood. A previous report emphasized the in vivo relevance of TNFR2 by showing that mTNF-induced adhesion molecule expression via TNFR2 was inhibited in mice lacking this receptor (TNFR2-/-), but not in mice lacking TNFR1 (TNFR1-/-), thereby inducing resistance against experimental induced cerebral malaria (7).
In addition there is increasing evidence that acute or chronic
liver disease is closely related to elevated TNF and TNFR (sTNFRs)
plasma levels as well as to a strong induction of in situ expression of
both TNFRs (8, 9, 10, 11). The direct hepatotoxic effect of TNF
has been studied in various experimental T cell- and
macrophage-dependent models (12). We recently described a
CD4+ T cell, TNF (13) and
IFN--dependent (14) model of liver injury in mice,
which is inducible by a single injection of the mitogenic plant lectin
Con A (15). The 26-kDa membrane-bound precursor form of
17-kDa sTNF and both TNFRs have been shown to be central for the
development of Con A hepatitis (16). Additional mediators
such as the Fas ligand/Fas system or perforin/granzyme have been
described either to contribute (17, 18) or not to
contribute (19, 18) to liver injury induced by Con A.
However, mTNF-, Fas ligand-, and perforin-mediated direct hepatotoxic
effects require cell-to-cell contact between hepatocytes and
leukocytes. A prerequisite for the direct action of effector cells is
their adhesion to and transmigration through the endothelial barrier.
The protective effect of anti-ICAM-1 mAbs (18) and
Arg-Gly-Asp mimetics (which block binding of 
1
integrins to several extracellular matrix glycoproteins)
(20) from Con A hepatitis confirmed this assumption.
Therefore, we designed a study, which had the objective to characterize the TNFR-dependent expression of adhesion molecules in Con A hepatitis and to evaluate the functional role of adhesion molecule up-regulation for Con A-induced CD4+ T cell recruitment and liver damage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male BALB/c and ICAM-/- mice were obtained from the institute’s internal animal breeding house. TNFR-/- mice were kindly provided by Dr. H. Bluethmann (Hoffmann LaRoche AG, Basel, Switzerland). The appropriate wild-type (wt) animals were used in all experiments in which knockout mice were used. Animals received human care according to the National Institutes of Health as well as to the legal requirements in Germany and were maintained under controlled conditions (22°C, 55% humidity, 12-h dark/light cycle) and had free access to standard laboratory chow (Altromin 1313) and water. When taken into the experiment, mice were 6–8 wk old and had a half weight of 18–25 g.
Con A and murine (mu) rTNF treatment
All reagents were injected in a total volume of 200 µl per 20-g mouse. Con A (Sigma, Deisenhofen, Germany) was injected i.v. in pyrogen-free saline. The dose of Con A was 20 and 18 mg/kg in BALB/c mice and TNFR-/- mice, respectively (because of the lower sensitivity of BALB/c mice toward Con A-induced liver injury; Ref. 21). rmuTNF (kindly provided by Dr. G. R. Adolf, Bender & Co, Vienna, Austria) was injected i.v. in pyrogen-free saline in a concentration of 10 µg/kg.
In vivo neutralization of adhesion molecules
The following mAbs were dissolved in pyrogen-free saline and injected i.v. 15 min before Con A application: 1) 200 µg of rat anti-mouse E-selectin mAb (UZ4) (22), 200 µg of rat-anti-mouse P-selectin mAb (RB40.34) (23), or a combination of both; 2) 150 µg of rat anti-mouse ICAM-1 mAb (YN1/1.7.4) and 150 µg of rat anti-mouse CD11a mAb (M17/4, PharMingen, Hamburg, Germany); 3) 150 µg rat anti-mouse VCAM-1 mAb (429, PharMingen). As isotype control, we injected an anti-endothelial Ab, which does not interact with adhesion molecules (BR2). In vivo binding was controlled with immunofluorescent staining of i.v.-injected mAbs (slides were solely incubated with the Texas Red-labeled goat anti-rat secondary Ab).
