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Produced by Kupffer Cells Protects Against the Nonspecific Liver Toxicity of Immunotoxin Anti-Tac(Fv)-PE38, LMB-2
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Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and
Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC 27106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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by these cells.
Following LMB-2 administration to mice, the levels of TNF- in the
liver increased to very high levels, whereas the rise in serum levels
was modest. In addition, the LMB-2-induced liver toxicity was blocked
by a specific TNF binding protein (TNFsRp55). Liver toxicity was also
blocked by indomethacin, which also blocked the rise of TNF- in the
liver. Both TNFsRp55 and indomethacin treatment protected mice against
a lethal dose of LMB-2. These data indicate that TNF- produced in
the liver by Kupffer cells has an important causal role in the
nonspecific liver toxicity of LMB-2. These findings have important
clinical implications for the use of immunotoxins in the therapy of
patients with cancer. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit of the IL-2 receptor (CD25) fused to a
38-kDa form of Pseudomonas exotoxin A (PE) (1, 2). In a recently completed clinical trial, LMB-2 produced eight
major responses in patients with leukemia or lymphoma, including one
complete remission lasting more than a year (3). A
prominent side effect in this trial that needs to be controlled is
liver toxicity (4). Recently, we reported the use of
site-directed mutagenesis and molecular modeling to decrease the
isoelectric point (pI) of the Fv portion of LMB-2 from 10.21 to 6.82
and found that a mutant of LMB-2 with a pI of 6.82, M1(scFv)-PE38, was
more than 3-fold less toxic to mice, yet had the same specific
cytotoxic activity and the same antitumor activity as the parental
LMB-2 (5). This approach represents one way to diminish
side effects of the immunotoxin. Although lowering the pI of the Fv
allowed the use of larger amounts of the immunotoxin, liver damage was
still the dose limiting toxicity in mice. The current study addresses
the mechanism of LMB-2-induced liver toxicity. PE is a bacterial toxin that binds to many types of mammalian cells. It inhibits protein synthesis by the ADP ribosylation of elongation factor 2 (EF2) (6). PE38, the fragment of PE used to make LMB-2, is missing the cell blinding domain of PE, and therefore should not be toxic to liver cells. Therefore, the liver toxicity of LMB-2 in mice must be caused by the nonspecific intake of LMB-2 by cells in the liver, because the Fv fragment used to make LMB-2 binds to human but not mouse cells.
Bacterial toxins play a central role in septic shock and sometimes
cause hepatic damage due to the systemic inflammatory processes. LPS
plays a major role in the processes. LPS is known to stimulate
monocytes to produce TNF-, IL-1ß, and other proinflammatory
cytokines that mediate multiorgan failure and cause cell death in
experimental animal models (7). PE has also been shown to
induce apoptosis in the liver, and this apoptosis is accelerated in the
presence of TNF- (8). Furthermore, acute hepatotoxicity
of native PE is partially mediated by TNF- (9).
In this study, we show that LMB-2 injected into mice accumulates in
Kupffer cells in the liver and that TNF- produced in the liver plays
an important role in LMB-2-induced nonspecific toxicity. Furthermore,
the toxicity of LMB-2 is blocked by a specific TNF- binding protein
or by indomethacin, which blocks the rise in TNF- produced by
LMB-2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LMB-2 was prepared as described previously (5).
LMB-2E553D, an inactive mutant form of LMB-2, was prepared by a similar
protocol to LMB2 using plasmid pRK79 M. LMB-2E553D did not induce liver
damage and was not toxic to mice when 100 µg/mouse was given by i.v.
injection (data not shown). LPS content of all immunotoxins used was
<10 Eu/mg and meet FDA criteria for human use. The amount of LPS in
0.9 mg/kg LMB-2 does not induce TNF- levels in mice (data not
shown). The LPS content of immunotoxins was determined using
Limulus amebocyte lysate (Associates of
Cape Cod, Woods Hole, MA). TNF--soluble receptor P55 (TNFsRp55) and
IL-1 receptor antagonist (IL-1Ra) were obtained from R&D Systems
(Minneapolis, MN).
Mice
Six- to 8-wk-old female NIH Swiss mice were obtained from the National Cancer Institute (Frederick, MD). Animals received humane care according to National Institutes of Health guidelines under an approved protocol.
