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*
Institute of Experimental and Clinical Pharmacology and Toxicology, and
Institute of Clinical Microbiology and Immunology, University of Erlangen-Nurnberg, Erlangen, Germany; and
Department of Anatomy and Cell Biology II, University of Heidelberg, Heidelberg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2, and IL-6 into the circulation. Most
strikingly, liver damage depended on T cells. Athymic nude mice or mice
depleted of T cells by anti-Thy1.2 mAb pretreatment failed to
develop acute hepatic failure, and survival was significantly prolonged
following T cell depletion. Neutralization of TNF or lack of TNF
receptors prevented liver injury. In the liver, TNF was produced by
Kupffer cells before hepatocellular death occurred. After T cell
depletion, Kupffer cells failed to produce TNF. Transaminase release
was significantly reduced in perforin knockout mice, and it was even
elevated in lpr/lpr mice. These results demonstrate that
PEA induces liver damage not only by protein synthesis inhibition but
also by TNF- and perforin-dependent, Fas-independent, apoptotic
signals. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The two bacterial products most likely implicated in the systemic toxicity of P. aeruginosa are its LPS and exotoxin A (PEA).3 Purified exotoxin A is highly lethal for animals, including subhuman primates, and produces shock in dogs and rhesus monkeys. The biologic significance of exotoxin A for the pathogenicity of P. aeruginosa became evident by studies showing that patients with high levels of serum Abs to exotoxin A at the onset of P. aeruginosa septicemia have a better chance of survival than those with low Ab titers (1, 3). Moreover, in a mouse model it was shown that PEA-producing P. aeruginosa strains were more toxic than nonproducers, and that their toxicity could be weakened by Abs to PEA (4).
Hepatic injury due to systemic inflammatory processes has been reported to occur in the pathophysiology of septic shock (5). LPS from Gram-negative bacteria have been intensively studied with respect to their capacity to induce shock by stimulation of monocytic cells. These cells release TNF and other proinflammatory cytokines that mediate multiorgan failure and lethality in experimental animal models of endotoxic shock. More recently, it became evident that activation not only of macrophages but also of T cells may result in a systemic inflammatory response syndrome and organ injury (6). We recently described two models of T lymphocyte-dependent apoptotic and secondary necrotic liver injury induced by either the anti-mouse CD3 mAb 145 2C11 or the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) in D-galactosamine (GalN)-sensitized mice (7, 8). In these models the T cell stimuli evoke a cytokine release syndrome. TNF was found to be the central mediator of hepatocellular apoptosis and the ensuing severe liver failure (7). The amino sugar GalN depletes uracil nucleotides selectively in the liver, thereby inhibiting hepatic transcription and translation (9) and sensitizing the liver toward T cell stimuli (7, 8), LPS (10, 11, 12), or TNF (13, 14).
Like GalN, PEA inhibits protein synthesis in mammalian cells, e.g., in hepatocytes (15, 16), thus being a bacterial toxin that possibly sensitizes the liver toward proinflammatory cytokines such as TNF. In human ovarian tumor cell lines, PEA induced apoptosis that was accelerated by TNF (17). This property of PEA led to the use of PEA-derived fusion proteins as immunotoxins for cancer treatment (18). In clinical trials in humans, the major side effects of immunotoxins are a vascular leak syndrome, fever, and hepatotoxicity (18, 19).
In the past, several reports have tried to draw a connection between PEA and T cells. In vitro, PEA led to mitogenic activation of T lymphocytes in murine whole splenocyte cultures and generated cytolytic T lymphocytes active against EL4 target cells (20). This apparent lack of an apoptotic signal on T cells may be explained by a minor sensitivity of lymphoid cells to inhibition of protein synthesis by PEA compared with the sensitivity of other mammalian cells (20). Moreover, PEA exhibited properties that are similar, but not identical with those of microbial superantigens; PEA selectively stimulated the proliferation of murine thymocytes expressing the Vß8.2 chain in their TCR (21), and thymocyte proliferation depended on the presence of APCs (22, 23). However, in contrast to conventional superantigens, lymphoproliferative activity required intracellular processing (24), and no superantigenic activity of PEA was found by others (25). Hence, the objective of this study was to further investigate the role of TNF and T cells in the toxicity of PEA in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old BALB/c mice were obtained from the institute’s internal animal breeding house. TNFR-deficient and the corresponding wild-type (C57BL/6 x 129/Sv) mice were provided by Dr. H. Bluethmann, Hoffmann-La Roche (Basel, Switzerland). Perforin knockout mice were provided by Drs. J. Tschopp and M. Schröter, University of Lausanne, Institute of Biochemistry (Epalinges, Switzerland). Athymic BALB/c-nu/nu mice, Fas-deficient lpr/lpr mice, and mice of the corresponding wild-type strain MRL/Mp, as well as C3H/HeN and C3H/HeJ mice, were purchased from Harlan Winkelmann (Borchen, Germany). Animals received humane care according to National Institutes of Health guidelines as well as the legal requirements in Germany and were maintained under controlled conditions (22°C, 55% humidity, 12-h day/night rhythm) and were fed a standard laboratory chow (Altromin 1313, Altromin, Lage, Germany) ad libitum.
