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IMMUNOBIOLOGY
From the Departments of Pathology, Medicine, and
Surgery, Arthur G. James Comprehensive Cancer Center, The Ohio State
University, Columbus, OH; Children's Hospital, Columbus, OH; Vertex
Pharmaceuticals, Cambridge, MA; Genetics Institute, Andover, MA; and
Roswell Park Cancer Institute, Buffalo, NY.
The administration of therapeutic doses of recombinant cytokines to
patients with malignant disease can be complicated by systemic
toxicities, which in their most severe form may present as a systemic
inflammatory response. The combination of interleukin (IL)-18 and
IL-12 has synergistic antitumor activity in vivo yet has been
associated with significant toxicity. The effects of IL-18 plus IL-12
were examined in a murine model, and it was found that the daily,
simultaneous administration of IL-18 and IL-12 resulted in systemic
inflammation and 100% mortality within 4 to 8 days depending on the
strain employed. Mice treated with IL-18 plus IL-12 exhibited unique
pathologic findings as well as elevated serum levels of proinflammatory
cytokines and acute-phase reactants. The actions of tumor necrosis
factor- Natural killer (NK) cells are large granular
lymphocytes that compose approximately 10% of peripheral blood
mononuclear cells and, along with macrophages, granulocytes,
eosinophils, and basophils, form the cellular arm of the innate immune
system. NK cells can mediate the lysis of malignant and virally
infected cells without prior sensitization and are also an important
source of immunomodulatory cytokines such as interferon (IFN)- IL-12 is produced primarily by macrophages and appears to play a
critical role in determining the outcome of infection through its
ability to coordinate and activate the various compartments of cellular
immunity.11,12 IL-12 induces the secretion of IFN- IFN- In the present report, we have employed a murine toxicity model to
characterize the lethal response to coadministration of IL-18 plus
IL-12. While doses of the individual cytokines were well tolerated, the
administration of IL-18 in combination with IL-12 induced a unique
fatal systemic inflammatory reaction characterized by elevated serum
levels of proinflammatory cytokines and acute-phase reactants as well
as by multiorgan pathology. In the present report, we demonstrate that
this toxicity is mediated by NK-cell production of IFN- Reagents
Mice
Analysis of cytokine-treated mice Serum levels of IFN- , IL-1 , and IL-10 were measured by
means of enzyme-linked immunosorbent assays (ELISAs) obtained from Endogen (Woburn, MA). TNF- levels were measured by means of an ELISA
obtained from Biosource International (Camarillo, CA). Macrophage inflammatory protein (MIP)-1, IL-6, and GM-CSF levels were
measured by means of ELISAs from R&D Systems (Minneapolis, MN).
Serially diluted serum was analyzed for haptoglobin and 1-acid
glycoprotein by immunoelectrophoresis, and the area under the
precipitation peak was quantified in arbitrary units with the use of
the National Institutes of Health Image program 1.61.40
The data for each peak were converted into milligrams per milliliter by
comparison with values obtained with calibrated mouse
acute-phase plasma.