Sampling of material and determination of alanine aminotransferase (ALT), cytokines, and sTNFRs
For determination of circulating cytokines (TNF and IFN-) and
sTNFRs by ELISA (see above), blood was taken from the tail vein at the
indicated time points. After lethal anesthesia, blood was taken by
cardiac puncture for ALT measurement as previously described (14, 24). Liver was then removed and frozen for determination of DNA
fragmentation as described elsewhere (14) or for
immunofluorescent analysis (see below).
Cytokine and sTNFR determination by ELISA
Detection of TNF, IFN-, and IL-2 was performed as previously
described (25). sTNFRs were measured using a commercial
ELISA kit (Genzyme, Cambridge, MA), which was exactly performed
according to the manufacturers instructions.
Abs used for immunofluorescence
As primary mAbs: YN1/1.4.7 reacts with ICAM-1, MK2/1 with VCAM-1; mAb 10E9.6 reacts with murine E-selectin; RM4-5 recognizes CD4 (both purchased from PharMingen); primary rat mAbs were detected using goat anti-rat IgG tagged with Texas Red (Dianova, Hamburg, Germany). TNFR1 and TNFR2 were detected with a polyclonal rabbit anti-mouse Ab (Hy Cult Biotechnology, Uden, The Netherlands); primary rabbit Abs were detected with Cy 3-labeled goat anti-rabbit Ab (Dianova). Clone 104 detected CD45.2 and was directly labeled with FITC (PharMingen). Mouse endothelial cell Ag 32 (MECA-32) recognizes an endothelial Ag and was used as positive, rat IgG (PharMingen) as negative control, to exclude unspecific binding of the primary Ab.
Immunofluorescent staining and confocal laser imaging
Cryostat sections were performed at 10 µm, fixed in acetone/methanol (1:1) at 4°C for 10 min. After washing in PBS, slides were blocked with PBS containing 3% BSA and then incubated overnight with the primary Ab, at 4°C in a moist, light-protected chamber. After rinsing with PBS, binding sites were detected with the secondary Ab dissolved in PBS/BSA 3% for 1 h at room temperature. After washing in PBS, slides were mounted with TBS/glycerol (1:1), pH 8.6. Sections were then examined by confocal laser scanning microscopy (Bio-Rad 1000).
Semiquantitative RT-PCR
Total liver RNA was isolated by Clontech Kit (ClonTech, Palo
Alto, CA) according to the manufacturer’s instructions. Two micrograms
of total liver RNA was then reverse transcribed into cDNA using the
Superscript II Polymerase (Life Technologies GmbH, Eggenstein,
Germany). The sequences of the oligonucleotide primers are: 5' TNFR1,
CTG CTG TCA CTG GTG CTC DTG; 3' TNFR1, CAC ACA CCG TGT CCT TGT CAG; 5'
TNFR2, GTC GCG CTG GTC TTC GAA C; 3' TNFR2, CAC TTG CTC AGC CTC ATG.
PCR was conducted in an automatic DNA thermal cycler Primus 96
Thermocycler (MWG-Biotech, Ebersberg, Germany). The cycle program was
set to anneal at 58°C for a total of 32 cycles. The expected fragment
lengths were 349 bp for TNFR1 and 413 bp for TNFR2. Amplicons were
visualized by agarose gel electrophoresis. The gels were scanned by
GelDoc 2000 (Bio-Rad, Richmond, CA). The relative quantities of TNFR1
and TNFR2 mRNA are presented as ratio between the intensity of TNFR1
and TNFR2 band relative to the intensity of the housekeeping gene
-actin.
Cell preparations
Mice were killed by exsanguination from the subclavian artery and vein and livers were then removed. Hepatic mononuclear cells (MNCs) were prepared as previously described (26). Briefly, the liver was pressed through a stainless steel mesh and suspended in RPMI 1640 medium with 10% FCS and 25 mM HEPES. The cell suspension was centrifuged at 1200 rpm for 5 min and resuspended in 20 ml medium and then centrifuged at 50 x g for 30 s. Supernatants were resuspended in 10 ml medium and cell suspension was overlaid on 5 ml histopaque solution (density: 1083, obtained from Sigma). After centrifugation at 600 x g for 25 min at 25°C, the visible interface was aspirated and resuspended in medium to perform cell counts. Cells were washed once again and resuspended in PBS/FCS 1% to perform flow cytometry.