Animal treatments
All reagents were injected in a total volume of 250 µl/25 g
mouse. PEA (Sigma, St. Louis, MO) was injected i.v. in pyrogen-free
saline containing 0.1% human serum albumin (HSA). The LPS content of
PEA was determined with the help of a commercially available
Limulus amebocyte lysate (LAL) kit (Coatest Endotoxin,
Chromogenix, Molndal, Sweden). In some experiments mice were treated
with one of the following combinations of reagents: 1) 30 mg/kg i.v. of
benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Bachem Biochemica,
Heidelberg, Germany) in saline/6% DMSO followed by PEA 15 min later;
2) 20 µl/mouse i.v. of the IgG fraction of a sheep anti-mouse TNF
polyclonal antiserum (provided by Dr. A. Wendel, University of
Konstanz, Konstanz, Germany) in saline/0.1% HSA followed by PEA 15 min
later; 3) 200 µl/mouse i.v. of rabbit anti-mouse IFN-
polyclonal antiserum in saline/0.1% HSA followed by PEA 15 min later;
4) 100 µl/mouse i.v. of anti-mouse Thy1.2 mAb (IgG2a; Bio-Yeda,
Rehovot, Israel; this treatment leads to depletion of 82% of T cells
24 h later (26)) in saline followed by PEA 24 h later; 5) 100
µl/mouse i.v. of control Abs, i.e., rat IgG2a (PharMingen, Hamburg,
Germany), in saline followed by PEA 24 h later; 6) 50 mg/kg i.v.
of cyclosporin A in saline/10% placebo (Sandoz, Nurnberg, Germany) 15
and 1 h before PEA; 7) 2.5 mg/kg i.p. of SEB (Sigma) in
saline/0.1% HSA followed by PEA 48 h later; 8) 2.5 mg/kg i.p. of
SEB in saline/0.1% HSA followed by GalN/SEB (GalN, 700 mg/kg i.p.;
SEB, 2.5 mg/kg i.p.) 48 h later; 9) 2.5 mg/kg i.p. of SEB in
saline/0.1% HSA followed by GalN/LPS (GalN, 700 mg/kg i.p.; LPS from
Salmonella abortus equi, S form (Metalon, Ragow, Germany),
10 µg/kg i.p.) 48 h later; 10) 10 µg/kg recombinant murine TNF
(provided by Dr. G. R. Adolf, Bender & Co., Vienna, Austria)
i.v. 15 min after injection of PEA in saline/0.1% HSA; 11) 10 µg/kg
LPS from S. abortus equi, S form, given i.p. 15 min after
injection of 3 µg/kg PEA in saline; and 12) 2.5 mg/kg SEB given i.p.
15 min after injection of 3 µg/kg PEA in saline/0.1% HSA.
Sampling of material
Mice were lethally anesthetized with 150 mg/kg pentobarbital i.v. containing a dose of 15 mg/kg heparin. Blood was withdrawn by cardiac puncture. Livers were perfused via the portal vein for 10 s with cold perfusion buffer (89 mM Tris-HCl, 89 mM boric acid, 2 mM EDTA, and 10 mM EGTA, pH 8.0) before excision. In some experiments, perfusion was performed for 20 s via the right ventricle of the heart before excision of liver, lung, cecum, kidneys, and heart. Cecum was luminally perfused with saline to remove contents. All organs were blotted dry and disintegrated in cold perfusion buffer with an Elvehjem-type homogenizer. The 20% organ homogenates were centrifuged at 13,000 x g for 15 min. One part of the supernatant was to detect oligonucleosome-bound DNA fragments (see below). The other part was used to precipitate DNA by addition of 1 ml of ice-cold ethanol plus 50 µl of 3 M sodium acetate.