Detection of IFN- -fluorescein isothiocyanate (FITC) or isotype control-FITC mAbs were added at a final dilution of
1/100, and cells were incubated for 30 minutes at room temperature. Cells were washed and resuspended in 1 mL of FACS buffer for analysis on a Coulter XL (Coulter, Miami, FL) flow cytometer as
described.26 NK cells were isolated from the spleens of
cytokine-treated SCID mice and analyzed for endonucleolytic cleavage of
cellular DNA via a flow-cytometric assay using propidium iodide, as
described.42
Analysis of IFN- transcript
levels in mouse spleens after treatment with
cytokine.26,43 Spleens were harvested, snap-frozen in
liquid nitrogen, and ground into a fine powder with a mortar and pestle
on dry ice. Total cellular RNA was extracted by means of the RNAqueous
total RNA isolation kit (Ambion, Austin, TX). Complementary DNA
(cDNA) was generated by means of standard methods,26 with
the use of 2 µg of cellular RNA and random hexamers (Perkin Elmer,
Norwalk, CT) as primers for first-strand synthesis. The PCR
mixture contained the following: cDNA template (2.5 µL); forward and
reverse primers for muIFN- (900 nm each);
6-carboxy-fluorescein-labeled probe for muIFN- (125 nm);
forward and reverse primers for 18S ribosomal RNA (rRNA, 50 nm
each); 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein-labeled probe
(50 nm) for 18S rRNA (internal control); and 2X TaqMan Universal PCR
Master Mix (PE Applied Biosystems, Foster City, CA). Reactions for
analysis of muIFN- transcript and 18S rRNA (control) were performed
in the same well of capped 96-well optical plates. The following amplification scheme was employed: 1 cycle at 50°C
for 2 minutes (AmpErase uracil-N-glycosylase
deactivation), 1 cycle at 95°C for 10 minutes (AmpliTaq Gold
activation), followed by 40 cycles at 95°C for 15 seconds
(denaturation), and 60°C for 1 minute (anneal/extension). Real-time
PCR data were analyzed with the use of Sequence Detector software
version 1.6 (PE Applied Biosystems). Final quantitation was derived by
means of the comparative CT method26 and is
reported as the n-fold difference in experimental cDNA
(IL-12-, IL-18-, or IL-12- plus IL-18-treated mice) as compared with a calibrator cDNA (PBS-treated mice).
Statistical analysis Statistical significance was analyzed by means of Student paired t test with P < .05 considered significant.
Concomitant administration of IL-18 with IL-12 is lethal in mice It has been previously demonstrated that IL-12 is well tolerated by inbred murine strains when administered at a dose of 1 µg/d.29 IL-18 exhibits antitumor actions in murine models of malignancy when administered daily at a dose of 0.5 through 1.0 µg/d.28 In order to establish the toxicity of this cytokine combination, rmuIL-18 (0.1 µg/d or 0.5 µg/d) was administered daily in combination with rmuIL-12 (1 µg/d) to C57BL/6 mice via the IP route (Figure 1A). Mice receiving IL-12 plus the lower dose of IL-18 exhibited only mild signs of systemic toxicity, and no deaths were observed even when injections were continued for a total of 14 days. In contrast, mice receiving IL-12 with the higher dose of IL-18 all died within 6 to 8 days. Similar results were obtained with IL-18 plus IL-12 in several different species of mice, including BALB/c mice, B6 x CBA mice, and C.B-17 mice bearing the scid/scid (SCID) mutation (Figure 1B). Indeed, SCID mice, which lack T and B cells,33 exhibited 100% mortality within 3 to 5 days of initiation of treatment. Of note, mice receiving daily injections of IL-18 or IL-12 alone exhibited minimal toxicity (Figure 1B and data not shown).
Histopathology C57BL/6 mice receiving daily injections of IL-18 plus IL-12 were subjected to histopathologic evaluation. Acute changes within the gastrointestinal tract were prominent and consisted of a diffuse degenerative enteropathy within both the small and the large intestines (Figure 2A-B). Atrophy of the lymphoid tissues was also noted. This may have resulted from apoptotic events within these organs, as apoptotic bodies were frequently observed (Figure 2C). Analysis of nonadherent splenic NK cells from cytokine-treated SCID mice by propidium iodide staining confirmed this observation (Figure 2D). Examination of the liver revealed diffuse moderate histiocytosis (macrophage proliferation) as well as diffuse hepatocellular vacuolization. Pulmonary pathology consisted of moderate diffuse perivascular and septal inflammation associated with mononuclear cell infiltrates and alveolar edema (data not shown).
Taken together, these findings suggest that administration of IL-18
with IL-12 induced an acute systemic inflammatory response with marked
injury to the liver, lung, and intestines. This interpretation is
supported by our analysis of serum acute-phase reactants, which revealed the presence of high levels of haptoglobin and Serum cytokine levels The combination of IL-18 plus IL-12 is a potent stimulus for NK-cell secretion of IFN-, TNF-, GM-CSF, and MIP-1/ in
vitro,26,45 and we considered whether overproduction of
these proinflammatory factors in response to coadministration of IL-18
plus IL-12 might be the origin of the observed toxicities. We therefore
measured serum levels IFN-, TNF-, GM-CSF, and MIP-1 in SCID
mice receiving daily injections of IL-18 plus IL-12. IFN- and
TNF- levels rose rapidly in SCID mice treated with IL-18 plus IL-12,
peaked at approximately 48 hours, and remained elevated until the death of the animal (Figure 3A-B). Serum levels
of GM-CSF were only modestly elevated following treatment with IL-18
plus IL-12 (Figure 3C), and levels of MIP-1 were not significantly
elevated at any time point (data not shown).45
Administration of IL-18 alone did not stimulate significant endogenous
production of IFN-, whereas modest serum levels of IFN- (below
500 pg/mL) were observed in mice receiving injections of IL-12 alone
(Figure 3A).28,46 Likewise, mice receiving injections of
IL-18 alone did not exhibit significant serum levels of TNF-,
GM-CSF, or MIP-1 (data not shown).