Animal treatment
All reagents were injected in a total volume of 200 µl/20 g mouse. LMB-2 (0.9 mg/kg, lethal dose) or LMB-2E553D (0.9 mg/kg) was injected i.v. in pyrogen-free PBS containing 0.2% human serum albumin (HSA) (10). In the experiments blocking the toxic effects of LMB-2, mice were treated as follows: 1) 10 mg/kg i.p. of TNFsRp55 in PBS/0.2% HSA 15 min before injection of LMB-2 and 24 h after LMB-2 (11); 2) 10 mg/kg i.p. of IL-1Ra in PBS/0.2% HSA 15 min before injection of LMB-2 and 24 and 48 h after LMB-2 (12); 3) 12.5 mg/kg or 1.25 mg/kg i.p. of indomethacin (Sigma, St. Louis, MO) in 100 µl of olive oil (Sigma) 24 h before injection of LMB-2 and 24 and 48 h after.
Liver homogenate preparation
Mice were anesthetized with methoxyflurane (Schering-Plough Animal Health, Madison, NJ) and the inferior vena cava severed to remove blood from the organs. Livers were removed from the mice, washed with PBS, and weighed. Livers were homogenized in cold buffer (10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 2 mM PMSF, 1 µg/ml antipain, 1 µg/ml leupeptin, 1 µg/ml pepstatin A; Sigma). The homogenates were centrifuged at 13,000 x g for 20 min. The supernatant was used to detect cytokines in the liver.
Analysis of liver enzymes and cytokines
Liver damage was assessed by measuring plasma enzyme activity of
alanine aminotransferase (ALT) measured by Anilytics (Gaithersburg,
MD). The cytokines TNF-, IL-1ß, and IL-6 were determined by ELISA
(R&D Systems).
Histological study
NIH Swiss mice were sacrificed 18 h after injection of immunotoxin. The livers and kidneys were fixed with 70% ethanol. Paraffin-embedded sections from each of these organs were stained with hematoxylin and eosin and examined histologically. For immunohistochemistry, the liver and kidney were frozen in liquid nitrogen and kept at -70°C. Six-micrometer cryostat sections were prepared, postfixed using 10% formaldehyde, then labeled using a primary immunohistochemical step consisting of the presence or absence of an affinity-purified rabbit Ab (2 µg/ml) reactive with PE. This was followed by affinity-purified secondary anti-rabbit IgG conjugated to HRP (25 µg/ml) and developed with diaminobenzidine without counterstain. The Ab incubations were conducted for 30 min (23°C) in the presence of 1% BSA.
Macrophage collection
NIH Swiss mouse peritoneal exudate cells (5 x
106) obtained 4 days following the i.p. injection
of 2 ml of 3% thioglycollate medium were plated in 96-well plates
(4 x 105 cells/well). Nonadherent cells and
nonviable cells were removed by washing with RPMI 1640 (Quality
Biological, Gaithersburg, MD) after incubation at 37°C for 1 h.
Adherent cells were found to be 
90% macrophages by light microscopy
(13). Also, indomethacin-pretreated exudate cells were
obtained 4 days following the i.p. injection of 2 ml of 3%
thioglycollate medium and 1 day following the i.p. injection of 100
µl of indomethacin in olive oil (2.5 mg/ml).
Cytotoxicity analysis
The cytotoxicity of LMB-2 was assessed on ATAC-4 cells by a protein synthesis inhibition assay (inhibition of incorporation of tritium-labeled leucine into cellular protein) in 96-well plates as previously described (14). The activity of the immunotoxin is defined by the IC50, compared with cells that were not treated with toxin.
Statistical analysis
The results were analyzed using Student’s t test. Survival estimates were calculated using the Kaplan-Meier life table method (15).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The i.v. injection of 0.9 mg/kg of LMB-2 into NIH Swiss mice
induces severe liver damage. Eighteen hours after injection, there is
an increase in the level of plasma transaminase (Fig. 1
). There are also severe morphological
alterations in the liver as determined by histological analysis of the
livers of mice sacrificed 18 and 24 h after immunotoxin injection
(Fig. 2). At 18 h, hepatocyte
necrosis is clearly evident. At the 24-h time point, severe hemorrhage
is also evident.