Analysis of liver enzymes and released proteins
Hepatocyte damage was assessed by measuring plasma enzyme
activities of alanine aminotransferase (ALT), aspartate
aminotransferase, and sorbitol dehydrogenase according to Bergmeyer
(27) using an automated procedure. The cytokines TNF, IFN-, IL-2,
and IL-6 were determined by ELISA (PharMingen). For determination of
TNF, a polyclonal sheep anti-mouse TNF capture Ab (28), purified on
protein G columns (Pharmacia, Freiburg, Germany) was used to replace
the PharMingen capture mAb. IL-10, IL-1
, and IL-1ß were
measured using commercial ELISA kits (IL-10: Genzyme, Cambridge, MA;
IL-1: Endogen, Cambridge, MA; IL-1ß: Paesel & Lorei Co., Hanau,
Germany).
DNA fragmentation
DNA fragmentation was quantified (29) by measuring cytosolic
oligonucleosome-bound DNA using an ELISA kit (Boehringer Mannheim,
Mannheim, Germany). Briefly, the cytosolic fraction (13,000 x
g supernatant) from approximately 200 µg of liver was
employed as Ag source in a sandwich ELISA with a primary
anti-histone Ab coated to the microtiter plate and a secondary
anti-DNA Ab coupled to peroxidase. DNA fragmentation was also
analyzed semiquantitatively after extraction of the 13,000 x
g supernatant (corresponding to 
80 mg of liver) by the
phenol/chloroform method, precipitation by ethanol, and electrophoresis
on 1.0% agarose gels (30).
Electron microscopic studies
For electron microscopic studies, the livers were fixed by perfusing the portal vein with a fixative containing 0.25% glutaraldehyde and 2% sucrose in 100 mM PIPES buffer at pH 7.4 for 5 min. Sections were postfixed with 2% aqueous osmium tetroxide, dehydrated in graded ethanol, and embedded in Epon 812. Ultrathin sections were counterstained with lead citrate and examined in a Philips EM 301 electron microscope (Eindhoven, The Netherlands).
Immunofluorescent staining and confocal laser imaging
Twelve-micrometer-thick cryostat sections of livers were thawed onto glass slides, air-dried, and fixed in acetone/methanol (1/1) for 10 min at 4°C before they were incubated in PBS containing 3% BSA for 30 min at room temperature. After the slides had been rinsed in PBS, incubation was continued with polyclonal rabbit anti-mouse-TNF Ab (Genzyme Virotech, Russelsheim, Germany; 1/750) together with rat mAb against murine pan-macrophage marker (clone BM8, Dianova, Hamburg, Germany; 1/100), or mouse CD4 (clone RM4-5, PharMingen; 1/50), or mouse CD8 (clone Ly-2, PharMingen; 1/50) in PBS containing 3% BSA overnight at 4°C. After rinsing with PBS, binding sites were detected using swine anti-rabbit IgG tagged with FITC (Dako, Hamburg, Germany; 1/30) and goat anti-rat IgG tagged with Texas Red (Dianova; 1/200) in PBS containing 3% BSA for 1 h at room temperature. After rinsing with PBS, sections were coverslipped with TBS/glycerol (1/1), pH 8.6. Sections processed for immunofluorescence were examined by confocal laser scanning microscopy (MRC 1000, Bio-Rad, Richmond, CA).
Statistical analysis
The results were analyzed using Student’s t test if
two groups were compared and the Dunnett’s test if more groups were
tested against a control group. If variances were inhomogeneous, the
results were analyzed using the Welsh test. The significance of
prolonged survival was tested by comparison of survival curves with the
log-rank test. All data in this study are expressed as the mean ±
SEM. p 
0.05 was considered significant.
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Results |
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The i.v. injection of PEA into BALB/c mice dose-dependently
induced liver cell damage within 12 h as assessed by an increase
in the activities of plasma transaminases as well as enhanced
activities of the liver-specific enzyme sorbitol dehydrogenase in
plasma (Fig. 1
). Administration of the
highest dose, i.e., 300 µg/kg PEA, resulted in lethality within
16 h. The release of liver enzymes was preceded by the appearance
of cytosolic oligonucleosome-bound DNA within the liver that was
significantly enhanced 6.5 h following PEA administration (Fig. 2). Internucleosomal DNA fragmentation as
a measure of programmed cell death was also demonstrated on an agarose
gel. The appearance of the DNA ladder again preceded the increase in
plasma ALT (Fig. 3, lanes
1–3). The unspecific tripeptidic caspase inhibitor
benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD.fmk), given in
a dose that protected mice from GalN/TNF-induced liver injury (31)
(data not shown), also significantly inhibited transaminase release
following PEA challenge (buffer plus PEA: ALT, 8970 ± 2355 U/l;
Z-VAD.fmk plus PEA: ALT, 990 ± 215 U/l (p
0.05); n = 6). The inhibitor had no effect on
PEA-induced TNF production, i.e., Z-VAD.fmk did not reduce the amount
of circulating TNF (data not shown). Together these data clearly
suggest a role for apoptotic proteases in PEA-dependent liver failure.