Elevated serum levels of IL-1 Role of IFN- or
IFN- can induce a shocklike state in animals and humans, whereas neutralization of these factors can markedly attenuate septic shock in
animal models.51 Given the presence of these factors in
the serum of mice receiving IL-18 plus IL-12, we proceeded to
investigate the toxicity of this cytokine treatment in mice that had
been rendered genetically deficient in the p55 TNF receptor (R) or the
IFN- ligand. TNFR p55 / mice were not protected from
the toxicity of IL-18 plus IL-12. In fact, mice lacking this receptor
component died slightly earlier than similarly treated mice of the
identical background (data not shown). In contrast,
IFN-/ mice were completely resistant to the toxic
effects of IL-18 plus IL-12, whereas normal mice of the identical
background all succumbed to this treatment within 5 to 9 days (Figure
4). Gross examination of the
IFN-/ mice revealed minimal pathology outside of
some minor changes within the spleen, which is in keeping with the
complete lack of measurable IFN- in the serum of these mice (not
shown). Importantly, serum levels of TNF- and IL-1 were still
significantly elevated at the 72-hour time point in
IFN-/ mice receiving IL-18 plus IL-12. These
experiments indicate that the endogenous production of IFN- in
response to IL-18 plus IL-12 is a critical component of the observed
toxicity and also suggest that IL-18 plus IL-12 can induce the
production of proinflammatory cytokines in an
IFN--independent fashion.
Intracellular staining of murine splenocytes and
measurement of IFN- in this toxicity model. Using an anti-muIFN- mAb
conjugated to a fluorescent moiety, we performed intracellular staining
of splenocytes obtained from cytokine-treated C57BL/6 mice at 24 hours.
These results are presented in Figure 5A
and reveal that NK cells are the major source of IFN- in mice
receiving IL-18 plus IL-12. More dramatic results were obtained in SCID mice in which the percentage of NK cells are increased relative to
normal mice (Figure 5B). In order to analyze the synergistic effects of
IL-18 plus IL-12 at the transcript level, we administered PBS, IL-18,
IL-12, or IL-18 plus IL-12 to SCID mice and analyzed splenocytes for
IFN- transcript via Real-Time PCR at 24 hours. IL-12 alone
stimulated significant transcription of the IFN- gene in comparison
with PBS (50-fold induction), while IL-18 alone was completely
ineffective in this regard (no induction). However, the combination of
IL-18 plus IL-12 resulted in a significant 140-fold increase in
transcript at the 24-hour time point as compared with PBS-treated cells
(P < .05). Taken together, these experiments suggest that
coadministration of IL-18 and IL-12 results in a marked and rapid
increase in the transcription of the IFN- gene within the NK-cell
compartment.