Flow cytometric analysis
FITC-labeled anti-CD45 (clone 104) and Texas Red-tagged anti-CD4 (clone RM4-5) were purchased from PharMingen. Cell suspensions of 105 cells were stained with mAbs and analyzed by flow cytometer (Coulter Pharmaceutical, Palo Alto, CA). Dead cells were excluded by forward sideward scatter. The number of CD4+ T cells per liver was calculated by multiplication of the percentage of CD4+ T cells with the percentage of CD45+ leukocytes of total isolated MNCs.
Statistical analysis
Data of ALT, cytokines, and sTNFRs are expressed as mean ± SEM and analyzed by one-way ANOVA; in case of differences among groups (at least p < 0.05), data were subjected to Dunnett’s multiple comparison test of the control against all other groups with the computer program INSTAT2 (GraphPad Software, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice deficient of either one of the two TNFRs and the
corresponding wt animals were i.v. injected with a dose of 18 mg/kg Con
A. TNFR1-/- and
TNFR2-/- mice failed to develop liver injury
upon Con A injection, as assessed by determination of plasma ALT-levels
(Table I
). To exclude the possibility
that this, also previously observed protective effect (16)
was due to a reduced production of the central mediators TNF and
IFN-, we detected TNF and IFN- plasma levels of
TNFR-/- mice. Circulating peak concentrations
of TNF (14) were significantly elevated in both,
TNFR1-/- and TNFR2-/-
mice (Table I). Maximum plasma levels of IFN- (14) were
significantly elevated in TNFR1-/- mice but
were comparably high to the wt in TNFR2-/- mice
(Table I).
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To investigate whether TNFRs are up-regulated within the liver
after systemic Con A injection, we performed RT-PCR and subsequent
semiquantitative analysis of total liver RNA. Both TNFR mRNAs were
constitutively expressed in the livers of healthy mice. Injection of
Con A did not significantly change overall hepatic TNFR1 (Fig. 1A) and TNFR2 (Fig. 1B) mRNA production during the observed time-span of 8
h. As internal control, we analyzed TNFR1 and TNFR2 mRNA production in
the spleen (Fig. 1, A and B), where both receptor
transcripts were significantly induced upon Con A injection. TNFR1 and
TNFR2 mRNA were undetectable in TNFR1-/- and
TNFR2-/- mice, respectively (Fig. 1C). Immunofluorescent staining of both TNFRs showed
constitutive expression of both TNFRs in livers of healthy mice.
However, TNFR1 and TNFR2 protein decreased as early as 1 h after
injection of Con A, reaching minimum expression levels at 2 to 4 h
after treatment (Fig. 2). Thereafter,
TNFR expression increased, reaching almost basal levels in case of
TNFR1 8 h after Con A challenge (Fig. 2). The expression level of
TNFR2 8 h after Con A injection was more prominent compared with
the constitutive expression of saline-treated animals (Fig. 2). No
staining of either TNFR1 in TNFR1-/- or TNFR2
in TNFR2-/- mice was detectable, confirming the
specific binding of the Abs (data not shown).
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). Con A injection induced a rapid
increase of both sTNFRs in plasma, which parallels the decline that we
observed by immunofluorescence (Fig. 3). We detected maximum levels of
sTNFR1 (1354 ± 12 pg/ml; Fig. 3, solid line) and of sTNFR2
(5776 ± 769 pg/ml; Fig. 3, dotted line) 8 h after Con A
injection. Thereafter, the levels of both soluble receptors declined
slowly. Twenty-four hours after Con A injection the serum levels of
both sTNFRs were still elevated. In comparison, TNF itself showed a
sharp transient plasma peak with a maximum at 2 h after Con A
injection (data not shown), as described previously
(14).