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To determine which cell types in the liver accumulated LMB-2, mice
were sacrificed 1.5 h after injection of a toxic dose of LMB-2 (2
mg/kg). This high dose of LMB-2 was necessary to obtain a detectable
signal by immunohistochemistry. Liver and kidney were removed at
various times after injection, rapidly frozen, and used for
immunohistochemical studies with an anti-PE Ab. In the liver, LMB-2
was detected in cells that had the location and morphology of Kupffer
cells (Fig. 3E, arrows). This
pattern was absent in livers from the PBS-injected control mice (Fig. 3F, arrows) and in LMB-2-injected mouse samples in which the
anti-PE Ab was omitted (Fig. 3D, arrow). In the kidney,
LMB-2 was detected in numerous small resorptive droplets in the apex of
proximal tubular epithelial cells (Fig. 3B, arrows). This
pattern was absent in both the PBS-injected control mouse kidney
stained with anti-PE (Fig. 3C) and in the LMB-2-injected
mouse kidney samples in which the anti-PE Ab was omitted (Fig. 3A).
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Because LMB-2 was found to accumulate in Kupffer cells, it seemed
possible that LMB-2 could have stimulated the production of
proinflammatory cytokines that are known to be made by such cells
(16). Therefore, we collected blood samples at various
time points after LMB-2 injection and measured the levels of TNF-
and IL-1ß in the serum. As shown in Table I, there was a small elevation of TNF-
and IL-1ß when compared with mice treated with diluent or a nontoxic
mutant of LMB-2 (LMB-2E553D). This mutant has undetectable ADP
ribosylation activity and does not produce animal toxicity (data not
shown). Table I shows the levels of IL-1ß. Both LMB-2 and LMB-2E553D
produced a rise in IL-1ß. However, there was no significant
difference in the elevations produced by LMB-2 or its nontoxic form
LMB-2E553D.
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To determine whether the levels of TNF- or IL-1ß were
elevated locally within the liver, liver tissue was removed at various
times after LMB-2 administration and homogenized. After centrifugation
to remove particulate matter, the supernatant was used for cytokine
analysis (Fig. 4). TNF- was found to
be elevated 8–10 h after administration of LMB-2. The peak level was
609 ± 56 pg/g at 10 h. PBS/0.2% HSA or LMB-2E553D did not
cause an elevation of TNF- levels in the liver (120 ± 55 pg/g
at 10 h). IL-1ß levels behaved differently from TNF- levels.
IL-1ß was elevated 4 h after LMB-2 injection (Fig. 4B, 3942.0 ± 2.0 pg/g) and remained elevated up to the
10-h time point. Although PBS/0.2% HSA did not increase IL-1ß,
LMB-2E553D did cause a rise in IL-1ß at both the 4- and 6-h time
points (2088.6 ± 332.2 pg/g). Overall, the effect of LMB-2 on
IL-1ß was more prolonged and of greater magnitude than that of the
nontoxic mutant.
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To assess the ability of cytokine production of Kupffer
cells by stimulation of LMB-2, we used peritoneal macrophages from NIH
Swiss mice as a substitute for Kupffer cells, because these cells are
the same lineage as Kupffer cells. Peritoneal exudate cells were
harvested 4 days following the i.p. injection of 3% thioglycollate
medium and were plated in 96-well plates. After incubation at 37°C
for 1 h, nonadherent cells and nonviable cells were removed by
washing with RPMI 1640 medium (see Materials and Methods).
Adherent cells were examined by microscopy for morphology and
viability. More than 90% of the adherent cells were macrophages, and
their viability was 99%. The cells were incubated with LMB-2, and
culture medium was collected at various times for the measurement of
TNF- and IL-1ß. Each measurement was done in triplicate. TNF-
was elevated 3 h after ex vivo stimulation by LMB-2 (Fig. 5A). The levels continued to
rise and reached a value of 1048 ± 30 pg/ml at the 24-h time
point. IL-1ß also was increased by LMB-2 incubation but lagged
slightly behind TNF- reaching a level of 206 ± 22.5 pg/ml by
24 h (Fig. 5B).
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as a mediator of LMB-2 hepatotoxicity
Because TNF- and IL-1ß have been identified as mediators of
hepatocellular apoptosis and liver damage in experimental mouse models
(17, 18), we investigated the possibility that these
proinflammatory cytokines could be mediators of LMB-2-induced liver
damage. Therefore, mice were pretreated with a specific TNF- binding
protein (TNFsRp55), to inactivate the TNF- produced following a
LMB-2 challenge, or with an IL-1Ra, to antagonize IL-1 action, or with
both. As shown in Fig. 6A,
TNFsRp55, but not IL-1Ra, significantly inhibited the rise of
ALT in the blood. TNFsRp55 combined with IL-1Ra also significantly
inhibited ALT release, but the combination was not more
effective than TNFsRp55 alone. These data indicate that TNFsRp55 by
itself can prevent the rise in ALT in the blood that is known to occur
when the hepatocytes are damaged.