We next analyzed splenocytes for IFN- Role of NK cells in the toxicity of IL-18 plus IL-12 Our results suggested that NK cells were the major source of IFN- in this model. To determine whether the toxicity of this model
was critically dependent on the actions of NK cells, we administered
IL-18 plus IL-12 to SCID mice depleted of NK cells by pretreatment with
an anti-asialo GM1 antibody. We have previously determined that this
regimen removes the majority of NK cells (approximately 98%) in most
murine strains while leaving the macrophage compartment largely
unaffected.30 This treatment completely abrogated the
toxicity of IL-18 plus IL-12 in both SCID mice and normal C57BL/6 mice,
whereas control mice all died within 5 days of the initiation of
treatment (Figure 6A and data not shown). Importantly, antibody-treated mice exhibited very low serum levels of
IFN-, TNF-, and IL-1 (less than 10 pg/mL) at all time points (data not shown). To confirm the role of NK cells in the lethal reaction to IL-18 plus IL-12, we next administered IL-18 and IL-12 to
CD3 transgenic mice that completely lack mature NK cells and T cells
owing to a developmental block.34 Control mice of the appropriate background died between 4 and 7 days, whereas CD3 transgenic mice all survived this treatment (Figure 6B). In keeping with our hypothesis, CD3 transgenic mice demonstrated very little reaction to IL-18 plus IL-12 at the histologic level (see Figure 9A-B) and exhibited a complete lack of serum IFN-. Taken
together. these data suggest that NK-cell-secreted IFN- is the
major mediator of toxicity in mice receiving IL-18 plus IL-12.
Depletion/deactivation of monocytes/macrophages Given the ability of NK-cell-derived cytokines to potentiate macrophage effector functions3,10 and the presence of activated macrophages in the liver of mice receiving IL-18 plus IL-12, the role of macrophages in the toxicity of this model was investigated. SCID mice can be depleted of monocytes and macrophages by 50% in bone marrow, spleen, and blood, and 100% in the peritoneal cavity by injection of the F4/80 mAb via the intravenous and IP routes 48 and 24 hours prior to the administration of IL-18 plus IL-12.44 Alternatively, macrophages can be deactivated via administration of IL-10.44,53 Our preliminary experiments revealed that these maneuvers led to partial protection in the present model (approximately 50% survival), which is consistent with our experience in other models of cytokine-induced inflammation (not shown).44 Therefore, in order to deactivate any macrophages remaining after the administration of F4/80 mAb, mice also received IP injections of muIL-10 beginning 48 hours prior to the initiation of cytokine treatment. This regimen afforded significant protection from the toxicity of IL-18 plus IL-12, and mice in this group exhibited 100% survival (Figure 7). Of note, NK cells obtained from the spleens of mice receiving F4/80 mAb and IL-10 were fully responsive to stimulation with IL-18 plus IL-12 in vitro, as measured by IFN-
production (data not shown). Taken together, these data suggest that
monocyte/macrophage effector functions play a significant role in
mediating the lethal toxicity of IL-18 plus IL-12.
The toxicity of IL-18 plus IL-12 is not abrogated in mice exhibiting deficiencies in iNOS activity Nitric oxide has been identified in the serum of mice receiving IL-18 plus IL-12 and has been implicated in the gastrointestinal toxicity observed in some murine models of infection.21 In addition, the fungicidal activity of murine peritoneal exudate cells is synergistically induced by treatment with IL-18 plus IL-12.54 This activity correlated with NK-cell production of IFN-, which in turn induced the production of nitric oxide. For
these reasons, we investigated the role of nitric oxide in the toxicity
of IL-18 plus IL-12 administration. Mice rendered deficient in the
enzyme iNOS were treated with IL-18 plus IL-12 as were control mice of the identical background. Contrary to what we might have predicted, both groups of mice were equally susceptible to the toxic effects of
this cytokine treatment. Similar results were obtained in SCID mice in
which the activity of iNOS was inhibited via the administration of
L-NAME (data not shown). Taken together, these results strongly suggest
that nitric oxide is not a critical mediator of the lethal reaction to
IL-18 plus IL-12.
Role of STAT signaling in the toxicity of IL-18 plus IL-12 The binding of IL-12 to its specific receptor complex results in phosphorylation of STAT4 as well as STAT1, STAT3, and STAT5.55-57 Mice rendered genetically deficient in STAT4 do not produce IFN- following administration of IL-12 and exhibit
distinct immunologic defects.38 In addition, costimulation
of T cells with IL-18 and IL-12 results in nuclear translocation
of phosphorylated STAT4 and AP-1, and these transcription factors must
bind to specific regions of the IFN- promoter in order for
transcription to be initiated.58 We were therefore
interested in the effects of IL-18 plus IL-12 in mice exhibiting a
genetic deficiency in the STAT4 transcription factor.