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Before the investigation of the effect of TNFR deficiency on
adhesion molecule expression in livers of Con A-treated mice, we
analyzed their expression in wt mice. Immunofluorescent staining and
subsequent confocal laser imaging revealed weak constitutive expression
of ICAM-1 (Fig. 4a) and VCAM-1
(Fig. 4d) in livers of healthy mice, whereas no constitutive
E-selectin (Fig. 4g) and P-selectin (data not shown)
expression was detectable. Time course analysis of the induction of
adhesion molecule expression revealed a strong increase of ICAM-1
staining on sinusoids, central and portal veins as early as 6 h
after challenge (data not shown), reaching high expression levels after
8–24 h (Fig. 4, b and c). VCAM-1 expression also
started to increase after 6 h (data not shown), reaching highest
expression 8 h after Con A injection (Fig. 4, e and
f). Expression of E-selectin also increased as early
as 6 h (data not shown) after Con A injection and was prominently
expressed 8 h after challenge with staining of central and portal
vein endothelial cells as well as of sinusoids (Fig. 4h).
Twenty-four hours after Con A, only minimal staining of E-selectin was
detectable on postsinusoidal venules (Fig. 4i). We failed to
detect any staining of P-selectin in livers of Con A-treated mice (data
not shown).
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Immunofluorescent staining of the CD4 Ag revealed accumulation of
CD4+ T cells as early as 6 h after Con A
(data not shown). Eight hours (Fig. 4
k) after Con A
injection, we found most of the CD4+ T cells in
the hepatic parenchyma. Twenty-four hours after Con A, most of the
CD4+ T cells were found as large cell clusters in
the periportal region and almost no CD4+ T cells
were detectable in the hepatic sinusoids (Fig. 4L). These
data are corroborated by the finding that 8 h after Con A
injection, CD4+ T cell accumulation is induced
6.1 ± 1.2 (6.7 ± 1.3 x 105
CD4+/CD45+ cells) fold vs
1.0 ± 0.2 (1.1 ± 0.2 x 105
CD4+/CD45+cells) in
saline-treated mice, as quantified by flow cytometric analysis of
hepatic MNCs.
Expression of ICAM-1, VCAM-1, and E-Selectin in TNFR1-/- and TNFR2-/- mice treated with Con A
TNFR-deficient mice are resistant against Con A hepatitis.
Therefore, we investigated whether deficiency in either one of both
receptors alters the expression of adhesion molecules induced by Con A,
thereby affecting the infiltration of the critical T cell population,
i.e., CD4+ T cells, into hepatic tissue. To this
end, we injected TNFR1-/-,
TNFR2-/-, and corresponding wt mice (all three
strains showed low constitutive ICAM-1 and VCAM-1 staining comparable
to untreated BALB/c mice, cf Fig. 4, A and D)
with Con A and analyzed the expression of ICAM-1, VCAM-1, and
E-selectin 8 h after challenge. We failed to detect any difference
in the staining intensity of ICAM-1 (Fig. 5, first column), VCAM-1 (Fig. 5, third column), or E-selectin (data not shown) between the
TNFR-deficient and the corresponding wt mice. Accordingly, there was no
difference in the accumulation of CD4+ T cells
(data not shown).
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To further elucidate the role of TNF in up-regulating adhesion
molecules in the liver, we injected TNFR1-/-,
TNFR2-/- as well as wt mice with 10 µg/kg
rmuTNF. Eight hours later, we removed the livers for immunofluorescent
staining analysis of adhesion molecule expression. ICAM-1 (Fig. 5, second column) and VCAM-1 (Fig. 5, fourth column)
expression was markedly induced in the livers of
TNFR2-/- and wt mice, whereas no up-regulation
was detectable in the livers of TNFR1-/- mice.
We were unable to detect E-selectin in the livers of TNF-treated
TNFR1-/-, TNFR2-/-, and
wt mice, respectively, most likely reflecting the fact that the
concentration of rmuTNF used was not sufficient to induce E-selectin
expression in the liver.