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and 7). All the mice that
were administered LMB-2 (0.9 mg/kg) died within 4 days
(n = 20). In contrast, the survival rate of
TNFsRp55-treated mice that received LMB-2 at the same dose was 60%
when measured 14 days after administration of LMB-2 (n
= 10). LMB-2-treated mice receiving IL-1Ra alone did not have an
improved survival rate (0% survival, n = 9), and the
survival of mice receiving TNFsRp55 and IL-1Ra (55% survival,
n = 9; data not shown) was not greater than when
TNFsRp55 was used by itself. These data indicate that one cause of the
liver damage produced by LMB-2 is TNF- and most likely the TNF-
produced locally in the liver.
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(19). To do this, mice were treated
with indomethacin (12.5 mg/kg) i.p. 1 day before, on the same day, and
1 day after LMB-2 injection. Indomethacin treatment inhibited the ALT
elevation produced by LMB-2 administration in NIH Swiss mice (Fig. 6A) and the morphologic damage to the liver (Fig. 2). The
livers from the mice treated with indomethacin 12.5 mg/kg and LMB-2
appeared relatively normal. We also investigated the effect of a lower
dose of indomethacin (1.25 mg/kg) and observed partial protection
against LMB-2-induced liver damage (Fig. 2). This result indicates that
indomethacin protects against LMB-2-induced liver damage in a
dose-dependent manner. Indomethacin by itself did not affect the
histology of the liver and was not toxic to mice in these doses (data
not shown).
We then examined the effect of indomethacin on the rise in TNF- in
the liver produced by LMB-2 and found that indomethacin inhibited the
elevation of TNF- in the liver produced by LMB-2 (Fig. 6). This
suggests that by suppressing TNF- production indomethacin prevented
the liver damage (Fig. 6, A and B) and increased
the survival of mice treated with a toxic dose of LMB-2 (Fig. 7). These
data about the indomethacin treatment support the conclusion that
TNF- is a mediator of LMB-2 hepatotoxicity.
Indomethacin did not prevent the specific cytotoxicity of immunotoxin on cancer cells
To study whether indomethacin affected the specific cytotoxicity
of LMB-2 on cancer cell line, we measured the cytotoxic activity of
LMB-2 on ATAC4 cells with and without indomethacin (0.0625 mg/ml).
Cytotoxic activity was determined by inhibition of protein synthesis
using [3H]leucine. This assay reflects the
ability of immunotoxins to kill cancer cells (20). There
was no difference in the cytotoxic effect of LMB-2 between
indomethacin-treated cells and nontreated cells
(IC50 = 0.07 ng/ml) (Fig. 8). Thus, indomethacin, by blocking
TNF- production, might improve the therapeutic index of LMB-2 by
decreasing its toxicity without blocking its antitumor effect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous hypothesis was that hepatocyte toxicity occurred because
LMB-2 nonspecifically bound to hepatocytes and that this nonspecific
binding was due to the high pI of the Fv portion of LMB-2. However,
when we injected LMB-2 into mice and determined its location in the
liver by immunohistochemistry, we were surprised to find LMB-2
concentrated in Kupffer cells rather than in hepatocytes. It is
possible that some LMB-2 is also taken up by hepatocytes, but the
method used for localization was not sensitive enough to detect it.
Having demonstrated the presence of LMB-2 in Kupffer cells, we
conducted a series of studies focused on the role of Kupffer cells in
LMB-2-based hepatotoxicity. Kupffer cells are macrophages and are known
to produce cytokines in response to a variety of stimuli, including
LPS. Therefore, we measured cytokine levels in the serum of
LMB-2-treated mice and found a modest rise in TNF-, whereas
substances like LPS produce large elevations of this cytokine. It was
only when we examined the liver directly that substantially elevated
levels of TNF- and IL-1ß could be demonstrated implicating one or
both of these cytokines in liver damage.