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To determine which cells in the liver died by apoptosis, we set up
transmission electron micrographs of mouse livers excised 12 h
following PEA injection. Micrographs clearly show that the dying cells
were hepatocytes (Fig. 4, a
and b). They died by apoptosis (Fig. 4, a and
b) and necrosis (Fig. 4b), whereas neither
apoptosis nor necrosis was seen in livers of saline-treated mice (33).
Necrotic liver areas contained infiltrated polymorphonuclear
neutrophils (Fig. 4b). As a cause of damage, perfusion of
the livers with the fixative was incomplete. Hence, blood cells were
still located in the hepatic sinusoids (Fig. 4c). Fig. 4c also shows a lymphocyte sticking to sinusoidal
endothelium. Some lymphocytes infiltrating into the hepatic tissue were
observed. One example is shown in Fig. 4d. Fig. 4, a–d, also demonstrates that hepatocytes of PEA-treated mice
contained numerous lipid droplets. The development of a fatty liver
most likely results from an inhibition of apolipoprotein synthesis by
PEA.
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PEA-induced cytokine release
As PEA has been described to stimulate the proliferation of T
cells in vitro (20, 21), it seemed feasible that the toxin also
stimulates cytokine production in vivo. By determination of plasma
cytokines following PEA administration to mice we found a
time-dependent release of the proinflammatory cytokines TNF and IL-6 as
well as of the T cell cytokines IL-2 and IFN- (Fig. 5). IL-1, IL-1ß, and IL-10 were not
detectable in plasma of PEA-treated animals (IL-1, 6 pg/ml;
IL-1ß, 1 pg/ml; IL-10, 15 pg/ml). Thus, cytokine analysis
suggests that PEA also activates T lymphocytes in vivo.
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Because TNF has been identified as a common mediator of hepatocellular apoptosis and liver injury in experimental mouse models (7, 29, 33, 34), we wondered whether TNF is also a mediator of PEA-induced liver damage.
Although TNF was not detectable in plasma until 12 h after
administration of PEA (Fig. 5
), i.e., at a point when liver damage had
already developed, TNF was locally produced within the liver as early
as 3 h after administration of PEA to BALB/c mice. This was shown
by means of immunofluorescent staining followed by confocal laser
imaging (Fig. 6). TNF was colocalized
with resident liver macrophages, which were stained with the BM8 murine
pan-macrophage marker (Fig. 6A, PEA 3 h), indicating
that TNF was produced by Kupffer cells at this early time point. CD4-
and CD8-positive T cells were present within the liver 3 h after
PEA injection in amounts comparable to those observed at time zero.
These cells did not produce TNF (Fig. 6, B and C,
PEA 3 h). TNF was also detectable on the surface of hepatocytes,
but not inside these cells, suggesting that macrophage-produced TNF
bound to liver parenchymal cells (Fig. 6, PEA 3 h). At later time
points, i.e., 6 and 9 h after administration of PEA, hepatic TNF
was not detectable by immunostaining (data not shown). Twelve hours
after challenge with PEA the number of CD8-positive T cells within the
liver was slightly increased (Fig. 6C, PEA 12 h),
whereas the number of CD4-positive T cells remained unchanged (Fig. 6B, 12 h). Macrophages had disappeared (Fig. 6A, PEA 12 h), and TNF was still undetectable in liver
sections (Fig. 6, PEA 12 h). This indicates that the systemic TNF
concentrations observed 12 h following PEA injection were produced
extrahepatically. T cells were essential for TNF production of liver
macrophages. In mice depleted from T cells by an anti-mouse Thy1.2
mAb (26) (see also below), there was no detectable TNF production
within the liver 3 h after administration of PEA (Fig. 6, anti-Thy1.2/PEA 3 h), whereas macrophage staining remained
unaltered (Fig. 6A, anti-Thy1.2/PEA 3 h). CD4- and
CD8-positive T cells were completely depleted from hepatic tissue in
anti-Thy1.2-pretreated animals (Fig. 6, B and
C, anti-Thy1.2/PEA 3 h). Because there was no
staining by the anti-TNF Ab within the livers of untreated mice and
anti-Thy1.2-pretreated mice 3 h after PEA administration,
unspecific binding of this Ab to macrophages can be excluded. Taken
together, these results show that T cells are required for Kupffer
cell-dependent early TNF production following PEA intoxication in mice.