These results clearly demonstrate that TNF-mediated ICAM-1 as well as VCAM-1 up-regulation in the liver is under the control of the TNFR1.
Functional role of adhesion molecules in Con A hepatitis
To elucidate the functional role of the different adhesion
molecules and their counterparts on leukocytes, we performed in vivo
blocking studies, using mAbs against either ICAM-1/LFA-1 or VCAM-1 or
E-selectin or P-selectin. But surprisingly, despite the strong
expression of the adhesion receptors (see above), none of these Abs
given either alone, i.e., anti-VCAM-1,
anti-ICAM-1/anti-LFA-1, anti-E-selectin,
anti-P-selectin (data not shown), or in combination (cf Fig. 6), or gene-targeted deletion of ICAM-1
(data not shown), prevented the development of acute hepatic failure
induced by Con A. Plasma ALT levels were unaltered in normal mice
passively immunized against E-selectin and P-selectin (Fig. 6A) or in ICAM-/- mice treated with
anti-VCAM-1 mAb before Con A injection (Fig. 6B)
compared with Con A controls pretreated with a control IgG Ab.
Moreover, the production of the central mediators TNF and INF-
as well as of IL-2 was not affected (data not shown). However, flow
cytometric analysis of isolated hepatic MNCs revealed that either
anti-E-selectin mAb injection (Fig. 7A) or the use of
ICAM-1-/- mice treated with anti-VCAM-1
mAb (Fig. 7B) before Con A treatment significantly reduced
total accumulation of CD4+ T cells 
45 and
55%, respectively, compared with control mAb pretreated mice. In
contrast, pretreatment of mice with either anti-ICAM-1 or
anti-VCAM-1 mAb alone insignificantly inhibited total Con A-induced
accumulation of CD4+ T cells by 20% (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown previously that TNFR1 and TNFR2 mRNA was rapidly induced in livers of mice suffering from TNF-dependent, carbon tetrachloride-induced liver damage (27). In contrast, in the Con A model, where liver injury depends on both TNFRs (16), we did not observe a transcriptional regulation of hepatic TNFR1 and TNFR2 mRNA, although both transcripts were significantly induced in the spleen within the same time-span after Con A injection. However, we observed a profound reduction of TNFR1 and TNFR2 expression in liver sections after Con A injection. This down-regulation is paralleled by a marked increase in both sTNFR serum levels. Elevated serum levels of soluble TNFRs, especially TNFR2 have often been described to correlate with TNF serum levels and disease activity in acute and chronic liver disease (i.e., hepatitis B or C or autoimmunohepatitis) in humans (8, 9, 11, 28, 29), which might point to a desensitizing mechanism, that protects hepatocytes from the high local levels of TNF. The contradictory observations between carbon tetrachloride- and Con A-induced liver injury are not yet clear, but it seems likely that the T cell mitogen Con A induces a different local cytokine milieu, thereby inducing a distinct transcriptional regulation of TNFR mRNA expression in hepatic tissue.
Leukocyte recruitment from the blood stream is a central feature of inflammatory responses, which is regulated by adhesion molecules expressed on endothelial cells and their counter receptors on leukocytes (2). Because expression of these adhesion molecules is induced during inflammation, we analyzed their expression pattern after Con A application. We found that, in contrast to selectins, ICAM-1 and VCAM-1 were constitutively expressed, which is in line with earlier reports. Upon Con A injection, a massive induction of hepatic ICAM-1, VCAM-1, and E-selectin expression was seen. This induction of adhesion molecules corroborates earlier reports showing an up-regulation of ICAM-1, VCAM-1, and E-selectin in the liver under inflammatory conditions in humans (30) and mice (31, 32). However, others described a lack of inducibility of selectins in human livers and excluded a role for selectin-mediated leukocyte recruitment via hepatic sinusoids (33, 34). Hence, the data concerning selectin expression and their functional role for leukocyte recruitment in liver microvasculature still remain to be elucidated. But one can conceive that selectin-mediated rolling mechanisms in hepatic sinusoids are not necessary for leukocyte endothelial interaction, because of the slow blood flow that allows continuous contact between leukocytes and endothelial cells.