A role for these cytokines in liver damage was supported by experiments
with indomethacin, which prevented the rise in cytokines in the liver,
the rise in blood ALT, the damage to the liver, and animal deaths
produced by LMB-2. To determine which of the cytokines was
involved, we used two specific inhibitors of cytokine function:
TNFsRp55 and IL-1Ra. When given by itself, TNFsRp55 prevented
LMB-2-induced liver damage and animal death, whereas IL-1Ra did not. We
conclude that the liver damage and subsequent death produced by LMB-2
is a consequence of TNF- production by Kupffer cells in the liver.
We wished to directly study the ability of LMB-2 to stimulate TNF-
production by Kupffer cells, but were unable to obtain sufficient
number of pure cells for this study. Therefore, we used peritoneal
macrophage as a substitute for Kupffer cells and found that the
macrophages produced TNF- after stimulation by LMB-2. Because tissue
macrophages and peritoneal macrophages are not functionally equivalent,
we plan to carry out in situ hybridization experiments in the future to
confirm this data. Our observation that TNF- levels peaked at 8
h, whereas ALT levels rose from 18 to 30 h, suggests that TNF-
produces progressive damage to liver cells that release ALT. It is of
interest that LMB-2 also caused a rise in IL-1ß, but IL-1ß did not
appear to have a major role in liver toxicity, because IL-1Ra did not
protect against liver toxicity and because IL-1ß levels in the liver
were also elevated by LMB-2E553D, a nontoxic mutant derivative of
LMB-2.
We also tested whether indomethacin prevents the LMB-2-induced
hepatotoxicity. Indomethacin treatment inhibited the LMB-2-induced ALT
elevation and TNF- elevation in liver. Indomethacin protects the
liver damage produced by LMB-2 by inhibiting TNF--mediated pathway.
Because indomethacin did not affect the level of TNF- stimulated by
LPS (21), the mechanism of TNF- production by LMB-2
should be different from the TNF- production by LPS. We also tested
the effect of indomethacin for TNF- production in peritoneal
macrophage cells in vitro. When macrophages were coincubated with LMB-2
(9 µg/ml) and indomethacin (0.125 mg/ml), the rise in cytokine
production (TNF- and IL-1ß) was markedly decreased (data not
shown). Because indomethacin enhances the LPS-induced TNF-
production (22), the mechanism of production of TNF- by
LMB-2 should be different from LPS.
Previously, indomethacin and other NSAIDs were shown to prevent the toxic effect of immunotoxin BR96(Fv)-PE40 in rats (23, 24). This immunotoxin, which contains a slightly higher m.w. form of PE than LMB-2, is directed against LeY, an Ag present on epithelial tumors. The major toxicity observed in these experiments was accumulation of fluid in the thoracic cavity and vacuolization of endothelial cells in the lungs. No damage to liver was reported in those studies. It is of interest that several nonsteroidal anti-inflammatory drugs as well as dexamethasone prevented toxicity in this rat model.
Agents such as TNFsRp55 and an Ab that neutralize the effects of TNF
(Infliximab) are currently in clinical use for the treatment of
rheumatoid arthritis and Crohn’s disease (25, 26). Our
data indicate that TNF- is the major cause of the liver toxicity
induced by LMB-2 in mice. Because both a TNF binding protein and an Ab
to TNF are already approved for use in the treatment of rheumatoid
arthritis and Crohn’s disease, these substances as well as
nonsteroidal anti-inflammatory drugs may be of use in preventing
some of toxic side effects of immunotoxin in humans. Clinically, we are
now evaluating whether an Ab to TNF- and a cyclooxygenase inhibitor
will protect patients against the toxic side effects of some
immunotoxins.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: LMB-2, anti-Tac(Fv)-PE38; PE, Pseudomonas exotoxin A; pI, isoelectric point; IL-1Ra, IL-1 receptor antagonist; ALT, alanine aminotransferase; HSA, human serum albumin.
Received for publication June 16, 2000. Accepted for publication September 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Immunol. 149:2089.[Abstract]
in rat-liver macrophages. J. Hepatol. 20:811.[Medline]
, rather than interleukin-1-ß, from human macrophages. Immunol. Lett. 31:85.[Medline]
monoclonal antibody combined with low-dose weekly methotrexate in rheumatoid arthritis. Arthritis Rheum. 41:1552.[Medline]
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),
TNF-
) or
TNFsRp55 (
) 24 h and 15 min before 0.9 mg/kg LMB-2.
B, The indomethacin treated animals received
indomethacin (12.5 mg/kg, 