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10 pg/ml) in the
Ab-pretreated group; n = 5). This Ab significantly
inhibited DNA fragmentation as well as ALT release (Fig. 7A). Inhibition of
internucleosomal DNA fragmentation in the liver by anti-TNF Ab
pretreatment was also demonstrated on an agarose gel (Fig. 3, lanes 3 and 4). Moreover, TNFR1 knockout
(tnfr1°) as well as TNFR2 knockout
(tnfr2°) mice were significantly protected from
PEA-induced liver failure (Fig. 7B). In contrast to
PEA-challenged tnfr1° mice, DNA fragmentation in livers of
PEA-treated tnfr2° mice was not significantly reduced
compared with that in wild-type animals, indicating that hepatic
apoptosis is primarily mediated via TNFR1. However, because
tnfr2° mice were significantly protected from severe
hepatic failure as measured by ALT release, it seems likely that either
costimulatory signals provided by TNFR2 (35) or ligand passing (36) are
necessary for TNFR1-induced hepatocellular death.
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0.05); n = 3). Similar results were obtained when mice
were treated with subtoxic doses of PEA (3 µg/kg) plus LPS (10
µg/kg) or PEA (3 µg/kg) plus SEB (2.5 mg/kg; PEA alone: ALT,
36 ± 8 U/L; LPS alone: ALT, 52 ± 6 U/L; PEA plus LPS: ALT,
4744 ± 2227 U/L (p 0.05); SEB alone:
ALT, 10 ± 3 U/L; PEA plus SEB: ALT, 8,663 ± 2,242
(p 0.05) U/L; n = 3). We
conclude that PEA sensitizes the liver toward TNF, e.g., induced by LPS
during systemic Pseudomonas infections or coinfections with
other bacteria. T cell dependence of PEA-induced toxicity
In vitro, PEA was shown to be a polyclonal activator of cytolytic
T lymphocytes, effective against Con A-treated target cells (20).
Furthermore, as described above, T cells were required for Kupffer
cell-dependent early TNF production following PEA intoxication in mice.
To test whether T lymphocytes accounted for part of the in vivo
toxicity of PEA, we pretreated mice with the T cell-depleting
anti-Thy1.2 mAb (26). This treatment significantly protected mice
from PEA-induced liver damage (Fig. 8A) and prevented hepatic
internucleosomal DNA fragmentation (Fig. 3, lanes 3 and
5). Pretreatment with control Abs, i.e., rat IgG2a, failed
to protect mice from PEA-induced liver failure (data not shown).
Similarly, athymic BALB/c-nu/nu mice lacking mature T cells
were resistant toward PEA-induced liver injury (Fig. 8A).
Circulating cytokine levels, including TNF, were significantly reduced
or were not detectable in anti-Thy1.2 mAb-pretreated or
nu/nu mice (Fig. 8B). In conclusion, the
production of TNF and other cytokines as well as liver injury clearly
depends on T lymphocytes.
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0.05); n = 3).
Recently, Muraille et al. described a Vß-unrestricted, T
cell-unresponsive state together with an APC defect and a selective
decrease in splenic dendritic cell numbers in mice 48 h after SEB
injection (37). A similar unresponsive state has been described by
Miethke et al., who observed that SEB pretreatment protected mice not
only from lethality induced by the very same superantigen, i.e., SEB,
but also by a third party superantigen with nonoverlapping Vß
specificity, i.e., toxic shock syndrome toxin-1 (38). Hence, we
wondered whether SEB pretreatment would also protect mice from
PEA-induced liver injury. Indeed, PEA-induced ALT release was
significantly reduced in SEB-pretreated mice (Fig. 8C). The
same SEB pretreatment also protected the animals from
GalN/SEB-dependent transaminase release but not from GalN/LPS-induced
liver failure (Fig. 8C), again backing up a prominent role
of T cells for PEA hepatotoxicity. The release of TNF into the
circulation, determined at the time of maximal systemic TNF production
(6, 39), was suppressed in SEB-pretreated mice challenged with PEA or
GalN/SEB, but not in SEB-pretreated mice challenged with GalN/LPS (Fig. 8C).