TNF is well known as a potent inducer of several adhesion molecules.
Hence, we wondered whether mice deficient in either one of both TNFRs
would exhibit a different pattern of adhesion molecule expression after
Con A treatment. However, we could not find any difference between Con
A-treated TNFR1-/-,
TNFR2-/- mice, and the corresponding wt
animals. Moreover CD4+ T cell infiltration was
unaltered in TNFR-/- mice compared with the wt.
These findings implicate that the resistance of
TNFR-/- mice toward Con A was not due to an
impaired induction of adhesion molecules by TNF. In contrast, a
resistance of TNFR2-/- mice against
experimentally induced acute cerebral malaria, which resulted from a
lack of mTNF-inducible ICAM-1 expression, was recently described by
Lucas et al. (7). However, treatment with rmuTNF instead
of Con A induced ICAM-1 and VCAM-1 only in
TNFR2-/- and wt mice, but not in
TNFR1-/- mice. Hence, our data provide evidence
that ICAM-1 expression in the liver is exclusively under the control of
TNFR1. These findings corroborate previous findings showing that in
other cell systems and tissues, TNF-inducible adhesion molecule
expression including ICAM-1 and VCAM-1 is mediated by TNFR1 (35, 36). The observation that Con A-treated
TNFR-/- mice express high levels of adhesion
molecules, although TNF-induced expression of adhesion molecules in the
liver is under control of TNFR1 suggest the involvement of other
endothelium activating cytokines such as IL-1, IL-12, or IFN-
(37, 38).
However, it remained unclear whether these adhesion molecules take part
in the hepatic accumulation of activated CD4+ T
cells, i.e., the critical cell population in this experimental model
(15). In this study we provide evidence that although
CD4+ T cell accumulation was significantly
inhibited by blockade of E-selectin or by lack of ICAM-1 together with
VCAM-1 blockade, these adhesion molecules play no functional role for
the onset of liver disease induced by Con A. This is in contrast to
previous reports describing inhibitory effects of either
anti-E-selectin and anti-VCAM-1 mAb (39) or
anti-ICAM-1 mAb but not anti-VCAM-1 mAb (18).
These controversial findings might have been due to different
pretreatment regiments, e.g., Ab pretreatment 24 and 48 h before
Con A injection, which might have depleted intrahepatic lymphocytes
critical for the development of liver injury. In contrast, in our
study, we have pretreated mice with the respective Abs shortly before
Con A administration and the pretreatment regiment was proven to be
functional with respect to a significant inhibition of
CD4+ T cell accumulation. Interestingly,
accumulation of CD4+ T cells in hepatic sinusoids
remained unaffected by blockade of adhesion molecules (data not shown).
Hence, E-selectin, ICAM-1, and VCAM-1 contributed to
CD4+ T cell recruitment via pre- and
postsinusoidal veins, whereas accumulation of
CD4+ T cells in sinusoids seems to be regulated
differently. The findings that adhesion molecules were strongly
expressed and that the accumulation of CD4+ T
cells was partially inhibited by adhesion molecule blockade, whereas
the development of liver disease remained unaffected, was surprising,
because CD4+ T cells have been shown to be
critical for the development of Con A hepatitis by Ab-dependent
depletion of CD4+ T cells (15). It
is conceivable that this procedure also depleted the
CD4+ proportion of liver resident V14 NK T
cells, which have been shown to be central for the onset of Con
A-induced liver disease (40, 41, 42). Hence, it seems likely
that Con A-induced stimulation of local immune cells, i.e., NK T cells
in concert with thymus derived T cells, which have been shown to be
abundant in the parenchymal space (43), and liver resident
macrophages are sufficient to induce hepatocellular damage. Recruitment
of CD4+ T cells from the circulation may then
contribute to the elimination of harmful activated intrahepatic
lymphocytes (i.e., T cells, NK T cells, or NK cells) (44).