STAT4/ mice and mice of the appropriate background
received IL-18 plus IL-12 daily via the IP route. Background mice all
died within 6 days of the initiation of therapy, whereas the
STAT4/ mice all survived (Figure
8A). In fact, these mice exhibited essentially no toxicity whatsoever even when injections were carried out for a period of 2 weeks. Histologic analysis of these mice confirmed the protective effects of the STAT4 mutation (not shown), and
analysis of serum from these mice demonstrated only very low levels of
IFN- (less than 50 pg/mL at all time points). These results suggest
that the toxic effects of IL-18 plus IL-12 are dependent on the
activation of STAT4 and confirm the importance of this transcription
factor in the induction of IFN- gene expression.
Binding of IFN-
We have demonstrated that coadministration of IL-18 and IL-12
results in a systemic inflammatory response that is fatal within a
matter of days. Analysis of mice treated with this cytokine combination
revealed histopathologic evidence of systemic inflammation and a
concomitant acute-phase response. The toxicity of this cytokine combination was abrogated in mice lacking NK cells, but not in T-cell-
and B-cell-deficient SCID mice. Production of IFN- NK cells appear to be central to the toxicity of cytokine combinations
utilizing IL-12. The combination of IL-2 plus IL-12 is similar to that
of IL-18 plus IL-12 in that it exhibits synergistic antitumor activity
in experimental systems, unfortunately at the cost of severe systemic
toxicity.59 We recently examined the effects of IL-2 plus
IL-12 in a murine model and found that this cytokine treatment induced
a lethal inflammatory response that was similar in many respects to
septic shock, as evidenced by the presence of increased pulmonary
edema, multiple organ system toxicities, and elevated serum levels of
proinflammatory cytokines and acute-phase reactants.44
Cellular-depletion experiments revealed that this cytokine-induced
toxicity reaction was dependent on the NK-cell compartment and its
ability to mobilize the effector functions of monocytes/macrophages, as
was the case in the present model. Interestingly, however, the lethal
reaction to IL-2 plus IL-12 was not critically dependent on any of the
known cytokine products or effector molecules of the NK-cell
compartment (eg, IFN- We have shown that the combination of IL-18 plus IL-12 induces a
severe enteropathy characterized by villus atrophy, crypt hyperplasia,
and abundant apoptotic cells in the crypt epithelium. Despite the
severity of these gastrointestinal changes, we cannot say with
certainty that they represent the primary cause of death. We observed
only mild degenerative changes in the gastrointestinal tract of mice
receiving IL-12 alone44 and believe that the addition of
IL-18 to this regimen may somehow exacerbate this process. Work by
other investigators suggests that overproduction of nitric oxide might
play a role in mediating the pathologic tissue damage associated with
dysregulated or inappropriate production of
IFN- To the best of our knowledge, the toxicity of an anticancer cytokine treatment has never before been ascribed to a specific signaling pathway. This discovery suggests a distinct strategy for ameliorating the toxicity of this cytokine treatment as well as characterizing the factors that might mediate its antitumor effects. Further study of NK-cell activation following costimulation is also warranted given the role of this cell type in the toxicity of the present model. Current efforts in our laboratory are now directed at a closer analysis of the immunobiology associated with the administration of cytokine combinations that induce systemic inflammatory responses.
Submitted December 1, 1999; accepted April 14, 2000.
Supported by National Institutes of Health grants CA68326 and CA68458 and in part by National Institutes of Health grant P30 CA16058.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: William E. Carson III, Arthur G. James Comprehensive Cancer Center, The Ohio State University, N924 Doan Hall, 410 W 10th Ave, Columbus, OH 43210; e-mail: carson-1{at}medctr.osu.edu.
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S. C. Whitman, P. Ravisankar, and A. Daugherty Interleukin-18 Enhances Atherosclerosis in Apolipoprotein E-/- Mice Through Release of Interferon-{gamma} Circ. Res., February 8, 2002; 90 (2): e34 - e38. [Abstract] [Full Text] [PDF]
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production and STAT-mediated signal
transduction
Top
Abstract
Introduction
) mice and C57BL/6 control mice (
)
received daily IP injections of IL-18 plus IL-12 and were monitored for
toxicity. These experiments are representative of 2 separate
determinations.
B).
cells.
J Immunol.
1998;160:4738