To study a positive effect of T cell depletion on survival, mice were
pretreated with the anti-Thy1.2 mAb and challenged with 30 µg/kg
PEA i.v. Survival was significantly prolonged by anti-Thy1.2 mAb
pretreatment (Fig. 8d). Mice treated with 30 µg/kg PEA
developed liver injury within 24 h (ALT, 1227 ± 271 U/L;
n = 5) that was again prevented by anti-Thy1.2
mAb-dependent T cell depletion (ALT, 139 ± 18 U/L;
n = 5; p 0.05). The enhanced
survival of the anti-Thy1.2 mAb-pretreated animals points to a
prominent role of T cells for PEA-induced toxicity. This finding was
corroborated by a significantly prolonged survival of SEB-pretreated
compared with nonpretreated mice following intoxication with 30 µg/kg
PEA (Fig. 8D).
Due to the fact that PEA was described to be a polyclonal activator of
cytolytic T cells in vitro (20) and our observation of a slight
increase in the amount of CD8-positive T cells in the liver 12 h
after challenge with PEA (cf, Fig. 6C, PEA 12 h), we
wondered whether effector molecules of cytolytic T cells might be
involved in PEA-induced hepatotoxicity. Perforin knockout
(perforin°) mice (40, 41) were significantly, but
not completely, protected from PEA-induced liver injury (Fig. 9). In contrast, mice lacking functional
Fas/CD95/APO-1 (lpr/lpr) were more susceptible to PEA
than corresponding wild-type mice (Fig. 9). Two of five
lpr/lpr mice did not survive the 12 h, while all
wild-type MRL/Mp mice were alive. These results suggest that CTL are at
least partially involved in PEA-induced hepatotoxicity using the
perforin-dependent pathway. Fas, in turn, expressed by the T cells
could possibly slow down the liver damage by mediating
activation-induced T cell death (42, 43).
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Discussion |
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-amanitin (11), which usually do not affect patients suffering from
septicemia. Our results presented here demonstrate for the first time that a bacterial toxin affecting the host during bacteremia, i.e., the protein synthesis inhibitory PEA, sensitizes the liver and probably also other organs toward either LPS or SEB or their common mediator TNF. This was shown by injection of subtoxic doses of PEA together with recombinant murine TNF, LPS, or SEB to mice and by demonstrating the development of liver damage. Moreover, toxic doses of PEA induced liver damage by themselves and stimulated the release of TNF and other cytokines by the immune system. Experiments using neutralizing Abs clearly identified TNF as a central mediator of this hepatic failure. Liver injury was characterized by early apoptosis that preceded the release of transaminases. A similar effect of TNF has been described in the case of coadministration of actinomycin D and TNF (29), GalN and TNF (33), or GalN and SEB (7). PEA-induced hepatocyte death was inhibited by the unspecific caspase inhibitor Z-VAD.fmk, as it was recently shown for GalN/TNF-induced apoptotic and secondary necrotic liver cell death (31).
In contrast to LPS or SEB, which induce systemic peak concentrations of TNF within 1 or 3 h, respectively (39, 6), and fulminant hepatitis in GalN-sensitized mice within 8 h (7, 12), PEA-induced plasma TNF was only measurable 12 h after injection of the toxin, i.e., at a time when hepatic necrosis was already detectable. However, circulating cytokine levels only insufficiently reflect local tissue concentrations, a concept that has been described as the tip of the iceberg (46). Indeed, after administration of very high doses of PEA to mice, plasma TNF was detectable by 90 min (data not shown). Furthermore, following injection of 300 µg/kg PEA, local hepatic TNF production was observed by 3 h after intoxication, as shown by means of immunofluorescent staining. This early appearing TNF was produced by Kupffer cells, but not by T cells. However, T cells were required for Kupffer cell-dependent early TNF production, providing evidence of cross-talk between T lymphocytes and liver macrophages. Such cross-talk has been previously suggested to occur upon Con A stimulation of lymphocyte/macrophage cocultures in vitro (47). Interestingly, we observed two phases of TNF production in PEA-treated mice. The first peak occurred after 3 h, and this TNF was found to be locally produced by liver macrophages. The second increase, measurable as systemic TNF, appeared after 12 h. A similar time course of TNF release was recently demonstrated by Toyabe et al. in a T cell-dependent hepatitis model in mice (48). It seems likely that Kupffer cells become activated by resident liver T cells upon PEA stimulation, because there was no additional infiltration of CD4- or CD8-positive T cells into liver tissue following PEA injection. Twelve hours after PEA challenge, Kupffer cells were completely depleted. This is probably a result of PEA-induced ongoing ADP ribosylation of translation elongation factor 2 in these phagocytic cells, which is the mechanism by which PEA inhibits protein synthesis. In contrast, T cells seem to be resistant to direct killing by PEA, because their cell number did not decrease in liver sections even 12 h after PEA injection. This is in line with earlier in vitro findings showing only marginal and short-lived inhibitory effects of PEA on protein synthesis in lymphoid cells (20). The number of CD8-positive T cells in the liver even increased 12 h after PEA challenge, providing a possible explanation for the dependence of liver injury on perforin. These findings support an earlier report describing PEA as a polyclonal activator of cytolytic T lymphocytes in vitro (20). TNF was not detectable in liver sections 12 h after administration of PEA, demonstrating that the systemic TNF measurable at this time point was produced outside the liver. This late TNF might be extensively synthesized by T cells in lymphoid organs such as spleen or lymph nodes.