Hence, blocking ICAM-1, VCAM-1, or E-selectin as therapeutic approach
to inhibit human liver disease has to be carefully considered.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gisa Tiegs, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany.
3 Abbreviations used in this paper: m, membrane-bound; s, soluble; wt, wild type; mu, murine; ALT, alanine aminotransferase; MNC, mononuclear cell.
Received for publication November 16, 1999. Accepted for publication October 24, 2000.
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References |
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 H. Kawamura, F. Aswad, M. Minagawa, S. Govindarajan, and G. Dennert P2X7 Receptors Regulate NKT Cells in Autoimmune Hepatitis J. Immunol., February 15, 2006; 176(4): 2152 - 2160. [Abstract] [Full Text] [PDF]
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B. Jaruga, F. Hong, W.-H. Kim, and B. Gao IFN-{gamma}/STAT1 acts as a proinflammatory signal in T cell-mediated hepatitis via induction of multiple chemokines and adhesion molecules: a critical role of IRF-1 Am J Physiol Gastrointest Liver Physiol, November 1, 2004; 287(5): G1044 - G1052. [Abstract] [Full Text] [PDF]
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B. John and I. N. Crispe Passive and Active Mechanisms Trap Activated CD8+ T Cells in the Liver J. Immunol., May 1, 2004; 172(9): 5222 - 5229. [Abstract] [Full Text] [PDF]
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A. Yamanaka, S. Hamano, Y. Miyazaki, K. Ishii, A. Takeda, T. W. Mak, K. Himeno, A. Yoshimura, and H. Yoshida Hyperproduction of Proinflammatory Cytokines by WSX-1-Deficient NKT Cells in Concanavalin A-Induced Hepatitis J. Immunol., March 15, 2004; 172(6): 3590 - 3596. [Abstract] [Full Text] [PDF]
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K. A. Muhlen, J. Schumann, F. Wittke, S. Stenger, N. van Rooijen, L. van Kaer, and G. Tiegs NK Cells, but Not NKT Cells, Are Involved in Pseudomonas aeruginosa Exotoxin A-Induced Hepatotoxicity in Mice J. Immunol., March 1, 2004; 172(5): 3034 - 3041. [Abstract] [Full Text] [PDF]
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E. Roth and H. Pircher IFN-{gamma} Promotes Fas Ligand- and Perforin-Mediated Liver Cell Destruction by Cytotoxic CD8 T Cells J. Immunol., February 1, 2004; 172(3): 1588 - 1594. [Abstract] [Full Text] [PDF]
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 G. Ramesh and W. B. Reeves TNFR2-mediated apoptosis and necrosis in cisplatin-induced acute renal failure Am J Physiol Renal Physiol, October 1, 2003; 285(4): F610 - F618. [Abstract] [Full Text] [PDF]
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J. Schumann, K. Muhlen, A. K. Kiemer, A. M. Vollmar, and G. Tiegs Parenchymal, But Not Leukocyte, TNF Receptor 2 Mediates T Cell-Dependent Hepatitis in Mice J. Immunol., February 15, 2003; 170(4): 2129 - 2137. [Abstract] [Full Text] [PDF]
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A. Massaguer, S. Perez-del-Pulgar, P. Engel, J. Serratosa, J. Bosch, and P. Pizcueta Concanavalin-A-induced liver injury is severely impaired in mice deficient in P-selectin J. Leukoc. Biol., August 1, 2002; 72(2): 262 - 270. [Abstract] [Full Text] [PDF]
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K. Streetz, B. Fregien, J. Plumpe, K. Korber, S. Kubicka, G. Sass, S. C. Bischoff, M. P. Manns, G. Tiegs, and C. Trautwein Dissection of the Intracellular Pathways in Hepatocytes Suggests a Role for Jun Kinase and IFN Regulatory Factor-1 in Con A-Induced Liver Failure J. Immunol., July 1, 2001; 167(1): 514 - 523. [Abstract] [Full Text] [PDF]
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0.05 vs saline control.