Besides the protective effect of anti-TNF Ab against PEA, further evidence of the critical role of TNF was shown by the resistance of tnfr1° and tnfr2° mice to PEA-induced hepatotoxicity. As described earlier, the p55 TNFR1 mediates liver injury and lethality in GalN/LPS-treated mice (45, 49) as well as TNF-induced hepatocyte apoptosis (50). In contrast, the blockade or the absence of p75 TNFR2 failed to protect mice from GalN/LPS-induced lethality (49, 51), and tnfr2° mice exhibited enhanced sensitivity to GalN/TNF-induced hepatic damage compared with the corresponding wild-type mice (G. Tiegs and G. Künstle, unpublished observations). However, we recently found in a different TNF- and T cell-dependent hepatitis model, which is induced by Con A and is independent of transcriptional inhibition (34), that tnfr1° as well as tnfr2° mice were protected from liver injury. This was explained by providing evidence of a critical role of transmembrane TNF (52) that has previously been described to cooperatively signal via both TNFRs (35). Hence, PEA-induced hepatocellular toxicity is possibly mediated by direct cytolytic action of Kupffer cell-derived transmembrane TNF and its activation of both TNF receptors (35). An alternative explanation may be given in view of the low TNF concentrations observed following PEA injection and the concept of ligand passing (36) from the high affinity TNFR2 to TNFR1, which, in turn, transduces cytotoxic signaling. We have in vivo evidence for the latter mechanism from experiments proving a significant hepatoprotection of tnfr1°, but enhanced sensitivity of tnfr2°, mice following administration of very high doses of PEA (data not shown).
The PEA-induced hepatotoxicity was clearly dependent on T cells. This was proven by the protective effect of T cell depletion by anti-Thy1.2 mAb, by the inability of nu/nu mice to develop liver injury, by the protective effect of cyclosporin A, and by the prevention of PEA-dependent liver failure following SEB pretreatment. Notably, a substantial inhibition of protein synthesis by PEA in mice was only observed in the liver, whereas the spleen was much less affected (15). Furthermore, T cells were not depleted from the liver by PEA in our experiments. These findings suggest that despite inhibition of hepatic protein synthesis, T cell stimulation by PEA was still possible in vivo. The lack of T cells in nu/nu mice or following anti-Thy1.2 mAb-dependent depletion correlated with significantly reduced levels of plasma cytokines upon PEA challenge, and T cell depletion abolished early TNF production by Kupffer cells, indicating that T lymphocytes are necessary for the production of TNF and other cytokines. Suppression of T cell activation by cyclosporin A provided protection from PEA-induced liver injury, as it did in the case of Con A-induced, T cell-dependent liver failure (26) or lethal toxicity of the superantigen SEB in GalN-sensitized mice (6). Moreover, the induction of an immunosuppressed state characterized by Vß-unrestricted T cell unresponsiveness and defective APC functions 48 h after SEB pretreatment (37) also provided protection from PEA-dependent liver injury accompanied by suppression of TNF release. Because SEB pretreatment only protects from the deleterious effects of T cell mitogenic agents, not from LPS, this result again emphasizes the key role of T cells in PEA-induced liver failure. Furthermore, the prominent role of T cells in PEA-induced toxicity was shown by the prolonged survival of anti-Thy1.2- as well as SEB-pretreated mice. However, T cell depletion was not life saving. Furthermore, immediately before death, high activities of plasma transaminases were detectable in T cell-depleted mice (data not shown). Hence, liver damage might be the cause of death in these mice also. This points to a multifactorial mechanism leading to liver injury and death in PEA-treated mice that includes T cell activation and production of TNF. The release of transaminases in T cell-depleted mice at the time of death could be explained in terms of ongoing ADP ribosylation of elongation factor-2 in hepatocytes (15). Nevertheless, it has to be emphasized that T cell activation significantly contributes to PEA toxicity. In athymic BALB/c nude mice, which were shown to be protected from acute liver injury, survival was not significantly improved compared with that in wild-type BALB/c mice (data not shown). Again, at the time of death, high amounts of plasma transaminases were detectable in nude mice (data not shown). Hence, the results obtained from nude mice corroborate the findings from T cell-depleted mice concerning the role of T cells in acute hepatotoxicity and the significance of liver failure for death. However, the lack of prolonged survival of nude mice points to the existence of additional mechanisms that might have developed in these mice as a consequence of a whole life without T cells. These differences include higher amounts of NK cells and the presence of extrathymically matured T cells, which possibly caused the increased susceptibility of nude mice compared with that of mice only deficient in responsive peripheral T cells.
Previous in vitro findings suggested that PEA is a superantigen, as it selectively stimulated the proliferation of murine thymocytes expressing Vß8.2 in their TCR (21). PEA induced the proliferation of murine splenocytes (20) (data not shown). However, we failed to observe specific accumulation of either CD4+ or CD8+ T cells expressing the Vß8.1 or Vß8.2 chain in their TCR (data not shown). Hence, our preliminary results together with previous findings by others (25) argue against PEA being a superantigen.
Besides TNF, perforin was found to comediate PEA hepatotoxicity, as suggested by a significantly reduced susceptibility of perforin knockout mice to PEA-induced liver injury. TNF and other proinflammatory cytokines may indirectly activate cytotoxic T cells (53, 54, 55), thereby being responsible for additional hepatocytotoxic signals by the perforin/granzyme system. In contrast, the second important mediator of cytotoxic lymphocytes, i.e., Fas ligand (40, 41), seems unlikely to induce hepatocellular damage in our model; lpr/lpr mice lacking functional Fas suffered even more from PEA toxicity than corresponding wild-type mice. This result suggests a functionally different role of Fas in PEA-inducible T cell-dependent liver injury compared with the results obtained by Kondo et al., who demonstrated a protective effect of soluble Fas-Fc in two different murine hepatitis models (56). One possible explanation might be a suppression of Fas-dependent activation-induced T cell death (42, 43) following PEA stimulation, thereby aggravating liver injury via the TNF and perforin pathways. Peripheral clonal deletion of T cells has been shown to be impaired in lpr/lpr mice following SEB (57) or Ag (58) injection in vivo. Our results are well matched with previous findings showing that Fas-deficient lpr/lpr mice were more susceptible than wild-type mice to GalN/SEB-induced lethality (59), which is mediated by T cells as well.
In conclusion, we propose a sequential stimulation of the immune system
by PEA resulting in hepatocytotoxicity (Fig. 10): T cell activation by PEA results
in the production of the central mediator TNF by Kupffer cells, which,
in turn, induces apoptotic signals in hepatocytes via the TNFR1,
whereas TNFR2 possibly passes the ligand or costimulatory signals to
TNFR1. The inhibitory effect of PEA on protein synthesis sensitizes the
liver to the cytotoxic action of TNF. TNF and other cytokines may also
activate cytotoxic T cells, providing additional hepatocytotoxic
signals by the perforin/granzyme system. The continual immune
stimulatory and hepatocytotoxic processes finally result in severe
liver failure, which causes lethality. Hence, protein synthesis
inhibition during Pseudomonas septicemia is likely to be a
relevant sensitization mechanism to the cytotoxic action of TNF, which
is produced not only upon direct stimulation of monocytes/macrophages
by LPS but also via T cell activation with PEA.
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Acknowledgments |
|---|
, and to Dr. A. Wendel (University of
Konstanz, Konstanz, Germany) for providing an antiserum that
neutralizes murine TNF. We thank Dr. W. Neuhuber (Institute
of Anatomy, University of Erlangen-Nurnberg, Erlangen, Germany) for
experimental support regarding confocal laser scanning microscopy. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Gisa Tiegs, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nurnberg, Universitätsstrasse 22, D-91054 Erlangen, Germany. E-mail address:
3 Abbreviations used in this paper: PEA, Pseudomonas aeruginosa exotoxin A; SEB, Staphylococcus aureus enterotoxin B; GalN, D-galactosamine; HSA, human serum albumin; LAL, Limulus amebocyte lysate; ALT, alanine aminotransferase.
Received for publication December 18, 1997. Accepted for publication July 10, 1998.
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